UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

An evaluation of leeches as in situ biomonitors of chlorinated phenolic compounds discharged from bleached… Prahacs, Steven Michael 1994

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1995-0097.pdf [ 8.45MB ]
Metadata
JSON: 831-1.0086733.json
JSON-LD: 831-1.0086733-ld.json
RDF/XML (Pretty): 831-1.0086733-rdf.xml
RDF/JSON: 831-1.0086733-rdf.json
Turtle: 831-1.0086733-turtle.txt
N-Triples: 831-1.0086733-rdf-ntriples.txt
Original Record: 831-1.0086733-source.json
Full Text
831-1.0086733-fulltext.txt
Citation
831-1.0086733.ris

Full Text

A N E V A L U A T I O N OF LEECHES AS IN SITU BIOMONTTORS OF CHLORINATED PHENOLIC COMPOUNDS DISCHARGED FROM B L E A C H E D KRAFT PULP MILLS by  STEVEN MICHAEL PRAHACS B . S c , Concordia University,  1986  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER' OF SCIENCE in THE F A C U L T Y OF GRADUATE STUDIES RESOURCE MANAGEMENT AND ENVIRONMENTAL STUDIES PROGRAMME We  Accept to  the  this  thesis  required  as  conforming  standard  The University of British Columbia November  1994  © Steven Michael Prahacs,  1994  In  presenting  degree freely  at  the  available  copying  of  department publication  this  in  partial  fulfilment  University  of  British  Columbia,  for reference  this or  thesis  thesis by  for  his thesis  or  of  this  of  ^€SauRO£  and study. scholarly her  of I  I further  agree agree  purposes may b e  representatives.  for financial  the  gain  shall  It  is  requirements that that  the  for  an  advanced  Library shall  make  permission for extensive  granted  by the  understood  not be allowed  that  without  head  of  my  copying  or  my written  permission.  Department  t>\ftsAGgMgAjT.  The University of British Columbia Vancouver, Canada  Datef3^ Z  DE-6  (2/88)  .  ~  -  it  w  11  ABSTRACT  Leeches discharged  were  from  evaluated  bleached  kraft  investigations.  Semi-static  out  to  determine  concentration,  water  p H , water  factors  as  in  order  such  evaluate during  leeches summer  Fraser  leech  downstream  There  was  contaminant  with  chlorinated  guaiacols  correlated ionized  with  increased  Water relative 2  also  =  a  between  in  bi-phasic  sediments  kraft  ug/L)  related  to  such  were  as  sediments  and  contaminant  as w e l l  as b i o t i c  In  order  field  monitoring  (February)  were  conducted  mills =  2  and  0.89 -  the  species  model  of  of  between  the  chlorinated  suspended although  weight  bioassays There  was  strong  obscura  11.8 to  bioconcentration  concentration o f 5%  chlorinated  increased  and b i o c o n c e n t r a t i o n  (Nephelopsis  -  presence  o f the  the  Bioconcentration  sediment the  of  weakly  i n d i c a t i n g that  fraction.  temperature  bioavailability  sediment  leech  water  Bioconcentration  compound,  bioavailable  for  conditions.  leech  o n the  to p H (5.1 - 9.0), but was o n l y  reduced  semi-static  trials  0.96) between  days).  undissociated  to  at P r i n c e George B . C .  bioconcentration 28  field carried  three  (r  >  duration  bioconcentration.  pulp  correlation  was o b s e r v e d ,  suspended  0.94) between  t w o different  marmorata).  and w i n t e r  o f the  week  factors  affect  rates  relationship  the  quiescent  0.90 -  laboratory  o f one  compounds  4.4 and 11.8 ° C , but d i d not show a significant change from  i n suspended  to  integrated  situ c o n d i t i o n s ,  10  contributed  N o clear  stirring  -  depuration  g / L ) and b i o c o n c e n t r a t i o n  material  (r  (0.1  phenolic  and suspended  species  in  concentration  indicating  relationship.  and  linear  using  chlorinated  environmental  temperature  was i n v e r s e l y  the  between  °C,  0.15  slow  compound  how  (October)  strong  of  bioassays  o f three bleached  concentration  guaiacols,  20.0  fall  River,  mills  laboratory  varying  (July),  biomonitors  pulp  weight  under  a  as  and  -  organic  phenolics.  bioconcentration  inverse with  (0  no  relationship clear  trend  Percymoorensis  Ill  Field ug/L). during  monitoring revealed  chlorinated phenolics  in pulp mill  and water (0.002 - 0.073  pg/L) and suspended  sediments  the  study  tetrachlorinated factors  ranging  proportions  of  periods.  guaiacols from these  465  Leeches  under -  diverse  6000  chlorinated  and  were  effective  seasonal were  contaminants  Fraser River, 40 km downstream of pulp mill  in  both  outfalls.  (0.36  -  biomonitors  conditions,  accurate  effluent  with  indicators pulp  mill  176 of  (0.6  -17  ug/kg) tri-  and  bioconcentration of  the  effluent  relative and  the  iv  T A B L E OF CONTENTS  Page  Abstract  1 1  Table of Contents  iv  List of Figures  •  •  viii  List of Tables  x  List of Abbreviations  •  Acknowledgments  Dedication  *  x  x y  ,  ii  xviii  1. General Introduction  1  2. Literature Review  5  2.1. Bleached Kraft Mill Technology  v  5  2.1.1. Introduction  5  2.1.2. Kraft Pulping, and Washing  5  2.1.3. Pulp Bleaching  8  2.1.4. Effluent Treatment  12  2.2. Environmental Fate of Chlorinated Phenolics  15  2.2.1. Introduction  15  2.2.2. Sources.:  16  V  Page  2.2.3. Partitioning to the Water Phase 2.2.4. Partitioning to the Sediment Phase  17 .'  2.2.5. Partitioning to Biota  18 20  2.2.5.1. The Effect of Octanol-Water Partition Coefficient  22  2.2.5.2. The Effect of pH  23  2.2.5.3. The Effect of Dissolved and Particulate Solids...  24  2.2.5.4. The Effect of Temperature  26  2.2.5.5. The Effect of Multi-Contaminant Exposure  27  2.2.6. Biotransformation  28  2.2.7. Fate of Chlorinated Phenolics in the Fraser River  31  2.3. Biomonitoring  35  2.4. Leech Biology  41  3. Methodology  43  3.1. Experimental Organisms  43  3.2. Laboratory Studies  44  3.3. Field Studies  47  3.3.1. Study Area  47  3.3.2. Study Periods  49  3.3.3. Sampling Procedures  50  3.4. Analytical procedures  52  3.4.1. Chemicals and Reagents  52  3.4.2. Extraction Procedures  53  3.4.2.1. General Rational  53  3.4.2.2. Effluent Sample Preparation  54  3.4.2.3. Water Sample Preparation  55  3.4.2.4. Sediment  56  Sample Preparation...  vi  Page  3.4.2.5. Leech Sample Preparation  57  3.4.2.6. Reference  58  3.4.3. Instrumental  Standard Preparation  analysis  58  3.4.4. Quality Control  59  4. Results and Discussion  65  4.1. Laboratory Studies  65  4.1.1. Bioconcentration as a Function of Contaminant Concentration  65  4.1.2. Bioconcentration as a Function of Leech Weight  76  4.1.3. Bioconcentration as a Function of Water pH  83  4.1.4. Bioconcentration as a Function of Water Temperature.  94  4.1.5. Bioconcentration as a Function of Suspended  Sediment  Load  102  4.2 Field Studies  113  4.2.1. Temporal Variations in Contaminant Concentrations in Effluent, Water, Suspended Sediments and Leeches  113  4.2.2. In Situ Bioconcentration as a Function of Contaminant Concentration  125  4.2.3. In Situ Bioconcentration as a Function of Leech Weight.  132  4.2.4. Interspecies Differences  136  in Bioconcentration  4.2.5. Estimation of In Situ Water Using  Laboratory  5. Summary, Recommendations 6. References  Contaminant  Bioconcentration  and Applications  Concentrations  Relationships  139  142 151  vii  Page  7. Appendices  167 Appendix 1:  Leech Identification  Appendix 2:  Calculations of Reported Chlorinated Phenolics Concentrations  168  170  Appendix 3: Raw Data - Laboratory Bioassays  171  Appendix 4: Raw Data - Field Monitoring Trials  181  Appendix 5:  Laboratory Predictions of In Situ Water  Contaminant  Concentrations - Sample Calculation  192  Vlll  LIST OF FIGURES  Page  1.1:  Chemical structures and abbreviated names of compounds tested  3.1:  Fraser River biomonitoring study area  3.2:  Separation of chlorinated phenolic compounds on a 30 m DB-5  3  48  capillary column  3.3:  60  Separation of chlorinated phenolic compounds on a 30 m DB-1701 capillary column  4.1:  61  The effect of chlorinated phenolic exposure concentration on leech (N. obscura) bioconcentration of 3,4,5-TCG and 3,4,5-TCVer  4.2:  The elimination of chlorinated phenolics from leeches (N.  68  obscura)  over a four week period  4.3:  73  Bioconcentration of 3,4,5-TCG and 3,4,5-TCVer by leeches (N.  obscura)  of differing weights  4.4:  79  The effect of water pH on the bioconcentration of 3,4,5-TCG and 3,4,5-TCVer by leeches (N. obscura)  4.5:  Dissociation and leech (TV. obscura) bioconcentration guaiacols plotted against water pH  85  of  chlorinated 87  ix  Page  4.6:  Dissociation and leech (TV. obscura)  bioconcentration  of  chlorinated  phenols plotted against water pH  4.7:  Relationship between leech. (TV. obscura)  88  bioconcentration  and  log Kow of chlorinated phenolics at pH 5.1  4.8:  92  Alternative interpretations of the effect of water temperature on the bioconcentration of 3,4,5-TCG and 3,4,5-TCVer by leeches (TV.  4.9:  97  The effect of suspended sediment concentration on leech (TV.  4.10:  obscura)  obscura) bioconcentration of 3,4,5-TCG and 3,4,5-TCVer  The effect of the presence of organic material in suspended sediments on the bioconcentration of chlorinated phenolics  4.11:  110  Relationship between chloroguaiacol pKa and the ratio of leech (TV.  obscura) bioconcentration in turbid bioassay  water of 0% organic  particulate content versus 5% organic particulate content  4.12:  105  Ill  Relative concentrations of chlorinated phenolics in leeches and water samples from Stoner, B.C. and effluents from two Prince George B K M outfalls, over three seasonal monitoring periods  4.13:  Leech (TV. obscura)  124  bioconcentration factor as a function of water  concentration for 4,5,6-TCG detected in field studies for Jul., Oct., 1991 and Feb. 1992 and laboratory studies  126  X  Page  4.14:  Leech (N. obscura) bioconcentration  as a function  of water  concentration for 4,5,6-TCG detected in field studies for Jul., Oct.,1991 and Feb. 1992 and laboratory studies  4.15:  Leech (N. obscura) bioconcentration  126  as a function  of water  concentration for 3,4,5-TCG detected in field studies for Jul., Oct., 1991 and Feb. 1992 and laboratory studies  4.16:  Bioconcentration of 3,4,5-TCG (a) and TeCG (b) by leeches (N.  127  obscura)  of differing weights, exposed under both laboratory and field conditions  :  134  xi  LIST OF TABLES  Page  2.1:  Major  classes  and  sources  o f chlorinated  phenolic  compounds  found  i n softwood B K M effluent  2.2:  The  effect  o f percent c h l o r i n e d i o x i d e substitution  c h a r g i n g order  2.3:  10  on the  formation  C o m p a r i s o n o f effluent  quality  and  chemical  o f chlorinated phenolics  parameters for  various  13  sequences  used  for bleaching softwood pulp  2.4:  2.5:  T h e effect  13  o f b i o l o g i c a l treatment on effluent  D i s s o c i a t i o n constants  (pKa)  and  percent  quality parameters  dissociation of  14  chlorinated  phenolics i n water o f p H 7.8.  2.6:  Bioconcentration  of  17  chlorophenols  by  various  aquatic  organisms  sampled from Canagagigue Creek, Ontario, Canada  3.1:  M e t h o d detection i n effluent,  4.1:  The after  water,  limit  suspended  bioconcentration seven  0 . 1 - 1 0 ug/L  days  ranges for  sediments  of chlorinated  exposure  the  to  analysis  39  o f chlorinated  phenolics  and leeches  phenolics  concentrations  by  64  leeches  ranging  (N.  obscura)  from 67  Xll  Page  4.2:  The average proportion of 4,5,6-TCG found in leeches after seven days of semi-static exposure at various concentrations  4.3:  69  Bioconcentration factors of chlorinated phenolics, after seven days exposure to concentrations ranging from 0.1 - 10 ug/L  4.4:  71  Bioconcentration of chlorinated phenolics by leeches (N. obscura) of different weights  4.5:  77  The effect of water pH on bioconcentration of chlorinated phenolics by leeches (N. obscura)  4.6:  84  The effect of water temperature on bioconcentration of chlorinated phenolics by leeches (N. obscura).  4.7:  95  The effect of suspended solids concentration bioconcentration of chlorinated phenolics by leeches (N. obscura).  4.8:  104  Field conditions for July 8-15, October 17-24, 1991 and February 19-26, 1992 at two monitoring stations on the Fraser River  4.9:  115  Pulp mill process conditions for July 8-15, October 17-24, 1991 and February 19-26, 1992 at Prince George, B.C  4.10:  Flow weighted average effluent  115  concentrations of chlorinated  phenolics discharged from Prince George bleached kraft mill outfalls.  ..  116  Xlll  Page  4.11:  Seven  day mean  water  concentrations  o f chlorinated  phenolics  detected i n the Fraser R i v e r at Stoner, B . C  4.12:  Suspended  sediment  concentrations  117  o f chlorinated  p h e n o l i c s and  percent organic content i n the Fraser R i v e r  4.13:  Predicted  sediment-water  118  p a r t i t i o n coefficients  ( K s w ) for 3 , 4 , 5 - T C G  and T e C G i n the Fraser R i v e r at Stoner, B . C . i n July 1991 and February 1992  4.14:  Mean  tissue  120  concentrations  (|ag/kg)  o f chlorinated  phenolics i n  leeches exposed i n the Fraser R i v e r at Prince George, B . C  4.15:  Cross  channel  phenolics  differences  i n leeches  (TV.  i n tissue  concentrations  obscura) exposed  122  o f chlorinated  for seven day periods i n  the Fraser R i v e r at Stoner, B . C  4.16:  T i s s u e concentration o f different  4.17:  Predicted leeches  o f c h l o r i n a t e d p h e n o l i c s i n leeches  weights, after  decreases  (TV.  129  (TV.  obscura)  seven days exposure i n the Fraser R i v e r  i n bioconcentration  obscura) between  o f chlorinated phenolics by  0.5 g and 1.0 g under both  laboratory  and field conditions  4.18:  135  B i o c o n c e n t r a t i o n s o f c h l o r i n a t e d p h e n o l i c s i n two leech  Nephelopsis  obscuraand  133  Percy mo or en sis  the Fraser R i v e r at Stoner, B . C  marmorata  species,  exposed i n 138  xiv  Page  4.19:  B i o c o n c e n t r a t i o n s o f c h l o r i n a t e d p h e n o l i c s i n two Nephelopsis  obscuraand  Percymoorensis  leech  marmoratq  species,  after  seven  days exposure to a water concentration o f 1.0 u g / L  4.20:  C o m p a r i s o n o f measured and  TeCG  to  those  seven day field  predicted  from  138  water concentrations o f  laboratory  leech bioconcentration  relationships  5.1:  140  S u m m a r y o f effects (N.  obscura)  3,4,5-TCG  o f environmental and b i o t i c  factors  on leech  bioconcentration o f 3 , 4 , 5 - T C G and 3 , 4 , 5 - T C V e r  147  XV  LIST OF ABBREVIATIONS  A O X  adsorbable  B C F  bioconcentration  B K M  bleached  kraft  m i l l  B K M E  bleached  kraft  m i l l  B C D  b i o c h e m i c a l  B S W  b r o w n  C  chlorine  bleaching  D  chlorine  dioxide  DtC  chlorine/chlorine  E  alkaline  extraction  o x y g e n  reinforced  E  0  organic  halide factor  o x y g e n  stock  stage bleaching dioxide  f  fraction  organic  gas  G P C  gel  H  hypochlorite  stage  stage extraction  chlorine carbon  c h r o m a t o g r a p h y p e r m e a t i o n  c h r o m a t o g r a p h y  bleaching  K  D  W  octanol  K  s  w  sediment  - water  m o d i f i e d  continuous  M C C  bleaching  alkaline  organic  C C  stage  bleaching  extractable of  d e m a n d  w a s h i n g  E C O o c  effluent  - water  stage  partition  coefficient  partition c o o k i n g  coefficient  x v i  M D L  m e t h o d  detection  N D  n o n e  P O X  purgable  SS  s u s p e n d e d  T O Q  total  limit  detected organic  organic  1°  p r i m a r y  2°  s e c o n d a r y  halide  solids chlorine  XVII  ACKNOWLEDGEMENTS  This from  research  academic  Parkinson, Civil  of  S.  samples. leech  Harper  species  knowledge  and  samples Inc,  pulp  paper  studies lent  pollution  his  abatement  people  industry. Dept.  acknowledged  of Soil  diffraction  British  North  field  of  and  Columbia  studies.  Vancouver  S.  kindly R.  Derksen,  shared  Parnell,  expertise  technology.  Sr.,  for  Dept.  of C i v i l  this  research  completion  of  on this research.  Canfor  in  providing  S.P.  Biosphere  committee  S p e c i a l thanks is extended  Engineering, University of British and this  E n g i n e e r i n g Research  science thesis. Council  in This  general  as  research  o f Canada  well was  operating  C o l u m b i a , for as  endless  funded  by  in  grant to  guidance in  National  Dr. K . J . Hall.  the  members, useful  to D r . K . J.  patience a  his  Company,  consultations  Thesis  expert  excellent  G.  instrumental  Prahacs  for  Ministry  N o r t h w o o d P u l p and Paper were  of  sediment  contributed  pollution chemistry.  staff  P.  Science, U n i v e r s i t y  analysis  branch,  River  periods. time  paper  Laboratory,  gratefully  Dept.  George  Protection,  technical  and  of  G . B e l l w a r d , D r . T . Northcote, D r . H . Schreier and D r . L . L a v k u l i c h p r o v i d e d  insight and commentary  of  are  D . Sutherland,  Fraser  River  pulp  efforts  University of Calgary, provided  and J. N y l u n d ,  mill  field  the  Engineering  X-ray  and  for  Fraser  Pulp Division  generously  and  the  Prince  aid  Environmental  during  as  Columbia,  of Biology,  Parks,  with  well  collaborative  Dr. L . Lavkulich,  W . Duncan  concerning  the  Environmental  British  advisory  along  as  performed  and  and  data  George,  of  of  kindly  Canada,  International area  Ma,  support.  Lands  P r i n c e George  effluent  T.  identification.  through  institutions  University  technical  Environment  Prince  possible  D r . R . W . D a v i e s , Dept.  logistical,  Dr.  and  Columbia,  Environment,  Ltd.,  made  government  a n a l y t i c a l , technical British  of  and  Engineering,  their  was  Hall,  i n a l l aspects awaiting Sciences  the and  XV111  DEDICATION  This  thesis  is  dedicated best  with' all m y friend  heart  anyone  and could  soul  to  have.  Brigita G r a z y s ,  the  1  1 . INTRODUCTION  On a broad scale, resource management can be defined as the development and execution of plans for the integrated, sustainable use of natural resources; a balance between  human use  and conservation.  Comprehensive management  and weigh social, economic and biophysical impacts of resource  should address  use, although the  nature and relevance of each aspect is unique to a given situation.  Management  decision making requires the integration of a vast array of needs with often complex and uncertain information.  In the case of water resources, both water quantity and  quality issues must be examined. water  demand  infrastructure, habitat.  water-deficient  protection  The latter  contaminants  track  of  regions,  drinking water  two concerns  are  municipal  quality  and  closely linked  sewage  maintenance  with  treatment of  management  aquatic of toxic  discharged by point and non-point anthropogenic pollution sources.  Water to  in  Areas of concern are diverse and may include:  the  pollutant spatial  management and  identify  the pollutants  periods  when  requires  temporal  monitoring  distribution of contaminants.  of concern as well  potential  a comprehensive  environmental  as determine  impacts  may  program  Monitoring  sensitive areas and time be  manifested.  Aquatic  monitoring data serve to aid in the assessment of the risks to both humans affected regulation efforts.  ecosystems  as  of pollutant There are  well  as  the  discharge,  effects  of management  industrial process  always many uncertainties  environmental decision making; therefore,  can  changes  decisions and site  concerning remediation  and unknowns to contend  it is desirable to have  and  with in  a comprehensive  data base from which to effect informed decisions. With the high economic cost associated with comprehensive field monitoring, it  is  necessary  to  select  monitoring techniques  information at the lowest cost.  which  yield  the  most  pertinent  In the case of bleached kraft mill (BKM) sources,  target areas for monitoring include: effluent discharges, receiving waters,  sediments  2 and aquatic organisms.  In most cases monitoring is carried out separately in one or  more of these areas.  Biomonitoring organisms can provide useful information in a  number of effluent  areas,  including seasonal  concentrations  (Phillips  This thesis involves monitoring  tool  contaminants  for  specific  (Figure  1.1)  purpose  to the as  of  discharge  in situ  routine  of bleached  biomonitors  through an integrated  monitor,  presence,  with  relative  the  of  program of  ability  proportions  compounds by assessment of In order to literature  review  abatement  provide  covering:  technology,  2)  as relative  water and  and assessment of an aquatic, biological monitoring  organochlorine  kraft pulp mills. chlorinated  Specifically,  phenolic  compounds  studies.  This  obscura, to be a hardy and sensitive  to  provide reliable  and  bioavailability  tissue  of  laboratory and field  thesis will show the leech species, Nephelopsis pollutant  well  1978).  the development  the  leeches are evaluated  bioavailability, as  information of  concerning  chlorinated  the  phenolic  bioconcentrations.  adequate  background information the  1)  of  state  the  art kraft  mill  thesis  process  environmental fate of chlorinated phenolic  contains  a  and pollution compounds,  3)  bioconcentration  of  biomonitoring theory, 4) leech biology. Many organic 1990),  environmental  contaminants, water  such  and as:  pH (Saarikoski  biotic  factors  contaminant  influence  concentration  and Viluksela 1981;  Hall  the  and temperature and Jacob  (Barron  1988), organic  particulate concentration (Lee et al. 1993), uptake/depuration rate (Ellgehausen et al. 1980) and body size (Connell 1991). spatial  variations,  environmental monitoring  sediment  is  factors  on  information.  bioconcentration laboratory:  it  of  concentration.  necessary  to  quantify  bioconcentration, The effect  chlorinated  contaminant  Since, in situ conditions show wide temporal and  of  the  phenolic  concentration,  in  the  order  nature to  following compounds  water pH, water  get  and  accurate,  environmental was  magnitude long  temperature  Ideally, routine biomonitoring should employ  uniform age, size and condition from a laboratory controlled population.  term  variables  investigated and  of  in  on the  suspended  organisms  of  However, in  reality, wild populations of differing life stage and origin must often be used.  3  Therefore,  it  is  bioconcentration.  necessary  to  identify  The effect of leech  and  quantify  interspecies  size on bioconcentration  was  variations  in  investigated  in  the laboratory.  Figure 1.1: Chemical structures of chlorinated phenolic  1)  Chlorinated phenols (CPs): OH  DCP  = dichlorophenol  TCP  = trichlorophenol  TeCP = tetrachlorophenol PCP  2)  = pentachlorophenol  Chlorinated guaiacols (CGs): DCG  = dichloroguaiacol  TCG  = trichloroguaiacol  TeCG = tetrachloroguaiacol  3)  Chlorinated catechols (CCs): DCC  = dichlorocatechol  TCC  = trichlorocatechol  TeCG = tetrachlorocatechol  4) Chlorinated vanillins (CVs): DCV = dichlorovanillin TCV  = trichlorovanillin  5) Chlorinated veratroles  (CVers):  DCVer  = dichloroveratrole  TCVer  = trichloroveratrole  TeCVer  =  tetrachlorveratrole  compounds.  4  Leeches under  a  were  variety  bioconcentration  of  seasonal  were  assessed  sediment  contaminant  primary  test  species, order  evaluated  species,  levels  laboratory  the  derived  accuracy  in  situ b i o m o n i t o r s  field  conditions.  relative  and v a r y i n g  Nephelopsis  Percymoorensis  to test  as  o f laboratory were  changing  was both  data,  compared  the  Temporal effluent,  environmental  obscura  marmorata,under  relationships,  to  in  field  water  against  a  bioconcentrations, field  and In  and l a b o r a t o r y  to measured  River  variations  conditions.  evaluated field  Fraser  system, in  leech  suspended  addition, second  conditions. predicted  values.  the  leech In from  5  2. L I T E R A T U R E  REVIEW  2.1. B L E A C H E D K R A F T M I L L T E C H N O L O G Y  2.1.1.  Introduction  A  variety  mechanical, sulphate, pulping (86%  of  and  the  total 1990).  loading  into  dominant  such  chlorinated  organic  the  chemical  used  2.1.2.  Kraft  The as  goal  benzene fibres.  is  being  a  large  rings The  wood, and  linked  1990)-,  a  summarized  world  1990).  acids  wide,  oxygen and  pollution  demand  kraft washing  along  with  produced  w h i c h was  bleached  of  pollutant  organic  pollutants (BOD),  plus  process  reprocessed  below,  of  kraft  tonnes  of  amount  terpenes  Columbia,  million  94%  including:  semi-chemical,  British  25.2  Classes  bleached  and  and  significant  pulping, pulp  recovered  organic  (Sinclair  biochemical  The  Canada with  for  fatty  wood  colour,  an  can  bleaching,  for  further  broken with  use.  descriptions  of  wood  assortment  be  and  discharged  the  down  much  The  of  of  major  technology  abatement.  Washing  o f pulping is  from  1989  compounds.  P u l p i n g and  lignins,  Lignin  and  used  process,  (Sinclair  resin  are  Throughout  responsible  (SS),  reagent  chlorinated  in  are  chemi-thermo-mechanical,  producing  waters,  stages:  involved  in  pulp  solids  as  following  processes  technologies  processes.  are  Canadian  suspended  the  pulping  production)  BKMs  extractives  into  kraft  pulp  (Celgar  include:  wood  thermo-mechanical,  sulfite is  of  to  to  remove  yield  complex together  p u l p i n g process  to  breaks  the  fibre  b i n d i n g organic  cellulose  fibres,  aromatic  polymer,  form  an  the  primary  composed  extensive  apart l i g n i n  molecules  support into  compounds,  known  ingredient  of  of  substituted  many  structure various  size  for  paper.  wood  fractions  6  ranging  from  single  ring  phenolic  ( O ' C o n n e r and V o s s 1992). a  substrate  (Voss  et  for al.  important  and  cellulose  is  is  an  The  the  a  each  can  spent are  exposed  to  last  pulp  continuous  decade,  lignin  delignification  the  in  or  lignin  that p r o v i d e  pulp  from  The  which  allow  the  integrity  maintaining  yields  cooking liquor  two  white  bleaching  pulp  major  for  the  and  is  an  focus  of  maximum strength  of  spent  and  system.  C h e m i c a l s such  to  which  batch  is  the  or  of  -  known 1140  and  sodium  termed  white White  containing  1983).  black  After  liquor,  is  The b l a c k l i q u o r , w h i c h  original cooking  continuous  pulping  Batch  as  kPa).  Na2S (McCubbin  recover  liquor.  pass  being  pulping  chemicals.  pulping  processes.  the  in  way  involves  the  which  one  time  through  cooking.  to  a  multi-zone  cooking  system,  conditions.  achievement  re-exposure as  soup,  sulphur  and  liquor,  forms  chips  ( M C C ) process  partially  NaOH  of pulping, in which  dose o f a l k a l i n e l i q u o r for a p e r i o d o f time, w h i l e  reduced  exposure  chemical  inorganic  p u l p i n g technology  pulp  and  caustic  either  exposure  This  longer  prolonged  the  wood  new  pulp.  are  by  a single large  characteristic  of  of  180 ° C ) and pressure (690  recycled  out  the  zone  with  -  cooking  between  process  effects  macro-molecules  during  effluent.  processes  a  various  is  carried  continuous  decreased  removal  desegregated i n a b l o w tank.  syrup,  be  o f w o o d to  residual  of  in  ingredients  w o o d fibres  are  In the in  organics  A O X from  while  fibres  mixture  the  p r i n c i p l e difference  exposure  lignin,  wood  a  odoriferous  chips  of  colloidal  of lignin breakdown  chlorinated  maximal  to  a l k a l i n e p u l p i n g , is a c h e m i c a l form  active  process,  pulping  wood  in  of  principle  thick,  Kraft  of  from  composed  and  of  the  h i g h temperature (160  cooking  drained  most  development  k n o w n as  separated  compounds; the  removal  also  l i q u o r , under liquor  the  up  various products  overall reduction  is  compounds  fibres.  Kraft, lignin  of  Therefore,  the  technology  degradation  is  in  It is the  synthesis  1980).  step  pulping  the  the  type  pulp black  is  anthraquinone  resulted  based  on  Normally strength, liquor  circumvents to  has  fresher and  i n 40  the  (Kocurek this  in  also been  a  in  degradative  The  through  extended  results  the  1989).  cooking liquor  ^ O ? have  cooking from  problem,  reductions  principle of  extended  stemming  - 50%  modified  removal  counter  introduced  of  current as  7  additives  to  1987).  increase  A d d i t i v e s are Following and  further  processing.  important,  decreases is  heavily  also  the  to  an  clean  from  the  organic pulp  an  initial  fibres, order  were  rapid  from  of  a  of  important;  as  such  regulatory  water  slow  an  primary desirable  that solute  washed prior  this  remove never  of  with  black  before  consumption perspective  the  all of  interfibre  bound  of  formation  of  the  solutes  Trinh  and There  surrounding  solutes  it  much  achieved.  liquor  in  bleaching  removes  substrate for  is  dark  out  to  environmental  to  this  very  chemical  since  Gratzl  r e m o v a l from pulp is bi-phasic.  associated  leaching  and  the  is  ( B S W ) generally  is  recovery  systems  volumes  and  much  a v a i l a b i l i t y o f water,  the  d i l u t i o n factor  stages pulps  discharged  requires  environment.  lasting  wood on  the  employed. have  resin  a  and  In  addition  relatively  fatty  acids,  the  high  of  of  the  o f the  of  than  material  effectiveness  rinse  characteristics  promote  greater  organic  Washing  concentration  which  water  used  of  the  is  and  the  pulp  are  saponifiable  foam  formation  in  B S W , upon  wood in  the  B S W efficiency. washing processes  situation.  a pulp  strategy  very  is  be  and  effect.  is  washing  possible,  i n practice  lignin  pulp  the  as  basic  specific  involves  of  as the  it  must  to  softwood  Two  from  the  reducing  several  lignin,  Ideally  brownstock,  which  From  thoroughly  and  called  increases  1983).  Allison  a relatively minor  perspective,  efficiency  to show  in-house  pulp  rinsing  extractives, system,  able  washing the  the  dependent u p o n number  liquor  1984;  days.  capacity  rinsed  black  b l e a c h i n g , but  removal  Brownstock the  to  followed by of  with  compounds.  prior  C r o t o g i n o (1987)  now  (McCubbin  pulp  have  pulp,  bleaching pulp  but  (McCubbin  the  economic  B O D , t o x i c w o o d extractives  chlorinated  is  From  removal  alternative,  impregnated  cleaner  important  of lignin  digestion,  since  with  rate  a cheaper  kraft  colour  is  the  air  cheap  been  older,  suction  moving  relatively  requirement,  have  The  vacuum mat,  strategies  a  and  entrainment  commonly  designed  more  driven  over  are  traditional  displacement  sequential effective, in  to  pulp,  set but  employed match  a  particular  strategy, of  vacuum  black  liquor  o f perforated, has  foam  certain formation,  economic drum  and  rotating  drawbacks increased  which and  washing,  rinse  water  drums.  This  such  as:  incidence  high of  8 spills and greater overall effluent  discharge  (McCubbin  1983).  which is employed in most modern mills is diffusion washing.  The second  strategy,  This process relies on  the positive pressure of water pumped through the pulp mat, to displace black liquor through  a perforated  wall.  Diffusion  washers  are designed  systems with minimal water usage and air entrainment. washing include: low incidence variant  of  of  and effluent  diffusion  washing,  discharge  termed  as  or no foaming,  and increased  pressure  operate  closed  The advantages of diffusion  circulating water requirement, little  spills  to  diffusion  decreased  chemical  recovery.  washing,  is  One  of particular  importance, because of its relatively low capital cost, ease of installation and ability to handle pulp directly from the digesters. method of choice more  stringent  Pressure diffusion washing has been the  for many mills required to upgrade their systems in response to  government  effluent  regulations.  2.2.3. Pulp Bleaching  Currently Canadian BKMs cater to a worldwide market demand for high quality fully bleached pulp, which can function on high speed printing machines up to the addition of various paper additives (B.C. Environment 1992).  and stand  Pulp which is  bleached with little or no chlorine is of inferior strength and at this time has limited market  with  environmentally  conscious  printing firms,  mostly  in  western  Europe.  Future markets for non-chlorine bleached pulp are expected to rise, but not enough, within the next decade,  to match the capacity of Canadian mills (B.C. Environment  1992). Effluent  content of  chlorinated  organic  compounds  individual compound basis or as a part of integrative organic halide organic  (AOX),  chlorine  purgable organic halide  (TOC1)  and extractable  may  be  on an  parameters such as adsorbable  (POX) for volatile  organic  reported  chlorine  (EOC1).  compounds,  total  TOC1 normally  ranges 10 - 30% below AOX levels, while POX is usually 5% of AOX values (McCubbin et al. 1990).  A O X is the most common parameter used in chlorinated organic regulation  9  in  Canada,  and must  be  measured  periodically by  Canadian  BKMs.  Theoretical  loadings of AOX may also be calculated using the following formula (Celgar 1990):  AOX = 0.11 (Cl + 0.5 H + 0.2 C10 ) 2  where: A O X is in kg of C l (hypochlorite)  and  C10  2  2  per  2  tonne  (chlorine  pulp  dioxide)  bleaching chemicals per tonne pulp.  and  Cl  represent  the  oxygen  delignification  There is a danger associated  process,  nitrogen  out  on  several  organisms  fish (Brachydanio with N 0  with relying on a  pretreatment,  resulted  did  not  correspond  bacteria, daphnid species  to  even though A O X concentrations large  variety  bleaching process.  of  in a  However, toxicity tests carried  any  decrease  in  and zebra associated  lethal  (Brannland et al. 1989). In fact, toxicity to microtox bacteria increased  A  chlorine  rerio), revealed that the reduced A O X levels in effluent  pretreatment  2  including: microtox  of  H  For example a modification  dioxide  10% reduction in effluent AOX (Brannland et al. 1989).  chlorine),  consumption  sum parameter such as A O X to gauge environmental risk. to  (molecular  2  toxicity  significantly,  were lower.  chlorinated  organic  compounds  are  synthesized  in  the  Roughly 250 of these compounds have been identified (Suntio et  al. 1988), accounting for only 10 - 20% of the organically bound chlorine (Earl and Reeve 1989). daltons),  Of those compounds identified, most are of low molecular weight (< 1000  while  the  chlorinated  lignin  (Kringstad  and  proportions  of  majority of fractions  Lindstrom certain  of  the  uncharacterized compounds  varying molecular  1984).  Each  chlorinated  organic  size,  bleaching  upwards  stage  compounds.  are represented  In  of  1000  by  daltons  releases  characteristic  general  most  compounds of < 10,000 daltons are discharged in the first stage (CD) of  of  the  bleaching,  while the fraction of molecular weight > 10,000 daltons appears in the first extraction stage (E) effluent  (Heimberger et al. 1988a).  classes of compounds identified fatty  acids,  single  ring  from B K M effluents  chlorinated  chlorinated phenolics) and dioxins.  Some of the very low molecular weight  aromatic  include: chlorinated resin and compounds  (chlorobenzenes,  Dioxins and furans are a special case since they  10  are not believed originate  to be the products of lignin or wood extractive  from  non-chlorinated  dibxin  precursors,  present  as  defoamers, treated wood chips and ambient air (Voss et al. 1988). chlorinated phenolics 2.1.  along with their bleach  The chlorinated phenolics  represent  chlorination, but contaminants  The major classes of  plant sources are presented  only  in  2 - 4% of the  in Table  total effluent A O X  (Heimberger et al. 1988a)  Table 2.1:  Major classes and sources of chlorinated phenolic compounds found in softwood BKM effluent.  Chlorinated Phenolic  Number of Possible  Number of Isomers  Bleach Plant  Percent of Total  Class  Isomers  Detected in B K M E *  Sources**  Chlorinated Phenolics  Chlorophenol (CP)  29  1 4  D+C  7  Chlorocatechol (CC)  1 6  1 5  D+C  36  Chloroguaiacol (CG)  15  5  E 1  43  Chlorovanillin (CV)  7  7  E1  1 4  * From Suntio et al. 1988. "  From Gergov et al. 1988.  D+C = First chlorination stage of bleaching. E1 = First alkaline extraction stage of bleaching.  Bleaching is the chemical treatment of pulp used to increase desired  level.  residual  lignin  Bleaching consists of  two  and  chemical  colour  and  the  phases: the  dissolution  oxidization  chemicals  as  :  hydroxide (E), hypochlorite mills commonly follow represents chlorine  low dioxide  chlorine  molecular  chlorine  (C),  (H), hydrogen peroxide  a five  pulp.  addition of such  dioxide  (P) and oxygen  of  of  (D),  (O).  sodium Canadian  stage sequence such as C E D E D or DEEDED, where C D  dioxide  substitution  chlorine  to a  and removal  (brightening)  Bleaching is carried out in multiple stages, involving the sequential bleaching  brightness  substitution  (5  -  (>20%) (Pryke 1989).  20%)  and  represents  D  high  The first two stages work to  remove lignin and colour and generate the majority of chlorinated organic material. The  final three stages serve to brighten pulp.  E stages both represent alkaline  11  extraction  stages, but with slightly  different  functions;  the first E stage is used  to  extract acidic lignin fractions and the second E stage serves to increase the reactivity of  pulp to the  final  D stage brightening  (Axegard et  al.  1984).  Two bleaching  technologies are in wide operation, the multiple tower-drum washer process and the displacement  bleaching process.  The former employs  each stage, with pulp drum washers interspersed bleaching  takes place  reaction  stage.  in a single  Other  than  a  closed  lower  between the  tower,  fresh  a separate  with  water  bleaching  stages.  multiple  Displacement  chambers  requirement  tank for  for  for  each  displacement  bleaching, the two methods show no difference in the total amount of BOD, colour or AOX discharged (McCubbin 1984). There  are  several  bleaching  process  modifications  currently  Canadian mills to reduce AOX discharge to the environment. or pre-bleaching, prior to first stage chlorine bleaching is result in 30 - 50% effluent The process functions  employed  Oxygen delignification a technology,  which can  A O X reductions (Heimberger et al. 1988b; Presley 1990).  by removing up to 50% of the lignin from pulp before  reaches the bleach plant and has a chance to react with chlorine.  Since the  from  can  the  through  oxygen  the  mill  delignification recovery  process, called PRENOX , R  oxygen treatment.  by  process  system.  involves  is  One the  non-chlorinated,  variation  of  the  it  oxygen  addition of nitrogen dioxide  A further 10% reduction in effluent  be  it  effluent recycled  delignification (N0 ) 2  prior to  A O X can be achieved using  this modification, but pulp quality seems to be variable (Lindqvist et al. 1986).  The  dramatic reduction in A O X seen with oxygen delignification has a heavy capital cost, since  the  pumping  system  requires  a separate  reaction  tower  with  associated  mixing and  equipment.  Chlorine multiple is the amount of chlorine administered to pulp with a given percent  lignin  generation  content.  (Pryke 1989).  Increasing  chlorine  A reduction in the  multiple  results  in  higher A O X  loading of chlorine to pulp can be  achieved though, as long as machines used for mixing bleaching chemicals into pulp are optimized.  In particular, high intensity  to increase mixing efficiency  mixing technology  has been introduced  to the level where chlorine multiple may be reduced  12  by 25 - 40% (Earl and Reeve 1989). Increased chlorine dioxide substitution is one of the most common bleaching process pulp  modifications used to achieve  brightness  and  chlorine  dioxide  greatest  reduction  Intermediate 1986).  is  charged in  results  While  strength.  The to  the  lower effluent  A O X levels,  most  delignification  efficient  pulp before  A O X effluent  levels  chlorine  dioxide  is  achieved  molecular chlorine, however  occurs  when  the  are observed when chemical charging is  increasing  while maintaining  substitution  order  is  data presented  concentrations that the  in Liebergott et  may rise  situation is  with initial  al.  (1990)  increases  shows  the  reversed.  simultaneous (Axegard  generally  reduces  AOX, the effects on individual chlorinated compounds are more variable. from  if  that  effluent  Table 2.2,  chlorinated phenolics  in chlorine dioxide  substitution and  further complicated by chemical charging order.  From these  data we can see that the CGs and CVs are the most prominent chlorinated phenolics in bleachery effluents,  even  at high chlorine dioxide substitution.  characterized B K M effluents  for chlorinated phenolics  Voss et al. (1980)  and found that the following  CGs and CVs were present in the highest concentrations: 4,5-DCG, 3,4,5-TCG, 4,5,6-TCG, TeCG,  6-CV and 5,6-DCV.  These particular compounds make good candidates for  tracing the extent of pulp mill derived chlorinated phenolic contamination. colour BOD  decreases values  with increasing chlorine dioxide  are variable and resin acid levels  substitution are unaffected  (Pryke  Effluent  1989),  while  (Heimberger et al.  1988b).  Table 2.3 summarizes the changes in effluent colour, BOD and TOC1 as a  function  of  bleaching  sequence.  2.1.4. Effluent Treatment  Pulp mills employ a number of effluent  treatment techniques  BOD, resin and fatty acids, AOX and effluent toxicity. can  include  pretreatment  (screening,  neutralization)  to reduce TSS,  An effluent treatment program primary  (1°) clarification  remove suspended solids, secondary (2°) biological treatment (aerated lagoon,  to  13  o  CJ)  a  CO  ZJ Q.  LE TJ  o  M  o IO  O  O)  CO  g "5 c CD  o o o co O OX  X:  Q-  b  "O <D  CD C O  Chi  oS c 'i— o o sz cn  o  q  --  o  CD  CO  O  E  »  d  CM  CO  CO  CO CO  CO CM  O) CO  o  in  CJ  h-  O o  5  o T— H— : o CC o  cvi  CO  ° x i  S3  co d  CJ> O)  o  IO  II  °> to  CD  co  iq  CO  CM  d  OJ CO X O  - ~  <1)  "CJ  II CO  o  c\i  CD  CO  CO  CO  co n o  ° 6 •a c o CO  CJ CO r~-  CD  CO  6  II  Xt  x  CO  Q. T3  cj  CJ)  O  S  I-  CO  ^  CM  s  D>  —  x: c  •q-3CJ  -ad CM  CO  5  &  cn  CM CO CM  m o  Q-  CM  o>  in  15-  O CO  o cr nj  .2  iz xi CD  o •~ O m  e s  CO  0)  cc cn  2" o  c  Q) CO  "8 5 .s c\i CM  a>  JO  xi  CO CO  a> co  (A  o c  0)  o  JZ O  2 o CO  o O  o o  JZ  O  o O  TJ  SZ CO  CO CO D  C O  5? o o  6  JZ  O  O  |  CO  o  o in  o  o  m  |  CO  E  CD CD  CD  CD  c  cr CD CO  « co Q  o  c  cr  CD CO  o» c lc o CO  co  o  CO  ZJ  — 8 Eo  XI  T-  CD  I  co cvi  in  XI  CD  o  E o O  IO  *—  XI  CO Q.  co in  co  "5 c  Q-  T -  "&»  v_  CD  ss  o r~  -a  cn  z  CM CM  H—'  O O  0} II  O  o  ZJ CL  ZJ  £  ZJ  a.  a.  "3 CD S c  o o  „  >  CO CO  •<*  Jo  CO  _® m  a  Q  LU  LU  Q  Q  Q  o  o" LU  O  o LU Q  a  Q  O  LU  LU  LU  a  Q  O  o  LU  o O  Q  O Q  O  14  activated  sludge,  anaerobic  treatment),  and tertiary (3°) colour removal.  2°  clarification  The majority of Canadian BKMs treatment,  sludge facilities. treatment, become  but  contaminated  and 2° clarification are most  discharging to inland waters have  usually aerated lagoons  with  but research  more for  stringent  all  in  communication)  has  aerated  lagoons  contain  organic  compounds  effluent  BKMs.  A O X regulations,  Secondary  the  shown  that  to  are  biosolids  biosolids  adsorbed  tend  compound  area of  to  adsorbed  the  Since  1  partition also  entering  organic to  treatment on effluent  by  treatment  been  sludge  contamination  dioxins .  2°  clarification has  many  solids,  most  Derksen  (personal  from B K M  hydrophobic chlorinated it  discharged  used  will  systems (NCASI  Fraser River  clarification could be one solution to this particular problem.  Table 2.4:  some form of  Coastal mills have been historically remiss in providing 2° effluent  a necessity  chlorinated  effective.  alone or in combination with activated  commonly in the recycling of biosolids lost from activated 1989),  biosolids  For the removal of BOD, toxic wood extractives and  chlorinated organics, 2° biological treatment  biological  for  is  likely  biosolids.  that  other  Secondary  The effect of biological  quality parameters is summarized in Table 2.4.  The effect of biological treatment on effluent quality parameters. Data from Heimberger et al. 1988a.  |  Production (kg/adt pulp) Chlorinated Bleaching Sequence  TOCI  BOD  Colour  Phenolics  (CD)(E0)DED  5.5  25  200  0.10  With Bio-Treatment  3.3  2.5  200  0.08  O(CD)(E0)DED  3.5  20  100  0.07  With Bio-Treatment  2.1  2.0  100  0.06  Under optimal conditions  both aerated lagoon  and activated sludge  can reduce BOD (85 - 95%) (NCASI 1989), resin and fatty acids 1  treatments  (71 - 100%) (Celgar  George Derksen, Environment Canada, Environmental Protection Branch, 224 West Esplanade, North Vancouver, B C . V 7 M 3H7, 1992.  15  1990),  A O X (48  Heimberger currently  et  -  65%)  al.  being  (biofilter) of  Anaerobic  investigated  1989).  for  process  R  ENSO F E N O X  process.  chlorinated  chlorinated  1988a).  system, E N S O F E N O X (NCASI  and  future  has R  has  -  90%)  (Gergov  is  not  applications  to  B K M effluent.  been  attempted  a  in  t o x i c i t y and  been  (15  treatment  is  Reduction  phenolics  phenolics  dual  on  a  anaerobic  achieved  generally  mill  scale  (fluidized  mutagenicity  using  this  al  practiced, One  with  bed  as  et  but  some  as  is  Anaerobic success  reactor)  well  1988;  aerobic  dechlorination  system.  2.2. E N V I R O N M E N T A L F A T E O F C H L O R I N A T E D P H E N O L I C S  2.2.1.  Introduction  An the  integral  determination  distributed all  and  questions  that  cleared  In  be  Since  become  environment, partitioning  takes  physically  sediments  major  namely  water,  chlorinated  biological  interactions.  the  to  inputs?  determine is  the  variable  both  is it?  environmental  available variables  o f pollutants  H o w is it  H o w persistent  interacting  highly  three  are  and  many  fate  clearance  physical  are  information  o f the  interfaces.  chemistry,  What  impact  from  which  affecting  temporally  fate  and  is  transported, These fate to  are of a  answer  and  given  spatially,  the  complex.  compartment of  of environmental  i n order  limited  often  exclusive,  suspended  addressed  are  are  place,  risk  b i o p h y s i c a l system?  are  context  there  the  fate.  there  highly  the  the  only  b i o p h y s i c a l systems  Within  not  from  reality  questions.  answers  assessing  o f environmental  that must  pollutant. these  part o f  since  pore  transported  chemical  organic  environmental and  water  present  is  water,  characteristics  biota.  These  within  while biota  influencing compounds  compounds  compartments  sediments  within  Factors organic  of chlorinated  the  fall of  into the  aquatic  amoung  which  compartments  sediment  may  transport, four  i n the  be  present  distribution  categories:  receiving  beds  are and  at  all and  pollutant  environment  and  16  2.2.2. Sources of Chlorinated Phenolics  The from  inputs  of  chlorinated phenolics  anthropogenic  sources,  but  phenolics to the environment.  some  to  the  natural  aquatic  environment  processes  contribute  Grimvall et al. (1991) found elevated  of AOX (200 (j.g Cl/L) in Scandinavian lakes  considered  are  to  be  mostly  chlorinated  concentrations  unpolluted.  Their  laboratory studies gave strong evidence  for enzyme  substances  The only chlorinated phenolic detected  in natural soils and waters.  these reactions has been 2,4,6-TCP.  mediated chlorination of humic from  The O-methylation product of 2,4,6-TCP, 2,4,6-  trichloroanisole, was also detected in the same systems and is believed to be the result of in situ bacterial  transformation.  Anthropogenic processing facilities chlorinated processes  such  runoff  chlorinated phenolics  industries  municipal  1985).  effluent  from  particles.  as  al.  of  include: saw  using chlorinated phenolic treated wood (Hall  herbicide  (Paasivirta et industrial  sources  waste  facilities  carrying  incinerators  and  out  fossil  and wood  and Jacob 1988),  organic fuel  combustion  burning  plants  Vehicles for the input of chlorinated phenolics include:  discharged  directly  terrestrial sources  In B K M effluent  and  mill  into  receiving  and atmospheric  waters,  deposition  leaching  on the  and  surface  surface of  dust  receiving waters, direct discharge is the most important  vehicle. The major classes of chlorinated phenolics, CPs, CCs, CGs and CVs (Figure 1.1) are derived from different industrial" sources. combustion  processes  and pulp bleaching.  The CPs and CCs are formed in organic The CPs, particularly tetra  and penta  substituted forms, are also associated with wood treated with these compounds as an antifungicide.  Currently,  government  restrictions  on  wood  treating  reducing input from this source in North America and Western Europe.  agents  is  Air pyrolysis  of the ubiquitous PCBs has also been, shown to result in formation of mono and dichlorinated  phenols  (Paasivirta et  al.  1985).  A relatively  small but unquantified  percentage of CGs seem to be formed in the combustion of organic fuels, such as peat and coal, but for the most part the CGs along with the CVs are restricted to BKM  17 sources (Paasivirta et al. 1985), making these classes of chlorinated phenolics  good  tracers of B K M chlorinated organic pollution.  2.2.3. Partitioning to the Water Phase  The  chlorinated  contaminants  phenolics  differ  from  many  in that they are acidic and therefore  the phenolate  ion form.  behaviour of  chlorophenols  other  lipophilic  dissociate under alkaline pH, to  Therefore, water pH is one of the factors in receiving  waters.  organic  The undissociated  governing form is  the  more  lipophilic and tends to partition to organic phases, such as living organisms (Carey 1988). pH  Table 2.5 gives the dissociation constants and percent dissociation in water of  7.8,  which  phenolics. decreases  Table  is  Note with  2.5:  close that  to  the  increasing  Dissociation  of  acidity  Fraser River water,  of  chlorinated  chlorine  substitution.  constants  (pKa)  phenolics in water of pH 7.8.  COMPOUND  that  for  phenolics  and percent  PERCENT DISSOCIATION (pH 7.8)  2.4- DCP  7.8  50\0~  2,4,6-TCP  6.0  98.4  2,3,4,6-TeCP  5.4  99.6  PCP  5.3  99.7  4.5- DCG  9.0  5.9  3.4.5- TCG  8.0  38.7  4.5.6- TCG  7.4  71.6  TeCG  6.0  98.4  increases,  dissociation  Data from Xie (1983).  pKa  selected  of  chlorinated while pKa  chlorinated  18  Volatilization of chlorinated phenolics ambient temperature and mixing conditions. less  chlorine  water  surface  compounds  agitation.  Volatilization favours  (Jacob  1986).  by the molecular  There is a greater tendency  substituted  chlorinated phenolics  to  is influenced  volatilize  with  the  increasing  for lighter,  temperature  unbound, undissociated  In the Fraser River,  weight,  the heavy  and  form of  tri- and tetra-  chlorinated phenolics are mostly in the dissociated form and would not be expected to volatilize  at a significant  rate, but the  lighter  mono-  and di-chlorinated phenolics  may be lost to a measurable degree through volatilization. Chlorinated phenolate  form,  phenolics  undergo  which absorbs light  form (Boule et al. 1982).  photolytic  at different  degradation,  particularly  wavelengths than the  the  undissociated  Photolysis rate varies with geographical latitude, time of  day, season and light admitting characteristics of the particular water body.  In the  Fraser River photolysis reactions would be at a minimum in winter, under ice cover and may not be of great significance  in spring and summer, since  the  suspended  sediment load in the Fraser River hinders light penetration. Halogenated aromatic compounds are generally resistant type  of reaction is  not considered  important in the  fate  to hydrolysis and this  of  chlorinated  phenolics  with  sediment  (Jacob 1986).  2.2.4. Partitioning to the Sediment Phase  The  interaction  compartment Pollutant coefficient strong  is  one  of of  chlorinated  the  chemistry  and  (Kow),  and the  influence  on  the  key  processes  environmental fraction  of  sediment-water  organic compounds (Karickhoff  organic  compounds  influencing  factors organic  such  their  as  Jaffe  environmental  octanol  water  carbon in sediments  partition coefficient  et al. 1979;  the  1991).  (Ksw)  (foe)  fate.  partition exert a  of hydrophobic  Voice et al. (1983) also  pointed out a third variable, the concentration of the solid phase in the system, as a factor  influencing  Ksw.  Their laboratory research revealed that as suspended  19 sediment  concentration  compounds  increased,  appeared to decrease.  sediment  partitioning  This phenomenon was  of  chlorinated  aromatic  explained by the  presence  of a third phase in the system, colloidal macromolecules and microparticles, too small to  separate  increased, making  from  the it  process  phase.  that  there  As  suspended  sediment  concentration  was  binding microparticles introduced also increased,  was  an  inverse  relationship  between  sediment  and K s w . The contribution of the microparticle and colloidal phase in  environment  suspended  water  amount of sediment  appear  concentration the  the  is  difficult  biosolids requires  and  to  quantify  colloidal  further  at  lignins  this from  investigation.  but  effluent  Partitioning  compounds to the larger, settleable sediment the whole particle size range.  time,  with  the  treatment of  discharge lagoons,  chlorinated  of this  organic  fraction does not remain constant  over  Particles of the silt and clay fractions, < 63 um tend to  have the greatest adsorptive capacity, due to the greater surface area per unit mass (Schellenberg et al. 1984)  and in some cases a greater amount of associated organic  carbon (Voice et al. 1983).  Equations used to estimate K s w , from Kow and foe have  been  developed (Karickhoff et al. 1979);  Ks w = Koc * foe and  Koc =Kow* 0.63  where  Koc is  the carbon normalized partition coefficient.  These relationships  are  only valid where organic content is > 0.1% (foe > 0.001); for organic poor sediments, inorganic interactions  may be of overriding importance (Schellenberg  et  al.  1984).  The term sorption is generally used to describe the binding of compounds to the solid phase.  Sorption to a two dimensional surface is usually referred to as adsorption,  while  the  of  sorbent  a  term absorption describes (Schwarzenbach  partitioning into  1985).  Dissolved  the  three  nonpolar,  dimensional matrix  hydrophobic  organic  compounds tend to associate with solids of similar hydrophobic character (ie. organic  20  particulates)  in  the  thermodynamic ally  favourable  process  of  hydrophobic bonding,  a non-covalent form of bonding of moderate strength (von Oepen et al. 1991). The relatively organic  simple  compounds  phenolics.  is  Sediment  partitioning model describing the  only  partially  partitioning  of  water pH and ionic strength (Westall ionizable  functional  groups,  applicable  chlorinated 1985).  may interact  to  the  phenolics  Ksw for  hydrophobic  ionizable  chlorinated  is  also  dependent  Hydrophobic compounds, which possess  with both organic and inorganic  sorbents,  by processes such as ligand exchange, ion bonding (von Oepen et al. 1991) pairs, which can in turn, move to organic phases (Schwarzenbach anion sorption can take place on surfaces surfaces acidic  (Westall highly  on  with positively  and ion  1985).  Phenolate  charged sites, such a clay  1985), but may only be significant, under neutral pH, for the more  chlorine  substituted  chlorinated phenolics  However, the mono and di-substituted  (Schellenberg  chlorinated phenolics,  et  al.  1984).  which tend to be more  in the undissociated phase would expect to be attracted to inorganic binding sites to a much  lesser  degree.  Further  complexities  arise  between  the  various  groups  of  chlorinated phenolics. For example, the CCs were shown by Remberger et al. (1993), to form readily reversible  complexes with metal cations  (Fe^ , Al^ ), while the CGs +  +  showed evidence of increased proportion of covalently  bound compound.  many  chlorinated  studies  investigating  difficult to make to vary greatly composition,  sediment  generalizations  partitioning  composition  phenolics,  it  is  as to the nature of the partitioning, since it seems  among various studies and is  water  of  From the  probably highly specific  and laboratory  to  sediment  conditions.  2.2.5. Partitioning to Biota  Partitioning bioconcentration  of and  contaminants  to  bioaccumulation.  biota  takes place  Bioconcentration  through can  be  the  processes  defined  as  absorption of a substance by direct contact with the ambient environment through  of the  21 oral, percutaneous dietary sources it follows  or respiratory routes  (Jaffe  is termed bioaccumulation.  that contaminant  concentration  1991).  Inclusion of uptake through  From this definition of bioaccumulation, may be magnified  through higher trophic  levels of the food chain; this phenomenon is termed biomagnification. the  quantity  concentration (BCF).  of in  a  contaminant  the  Strictly  water  is  speaking,  the  bioconcentrated  related  through  B C F equals  the  the  to  the  term  steady  The ratio of  original  exposure  bioconcentration state  factor  equilibrium tissue  concentration divided by the water concentration (Ellgehausen et al. 1980). Since hydrophobic compounds tend to passively  partition into lipid phases, the  mechanism of uptake is usually passive diffusion through a lipid membrane, across a concentration gradient.  Uptake sites are most commonly associated  with respiratory  surfaces exposed directly to contaminated water, such as gills in fish and respiratory pores or gill like structures for invertebrates (Barron 1990). an important uptake route in some fish species. skin uptake accounted for 25  Skin absorption is also  Saarikoski et al. (1986) found that  - 40% of PCB bioconcentration in guppies  (Poecilia  reticulata).  Bioconcentration  is  a function  of  elimination (Ellgehausen et al. 1980). chemical factors  nature with  specific  of  both  biology,  the the  substance  competing  processes  of  uptake  and  In turn, these processes are controlled by the and  physical-chemical  specifically  the  uptake,  the  interaction  behaviour  distribution  of  of  external  a  substance  and elimination  environmental and  species  kinetics  (Barron  1990). Chemical molecular  size  Schmieder  properties (Barron  1991).  bioconcentration concentration  of  found  1990)  (Ellgehausen  environmental  contaminants et  influence  bioconcentration  and hydrophobicity (Neely  External organic  to  al.  1980;  in Hall  variables  aquatic  et  al.  1974;  documented  organisms  and Jacob  include  1988),  both  McKim and to  affect  include  contaminant  water  temperature  (Veith et al. 1979; Barron et al. 1987b; Sijm 1991), pH (Saarikoski et al. 1986; Hall and Jacob 1988) and suspended and dissolved solids (Opperhuizen and Stokkel 1988; Lee et al. 1993).  Biophysical characteristics such as lipid content (Ernst et al. 1991) and  22  surface  to  volume  ratio  (Saarikoski  et  al.  1986)  have  been  reported  to  affect  bioconcentration.  2.2.5.1. The Effect of Octanol-Water Partition Coefficient  The a  octanol-water  chemical  to  relationship  partition  between  to  the  organic  bioconcentration  lipophilic  organic  phenolic  compounds.  correlation  partition coefficient  compounds  Hawker  (Carey  Kow has  Connell  and log  1988).  A  positive  of  linear  been reported for both neutral  undissociated  and  between bioconcentration  phase  and  and the  (Kow) defines the relative tendency  form of  (1986)  K o w of  the  acidic  reported  a  chlorinated  strong  organochlorine  linear  pesticides  and  chlorinated biphenyls in fish, mollusc and daphnid species over the log K o w range - 6. in  Saarikoski et al. (1986) reported similar relationships  the  unionized form (i.e.  for chlorinated phenolics  pH > 1 unit below pKa), but found that the plot of  bioconcentration vs. log Kow started to level off at log Kow values > 4. of  the  relationship  compounds (Barron  appears  bioconcentrate 1990;  Jaffe  bioconcentration  and  2  The upper limit  to be limited by molecular size; large super-lipophillic to  less  than  1991). Kow has  predicted  The led  values  apparent  many  due  strong  investigators  to  steric  hindrance  correlation  between  attempt  use  to  to  this  relationship as a predictive tool (Mackay 1982; Isnard and Lambert 1988; McKim and Schmieder 1991). with caution. and  There are several reasons why such a practice should be regarded  Jaffe  (1991) pointed  out  that structural differences  bio-lipids lead to differing thermodynamics  of partitioning.  between octanol  Furthermore, close  inspection of large data sets (Mackay 1982; McKim and Schmieder 1991) reveals that the relationship is only of a general nature; compounds of similar Kow can differ in bioconcentration  by  bioconcentration  based  toxicokinetics 1990).  >  1.5  upon  and response to  orders  of  magnitude.  K o w fail  to  account  variations  in external  Finally,  for  interspecies  environmental  predictions  of  differences  in  factors  (Barron  23 2.2.5.2. The Effect of pH  The change  chemical nature of neutral organic compounds (e.g.  with  compounds  water  pH.  dissociate  approaches pKa.  However,  into  the  less  the  acidic  family  lipophilic phenolate  of  3,4,5-TCVer) does not chlorinated  anion form as  phenolic water pH  TeCG (pKa = 6.0) is more than 90% ionized at pH 7.0, while 4,5-DCG  (pKa = 9.0) is < 1% dissociated at the same pH.  The ionized species is water soluble and  therefore, less bioavailable to aquatic organisms (Barron 1990).  The Kow for PCP is  reduced by almost two orders of magnitude at pH > 7 (Carey 1988).  However, when pH  is > 1 unit below pKa, pH has a negligible affect on bioconcentration (Saarikoski et al. 1986).  The assumption that the ionized form of a chlorinated phenolic compound does  not represent  a significant  be  in  incorrect  several  pool of bioavailable compound has been demonstrated studies.  Laboratory  studies  have  relationship between water pH and both chlorophenol toxicity however  the  predicted reported reticulata)  magnitude  from  of change  chemical  decreased  of  tri-  curves. and  as pH increased (pH = 5 - 8 ) ,  at pHs corresponding virtually  unionized  increasing  toxicity  at elevated  at  all  pH.  Saarikoski  inverse  and bioconcentration,  and  pentachlorophenols  to  would be  Viluksela guppies  (1981) (Poecilia  with a significant toxic effect still apparent  to > 90% ionization.  remained  an  in bioconcentration is less than what  dissociation  toxicity  revealed  to  water  The toxicity pHs  Investigating  showed the  of 4-chlorophenol, which a  effect  slight of  trend  water  towards  pH on  the  bioconcentration of 17 phenolic compounds, Saarikoski et al. (1986) again found that bioconcentration  decreased  less  at  elevated  pH than  dissociation curves, indicating that the phenolate bioavailable compound.  predicted  ion represents  from  pH - pKa  a significant pool of  Support for this conclusion comes from the finding that fish  bioconcentrate relatively high levels of PCP (pKa = 5.0) and TeCG (pKa = 6.0) in the Fraser River (pH = 7.5 - 8.5), even though they are present at > 92% in the ionized form (Carey 1988; Rogers et al. 1988; Servizi et al. 1988; Dwernychuk et al. 1991). Variation in external pH, not only  alters chemical  but also affects bioaccumulation physiology.  speciation  of acidic compounds,  Increased water pH resulted in  24  decreased  metabolic  clearance  of PCP by goldfish  (Carassius  auratus) (Stehly  and  Hayton 1990).  A similar affect would explain the trend towards increased toxicity of  4-chlorophenol  to  guppies  at  increased  water  pH, observed  Viluksela (1981) and the increase in leech (Nephelopsis  by  Saarikoski and  obscura) bioconcentration  2,4-DCP over the pH interval 5.0 - 7.5 (Hall and Jacob 1988).  of  Decreased bioavailability  of chlorinated phenolic compounds at high water pH may be offset to some degree by reduced  elimination  including  a  rates.  neutral  Theoretically, such  organic  compound,  such  an effect as  could  3,4,5-TCVer  be in  monitored by the  laboratory  assessments.  2.2.5.3. The Effect of Dissolved and Particulate Solids  Adsorptive organic  partitioning  (Schellenberg  et  al.  of  neutral  1984;  and ionized  Schwarzenbach  organic  1985)  compounds  and inorganic  to  both  (Remberger  et al. 1993; Xing et al. 1993) particulates as well as colloidal and dissolved organic material (Kukkonen and Oikari 1991) material  represent  phenomenon dependent  has  potentially  character  particle  (Schwarzenbach  character of  significant  only received  upon  suspended  has been documented. competitors  the  percentage  1985). and  and dissolved  water  of pH  and difficult  bioavailability  of  to  generalize.  neutral  acidic (dehydroabietic  bioconcentration,  The degree of the organic  (Westall  could  hydrophilic  be  material, each  into  naphthalene;  with  Partitioning of benzo(a)pyrene  hydrophobic  a characteristic  is  chemical  1985).  The  variable  and Oikari  in natural receiving  (1991)  acids, affinity  assessed  tetrachlorobiphenyl)  acid) organic compounds to Daphnia  divided  effect  its  magna in the  of surface waters from 20 different locations in Eastern Finland. material  the  effects upon bioconcentration highly  Kukkonen  (benzo(a)pyrene;  but  material,  organic material present  waters makes the task of assessing competitive complex  to  limited investigation.  size,  Suspended and dissolved  and  presence  Dissolved organic  hydrophobic for  the  organic  neutrals  and  contaminants.  to dissolved organic material was correlated to the  25 hydrophobic  acid  between  other  the  content test  material was evident. hydrophobic  acid  (humic  content  dissolved  of  no  and  measured  characteristic  any  Bioconcentration by Daphnia  Physical-chemical magnitude  however  compounds  for  benzo(a)pyrene  weak correlations for naphthalene  the  material),  material.  magnawas  and  of  tetrachlorobiphenyl  a contaminant,  inhibition  of  Opperhuizen and Stokkel  phase  bioconcentration soluble, not  (Chromosorb),  by  guppies  (Poecilia  moderately lipophilic chemicals  affected  significantly  by  of  dissolved  strongly correlated to  such  as  and  Kow, can  bioconcentration  by  showed  resulting  in  reticulata)  particulate  to partition to the  significant  decreases  material.  This  is  consistent  with  magna and zebrafish (Brachydanio  aimed at predicting the fate of chlorinated phenolics  with  suspended  particulates  bioconcentration.  However, colloidal lignins  effluent  lagoons may reduce  treatment  the  In studies  in the Fraser River by Carey  causing  studies  association  of  inhibitive  chlorinated  with  very  centrifuged out of B K M effluent from mills in Prince George, B.C . 2  that  adequate  their  effect  characterization on  of  bioconcentration,  the in  amount  receiving  prediction of partitioning to biota.  2  and  George Derksen, Environment Canada, 1992.  nature  waters  is  of  on  from pulp mill  found evidence  dioxins  in the  effect  amount of chlorinated organics  Derksen (personal communication)  the  little  and organic particulates  to aquatic organisms. examining  the  rerio)is  (1988), it was concluded that most of the compound would remain dissolved column,  in  Bioconcentration of more water  not affected by the presence of dissolved humic material (Lee et al. 1993).  water  and  such as di- and trichlorinated benzenes was  particulate  observation that PCP toxicity to Daphnia  influence  (1988) reported that highly lipophilic  compounds such as chlorinated biphenyls have a greater tendency particulate  correlation  and dehydroabietic acid.  properties  competitive  significant  available  of this in  fine  biosolids  Evidence suggests  organic  necessary  material and for  accurate  26  2.2.5.4. The Effect of Temperature  Ambient  temperature  may  influence  Since fish and aquatic invertebrates rate  of  physiological  processes.  elimination usually increase  bioconcentration  in  a number of  are poikiolothermic, temperature will Rates  of  contaminant  with temperature increases  uptake,  (Barron  ways.  affect  metabolism  the and  1990).  Over the temperature range 4 - 22 °C, Hall and Jacob (1988) observed a strong positive  correlation between leech  and temperature.  (N.  obscura) bioconcentration  The time to reach steady  related to water temperature.  of  chlorophenols  state tissue concentrations was inversely  At 4 °C equilibrium bioconcentrations of 2,3,4,6-TeCP  were reached after four days, while steady state was not reached after seven days at 22 °C.  The effect of temperature seems to be dependent upon the species specific  optimal tolerance range.  Veith et al. (1979) tested the effect of temperature (T = 5 - 25  °C) on PCB bioconcentration by three fish species.  Rainbow trout (Salmo  gairdneri)  showed no increase in bioconcentration between 5 and 10 °C, with a relatively sharp increase  over  (Pimephales  the  temperature range  promelas)  opposite trend.  and  the  of  green  10  - 20  sunfish  (Lepomis  Species  specific  be linked to the  relationships  cyanellus),  effect  reticulata)at1  between  showed  the  = 13 - 33°C.  bioconcentration  and temperature  can  Barron et al. (1987a) observed linear increases in both cardiac output and  6, 12 and 18 °C. mostly  gairdneri) exposed  to water temperatures of  Uptake of organic contaminants by many aquatic organisms takes  across  (Barron 1990).  highly  blood  profused  respiratory  surfaces  the  gill  (e.g.  fish  gill)  Therefore, physiological variables such as cardiac output, ventilatory  volume and blood flow rate are potential uptake rate limiting factors. to  minnow  of temperature on physiological function and accumulation  blood flow rate for rainbow trout (Salmo  flow  fathead  For example, Sijm (1991) reported only small differences in  bioconcentration of PCBs in guppies (Poecilia  place  Both the  In some cases, very slight effects of temperature on bioconcentration  have also been observed.  kinetics.  °C.  can result  in  a greater  uptake rate,  volume increases apparent exposure at the uptake site.  while  Greater blood  increased  ventilatory  Elevated temperature also  27 generally  leads  (Barron 1990). an  increase  trout.  to  increased  metabolic  elimination  rates  of  organic  compounds  Over the temperature range 6 - 18 °C Barron et al. (1987b) observed  in  the  total  body  clearance  of  di-2-ethylhexyl  phthalate  in rainbow  However, uptake rate and distribution to deep storage compartments remained  dominant  over  the  enhanced  elimination  rate.  Similarly,  Jiminez  et  al.  (1987)  observed 5.8 times increase in uptake rate and 3.6 times increase in elimination rate of benzo(a)pyrene  in sunfish species, over the temperature range 13 - 23 °C.  The  lack of increase in bioconcentration of PCBs by rainbow trout over the temperature range 5 - 1 0  °C, observed by Veith et al. (1979) is consistent  increased uptake rate due to elevated rate of elimination.  with the idea that  cardiac output was balanced by an increased  A sharp increase in bioconcentration of PCBs above 10 °C can be  explained by dominance of uptake and deep compartment storage over elimination. Temperature  affects  other  physiological  determinants,  permeability and lipid composition, which changes temperature  changes  (Barron  such  as  membrane  to maintain a constant fluidity as  1990).  Furthermore, the  physiological process will be affected  will vary with size,  degree  to  which  each  age and species (Barron  1990). Temperature can affect  the  chemistry  of  organic compounds.  Solubility of  compounds is generally increased at higher temperature (Phillips 1978), resulting in more available compound, but this is not expected since  chlorinated phenolics, discharged from BKMs,  to be environmentally relevant, are not expected  to reach high  enough concentrations to be in the undissolved form.  2.2.5.5. The Effect of Multi-Contaminant Exposure  Investigators simultaneous individual  have  exposure  to  been  trying  many  toxic  to  contaminants  compounds with conflicting results.  shown synergistic,  elucidate  the on  the  potential  effects  of  bioconcentration  of  Exposure to chemical mixtures  additive or independent effects,  has  depending upon the compounds  28  and  animal  when  species  white  tested.  suckers  dieldrin,  both  Atlantic  salmon  Frederick  (Catostomus  individually  higher  fry (Salmo  (Salmo (1978)  levels  gairdneri) suggests  should  any  field  that  the  since  bioconcentration. exposures  2.2.6.  were  to  bioconcentration  Arochlor  result  to mixtures  were  added  when  o f the pesticides  aldrin  with  respect there  realistic  to water  always  approach  be  m a y be  as  a  Phillips  established. occurring  pollution,  this  interactions  to  conduct  trout  biomonitor,  be  interactions  B K M water  will  rainbow  as a m i x t u r e .  organochlorines  to  ( P h i l l i p s 1978)  by  consideration  contaminant  1232 and  was observed  accumulated  from  between  possible  likely  more  exposed  on  in  seems  affecting  multi-compound  at levels w h i c h are l i k e l y to occur i n the field.  Biotransformation  Chlorinated mediated most  of  especially  A  exposed  effects  spectrum  is  effect  A similar  be e l i m i n a t e d  interactive  it  were  D D T and d i e l d r i n  the c o m p o u n d s  wide  no  Contrary to these results i t was reported  an o r g a n i s m  situation,  unrealistic,  commersoni)  salar) were  both  when  known  Considering any  of  found  and i n c o m b i n a t i o n .  and D D T ( A d d i s o n et a l . 1976). that  (1975)  phenolics  transformation  o f the  microbial  degradation  is  chlorophenols  In by  highly  such  one case,  stream  by  Microbial  o f chlorinated  variables,  bioavailability.  metabolized  reactions.  the r e m o v a l  environmental  are  site  as  periphyton;  et half  organisms  transformation  phenolics  from  specific  temperature,  Carey  aquatic  light  a l . (1984) lives  were  probably  aquatic  and  dependent  reported  rapid  the  order  enzyme  accounts  systems.  penetration,  on  through  T h e rate  upon  water  for  many  flow  and  dechlorination o f o f four  to s i x  hours. One  of  transformations  the involves  most  environmentally  microbial of  toxic  significant  O-methylation  concomitant  formation  anisoles  derivative).  T h e resulting derivatives  of  phenolic  ( C P derivative)  are more  chlorinated  lipophilic  and  compounds, veratroles  and t o x i c to fish  phenolic with  the  (CC,C G than  the  29 parent compounds (Neilson et al. 1984).  These transformations are performed by a  number of classes of microbes including gram positive and gram negative bacteria as well as several species of fungi (Neison et al. 1987; Harper et al. 1989).  Allard et al.  (1988) investigated the rate of transformation of 3,4,5-TCG to 3,4,5-TCVer in a number of bacterial strains. resulted  It was discovered that high concentrations (100 ppb) of 3,4,5-TCG  primarily  substrate  in  the  concentrations  formation  led  chloromethoxybenzenes).  to  of  the  a complex  However,  veratrole array  metabolism  of  derivative,  but  metabolites  seemed  to  decreasing  (chlorosyringols,  shift  back  towards  chloroveratrole production as cell densities decreased to low concentrations, as would be expected in the environment.  Relatively rapid O-methylation of CCs, to CGs and  veratrole derivatives has also been observed in natural sediments et al. 1992; Remberger et al. 1986).  and soils (Brezny  Incubation of cell cultures with B K M derived  lignin has also  yielded both tri- and tetra-chloroveratrole derivatives  1988), however  it is  cleavage methyl  conditions  in  environmental  (de-O-methylation) sediments  (Neilson  significance  Dechlorination  has  reactions  derived from  has et  not  been  al.  yet  are  reported  to  Allard  et  1984;  occur al.  attack and Removal of  under  1988),  anaerobic  however  the  been determined.  carried  out  by  both  aerobic  and  anaerobic  Aerobic processes most commonly proceed by hydroxylation followed by  reductive dehalogenation were  products were  of lignin precursors or from adsorbed C G and CC molecules. groups  bacteria.  not certain whether  (Allard et al.  carried  out  chlorophenolicus  by .  and aromatic ring cleavage. Haggblom et  al.  (1988),  Investigations  using  the  of this  bacteria  process  Rhodococcus  This species prefers to attack the chlorophenols, but will  also  degrade other related compounds possessing a methoxy group in ring position 2 or 6. Therefore it is not a good degrader of the CCs. Fastest reaction rates seem to be for the more highly chlorine substituted compounds, but is also dependent upon the position of the chlorine atoms (eg. rate of reaction for 3,4,6-TCG > 4,5,6-TCG). Anaerobic  dechlorination  of  CCs has  mesocosm studies (Neilson et al. 1989).  been  observed  in  natural  sediment  The CGs can also be metabolized by this route,  provided they undergo de-O-methylation to yield the corresponding C C .  Unlike  30  aerobic  dehalogenation,  which  can completely  parent  molecule,  anaerobic  dechlorination  remove  all chlorine  appears  to  halt  atoms from after  a  the  single  dechlorination (Neilson et al. 1987). Higher  organisms,  biotransformations  of  such  as  fish  tend  chlorinated phenolics  the  urine  also excreted,  or intestinal  bile  soluble  secretions.  but in concentrations  carry  in detoxifying  the liver and kidneys, yielding more water through  to  roughly  ten  Conjugation, in fish  enzyme  times lower  1989).  species,  enzyme  catalysed  (UDP-glucuronyl  molecule  (glucose derivative) to a reactive hydroxyl site.  phenolics  than the  most often  transfer  (Brachydanio  rerid),  with  of  a  Sulfate  by  zebra  quantities  fish,  are  involves  glucuronic  the acid  group conjugation For example,  to glucuronide and sulfate conjugates by zebra fish  times greater (Neilson et al. 1989). metabolized  in  transformed  is also found in most fish species, but to a lesser extent (Kennedy 1989). CGs and CCs were metabolized  mostly  which are eliminated  Parent chlorinated  (Kennedy  mediated  tissues located  conjugates,  products  transferase)  out  first  of  the  former  ranging  from three  The more lipophilic chloroveratroles by  single  or  double  to  seven  were also  de-O-methylation  to  the  corresponding C G or C C , followed by conjugation and excretion (Allard et al. 1988). Half lives of chlorinated phenolics in fish vary with species, but generally appear to be < 3 days. (Salmo  Kennedy (1989) reported a half life of 65 h for PCP in rainbow trout  gairdneri),  while a half life of 6.2 - 23 h was reported for PCP for the same  fish species in another study (Metcalf et al. 1988). half  life  of 4,5,6-TCG  alburnus)as  and TeCG  being less than 48 h.  in the  Renberg et al. (1980) reported the  brackish water  aquatic  trophic levels.  species  (Alburnus  These results are of importance, because they  indicate that there is little likelihood of biomagnification higher  fish  of chlorinated phenolics  at  31 2.2.7. Fate of Chlorinated Phenolics in the Fraser River  In the Fraser River upstream of Hope B.C., the major source phenolics  are five BKMs, discharging approximately  15,000 kg of A O X and 56 g of  chlorinated phenolics per day into the river (Schreier et al. 1991). industrialized Lower Fraser Valley there sources of chlorinated phenolics,  are a wide  that during spring and summer high river flow  winter low  flow  in higher  concentrations  periods concentrations  The relative contribution  to the Fraser River Estuary is of  the greatest overall importance, but varies seasonally.  were present  In the heavily  variety of point and non-point  most commonly the CPs.  of upstream B K M sources of chlorinated phenolics  phenolics  of chlorinated  Carey and Hart (1988) found  periods, in the  of chlorophenols  B K M derived chlorinated  Estuary.  However, during  in the Fraser Estuary area,  the north arm of the Fraser River in particular, showed sporadic high episodes due to runoff  from riverside  lumber treatment  fascilities.  Fraser River waters and sediments show measurable levels of CPs (2,4,6-TCP, 2,3,4,6-TeCP, PCP), CCs (3,4,5-TCC, TeCC) and CGs (3,4,5-TCG, TeCG) throughout the year (Carey 1988; Dwernychuk et al. 1991).  Water concentrations of 3,4,5-TCG in the Lower  Fraser River were found to range from 0.03 ug/L at high flow to 0.07 pg/L at low flow (Carey and Hart 1988). 3,4,5-TCG,  which is  TeCG concentrations were roughly 50% of those recorded for  in agreement  compounds in bleachery effluents  with the  estimated  (Voss et al. 1980).  River sediments (Dwernychuk et al. 1991)  relative  proportions of these  Recent  monitoring of Fraser  from both the middle and lower reaches  revealed concentrations of 3,4,5-TCG, TeCG, 3,4,5-TCC and TeCC ranging from 1 - 19 ug/kg, however  very little TeCP was detected in middle Fraser River sediments and  levels up to 6 pg/kg were measured in Lower Fraser River sediments. A  variety of fish species have been shown  chlorinated macrocheilus) oregonensis,  phenolics. and  Both  open  rainbow  water  trout),  bottom  fish  predatory  fish  sampled  from  to bioconcentrate (largescale  (northern Prince  sucker;  squawfish;  George  tri- and tetra-  south  Catostomus Ptycocheilus 150  Marguerite, B.C., showed CP, CC and C G contamination in liver and white muscle  km  to  32  (Dwernychuk et al. 1991;  Schreier et al. 1991).  highest, ranging from 5 - 4 0 higher (20  - 200  concentrations  ug/kg)  Liver concentrations  pg/kg for tri- and tetra-chlorinated phenols  for  were generally  tri- and  tetra-chlorinated  guaiacols.  an order of magnitude lower.  species may be especially  Rogers et al. (1988a)  at risk, since  they  are present  during the winter,  dilution is at a minimum and environmental conditions  In  spring, Pacific to  chlorinated  phenolic  bioconcentration  statistically  and  concentration  fish  (Thaleichthys  These  liver  estuary.  eulachons  spawn.  bioconcentrated their  (Oncorhynchus  Over wintering stages of salmonid  effluent  estuary  tissue  over wintering in the Middle Fraser, and 3,4,5-TCG and TeCG in the  same species, in the Lower Fraser at Agassiz, B.C.  the  and even  Muscle  detected 3,4,5-TCG, 4,5,6-TCG and TeCG in juvenile chinook salmon tshawytschd),  were usually  found  to  The toxicological  investigated  for  Rogers  al.  by  significant  reproductive  was  were  greater  organs. increase  pacificus)  In  et  amounts  of  the  related  (1988b). of  addition,  the  these findings  Male  the  phenolic time  pacific  in  eulachons  and TeCG  residence to  Fraser River differences  3,4,5-TCG  chlorinated  in proportion to  significance  are at an extreme.  enter  sex  when  in  tissue in  the  eulachon  were not determined. At  the  average  phenolics are present  pH of  the  Fraser River  Fraser  at pH 7.8.  or hydrolysis are expected  River.  The  7.8  (Carey  1988), chlorinated  mostly in the ionized, water soluble form, except for 3,4,5-TCG,  which is only about 40% dissociated photolysis  of  suspended  Also, as explained earlier, neither  to be significant  sediment  degradative processes in the  concentrations  in  relatively high, but vary considerably between high and low  the flow  Fraser River  are  periods, ranging  from 30 - 130 mg/L in the Lower Fraser at Hope, B.C. (Hall et al. 1991).  The foe,  calculated as 40% of the loss on ignition, is estimated to range from 1.6% at high flow to 6.0% at low flow (Carey 1988). high flow freshet.  The lower percentage of organic particulates during  is due to dilution with inorganic sediments introduced during the spring Partitioning of various CPs to the sediment phase in the Fraser River was  found to be moderate (Ksw = 100 - 1000) by Carey (1988).  Considering the chlorinated  phenolics tend to be in the ionized, water soluble form at the ambient pH and that the  33 actual sediment load in the Fraser is low (30 - 130 ppm) relative to the water volume, it is expected that most of the chlorinated phenolic load would be carried in the water phase;  environmental  fate  would probably be  governed  by processes  affecting  the  water phase more than the sediment phase (Carey 1988). For similar reasons discussed above, it does not seem likely that a very large portion of the total chlorinated phenolic loading from BKMs would partition into the biota, which does not, however, imply that exposure levels would not be biologically significant.  The primary mode of uptake of chlorinated phenolics  biota would be direct absorption from the  water,  by Fraser River  of undissociated  and ion paired  forms, rather than accumulation through the food chain (Carey 1988).  Many aquatic  organisms clear these compounds rapidly from their bodies, thus there may never be a build up of chlorinated phenolics. water, sediment residence summer  organisms phenolics  of chlorinated phenolics in  and biota should be short, considering the relatively short hydraulic  times in the high  Overall, persistence  flows  Fraser River,  and the  (Carey 1988).  rapid  However,  the  metabolism since  from the five upstream BKMs,  long-term chronic exposure Direct evidence  flushing  there  action of  of  chlorophenols  are constant  invertebrate  species has  (Schreier et al. 1991). one  benthic been  most  aquatic  of chlorinated  population documented  structure  Benthic invertebrate sampling  to  downstream  pollution, from  of  above more the  and below B K M pollution  tolerant  Prince George  mills  However, it is impossible to link any observed effects to any  class of compounds  known toxins  inputs  impact of chlorinated phenolic  is done routinely on the Fraser River, at various locations in  by  there is still concern about the impact of  B K M sources, in the Fraser River has not been found.  Shifts  spring and early  to the biota.  of ecological  outfalls.  the  present  in B K M effluent,  as well as uncharacterized components  since  there  are so  in B K M effluent.  many other Cytochrome  P-450 enzyme induction is another impact monitoring parameter which may be used in the future, but also suffers the same uncertainties.  Assuming that there are some  impacts attributable to chlorinated phenolics or B K M effluents in general, the broad ecological  relevance  remains  uncertain.  34  The  uncertainties  phenolics,  together  this  of  class  Though halides of  acutely wide as  from  point  variety  of  liver  imbalances  and  environmental  chlorinated  (Schreier  effects and  since  are  they  highly  vertebrates,  it is  focusing  to  toxic, also on  that  the  prudent  chloroguaiacols  shown  1991).  Chlorinated  1000  et  1988).  sound  and  high  fate  cheap  to  programs  Since  by  in  would  3,4,5-TCG,  Much to  in on  focus  make  4,5,6-TCG,  TeCG),  on  the  chemical the  variety  of  B K M sources  from  good  such  making  wide  target  microbes  of  compounds, and  seem  are  readily  and  higher  Based  upon  B K M sources,  choices  a  metabolism  detailed  a  also  species,  in monitoring.  phenolics  are  manifest  B K M effluents  both  kilometres  and  chloroguaiacols  these derivatives  of chlorinated  of  fish  promising  the  classes  techniques,  focusing  especially  in all  glycogen  lead  monitor  concentrations  compounds  chloroveratrole ( 3 , 4 , 5 - T C V e r ) .  also  analytical  chloroveratroles,  to i n c l u d e  in  (Oikari  has  organic  phenolics  1987)  hormones,  that  total  hundreds  ug/L)(McLeay  al.  suggest  the  bioconcentrate  sampled  C V s make  (4,5-DCG,  to  chlorinated  situ m o n i t o r i n g .  of  species,  B K M industry.  following  fraction  of  waters  routine in  steroid  monitoring  and  for  small  phenolics  easy  lipophilic  receiving  and  of  relatively  discharge the  rate  C G s and  in  specific to  DCV),  the  present  transformed  suggested  For  al. -  in  effects  p h y s i o l o g i c a l disturbances  chlorinated  media.  fish  bilirubin  relatively  a  been  (200  growth  ecological  targets  only  et  development  phenolics  phenolics,  monitoring:  of  of  good  to  metabolic,  and  presence  have  sources  chlorinated  is  they  microbes  metabolism  fate  up  from  decreased  environmental  studies  make  make  BKMs,  sublethal  characterization  be  would  t o x i c i n h i g h concentrations  altered  to  the  measurable  phenolics  organisms,  known  their  compounds  discharged  from  with  chlorinated  aquatic  concerning  for  it  routine  chlorovanillin  (5,6-  35 2.3. Biomonitoring  Aquatic environmental pollutant monitoring can be as defined of  the  spatial/temporal distribution of  pollutants  of concern as well  contaminants.  as determine  Monitoring  aid in the  assessment of  the  associated  risks  are  always  many  environmental  uncertainties  decision  changes  and  it  data base from which to draw conclusions. with comprehensive which  yield  the  and site  knowledge  making, therefore  is  pertinent  and  remediation effort.  desirable  affected  concerning regulation of  gaps  to  to  contend  have  There with  in  a comprehensive  With the high economic cost associated  field monitoring, it is necessary  most  the  Aquatic monitoring  to both humans  ecosystems as well as the effects of management decisions pollutant discharge, industrial process  can identify  sensitive areas and time periods where  and when potential environmental impacts may be manifested. data  as the tracking  information  at  to select monitoring techniques  the  lowest  cost.  However,  the  variability and complexity of temperate aquatic systems conspire against this goal. The primary tool employed in aquatic pollutant monitoring is water sampling, often  conducted by periodic single  monitoring is,  however,  of low  sample (grab sampling) collection.  sensitivity,  since  present in very low (ppb - ppt) concentrations. discharged  from  B K M sources,  target  Direct water  organic contaminants  are usually  In the case of chlorinated phenolics  compounds  are detected  with  only variable  success, often only within several kilometres of the point source (Oikari et al. 1985; Metcalf and Hayton 1989). pollutant concentrations, in pollutant discharges.  Further complications occur due to temporal variations in  arising from  seasonal  water  flow  changes  and  For example water flow on the Lower Fraser River varies, on  the average, from 800 m^/sec to 5700 m^/sec between spring high flow low flow water  periods (Hall et al. 1991), resulting in possible  contaminant  distribution  between  fluctuations  concentrations. water  and  Further suspended  seven fold differences  complications sediments  sediment loads shift seasonally in quantity and character.  and winter  may  arise, change  in  since  pollutant  as  suspended  Temporal variations in  36 point  source  depending  pollutant  upon  substitution, weekly et  is  are  In  related  place take  examples  was at  late  place  to  hours  (Hall  contaminant  water  appropriate  timing  travel  that  the  and  important Spatial  with  are  depositional adequately  detecting  flow  the  in  the  great  of  main  the  as  time  of  variations  in  and  of  wash  down  water  to  of  water  many  output  filtrates  as  in  as  pollutant  activities, not  whole  discharge.  from  which  have  a  (Liebergott  well  chlorophenols,  on  treated  often  been  took  likely  to  sources  of  temporal  variations  in  necessity  to  carefully  consider  the  take  until  as  into  account  phenolics,  habitats  under  the  and  repositories may  also  differing  for  distribution  the  Fraser  the  with  ice  the  conditions  may part,  Estuary.  which  serve  contaminated  change  flow  most  River  sloughs,  for  under  spatial  such as the Fraser, we  reaching  example,  variations  along  pollutant  simple  dioxide  as  sediments.  season,  due  dynamics have  not  to and  been  River.  with  systems,  results,  data  chlorine  basis  implemented  phenolic  s a m p l i n g might  the  as  be  variations  release  contaminants  aquatic  integrated  monitoring  effects  well  For  concentrations  generating  through  can  bleaching  chlorinated  water  spatial/temporal  of  achieved  needs  back  Fraser  monitoring of  water  such  monthly  monitoring.  flow,  sediments  comprehensive  linking  out  changes, which  above  soluble  dynamics.  studied  low  of  water  habitats  tendencies  With  uptake  point  also  numerous  aquatic  river  The  w e e k l y or  term  water  1988).  d i s t r i b u t i o n o f r i v e r borne  changing  and  on-site  a daily,  chlorinated  short  when  monitoring  in  sporadic  frequency  relatively  there  change  pulping  to  night,  on  In the case o f d y n a m i c r i v e r systems,  downstream  However,  related  concentrations  of target pollutants. expect  of  sporadic  Jacobs  Comprehensive  process  industry,  i n the  occur,  variations  more  be  and  also  B K M process  a  spills  of  lumber  found  of wide  Accidental  effluent  lumber  in  can  cause.  example  resulting  1990).  the  specific  an  basis,  al.  the  discharge  the  covering,  biological  on  monitoring,  organic  risk since  pollutants  are  complexities, high  there i s  concentrations.  to  and  a  cost  need  average,  Also  needed  and as  effect.  a low the is  on  in  whole  is  method spectrum  method not  section  in  with  cost  some  This  described  dependent  associated  for  the  difficulties  of  directly  2.2.5.  environmental  the  37  variables  other  than  water  that,  as  a compliment  used  as  reliable  method,  to  effects on organisms  This  thesis  focuses  on  tracking  of  Biomonitors,  in  tissues,  excess  the  of  were too low time  samples  the  relatively  short  bioavailability changing  indicators,  advantage  pollutant  of  a  phenolics,  since  or  given  compound,  which  exist  phenolic  measurement  by  taking  can  up  contaminant  binding  effective  concentration  a single  water  that  specific  site  of  entities,  such  to  detect  they  are  which  phenomenon  also  spatial  may  in  the  more  organic  competitive  of  a  -  particulates partitioning.  appropriate for every  aid in development  vary  such  may  potential  the  under as  the  presence the  implies  that  aquatic  a  of  a  decrease  This  adjustable  on  non-dissociated  Also the  of  small  greatest  constant  bioavailable  far  which  up  the  compounds  in  effective  measure  remain  for  levels  taking  conditions  not  important  tissue  are also  is  provide  is  quality criterion may not be biomonitoring could  both  waters.  contaminants  constantly  environmental  as  a compound by  This  of  and  be  contaminants  concentrate  form or the less bioavailable ionized form; depending on water p H . of  could  distribution.  Biological organisms  organisms  This  may  organisms  situ identification  possible  This  Living  conditions.  in  they  ambient water.  discharge  suggests  of pollutant levels in receiving  function  making it  levels,  biological  which  contaminants.  that  scale.  environmental  chlorinated  being  evidence  broadly include  for  environment.  pollutant time  leeches  pollutant  ambient  when  of  strong  chlorinated  as measures  phenolic  to detect in the  of  importance  use  is  monitoring,  biomonitoring, can  concentrations,  of  water  chlorinated  major  integrators  There  B K M derived  as well  the  as  water  of  as  toxic  their  standard  indicators  properly termed  temporal  concentration.  system and  water  quality  criteria. There chlorinated the  use  are  numerous  phenolic of  species  from  contamination.  bioindicator  Phillips  organisms  provided a useful set of guidelines  which  to  to  (1978),  quantitate  select in  a  comprehensive  organochlorine  to aid in the selection  biomonitor review  of of  contamination,  of an appropriate  38  biomonitor.  The selection criteria suggested that the potential biomonitor should be:  1) representative  of the region to be monitored,  2) hardy enough to be maintained and tested under laboratory conditions, 3) long lived enough to allow sampling of more than a single age class, if desired, 4) large enough to provide an adequate sized tissue sample for analysis, 5) demonstrate  a high bioconcentrating capacity, without being killed under a wide  range of exposure  conditions,  6) show a direct correlation between bioconcentration and average  pollutant  concentration.  Phillips  also  suggested  that  the  biomonitor  should  be  tolerant  of  brackish  water,  which adds to the versatility of the biomonitor, but is not strictly necessary under freshwater  conditions.  characteristic  of  To  a slow  rate  the more a compound is and the better the  this of  list  it  is  also  contaminant  important  to  transformation  the  additional  and elimination,  retained in a biomonitor, the greater  correlation between water concentration  add  the  since  bioconcentration  and tissue concentration.  It would also be desirable to select an organism which can be propogated at low cost, under laboratory conditions,  in order to provide a homogeneous and uncontaminated  population. Various  aquatic  species  phenolics in the field. Ontario,  revealed  aquatic  organisms  informative, field varies  have  2.6)  serving  considerably  to  in  (Metcalf  since there were a wide  conditions,  shown  to  bioconcentrate  chlorinated  Comprehensive sampling of an industrially polluted creek in  marked differences (Table  been  illustrate  between species  the et  bioconcentrations  al.  1984).  This  of  chlorophenols  study  was  in  especially  variety of organisms  sampled, under identical  that bioconcentration  of  and that leeches  indicators of chlorophenol contamination.  chlorinated  phenolics  may be particularly sensitive  Further investigations by Metcalf et al.  39 (1988),  comparing biomonitoring potential  Erpobdella  punctata and Helobdella  the  stagnalis,  of  three  leech  species,  Dina  dubia,  revealed that all three species of leech  showed a high bioconcentrating capacity (BCF = 600 - 16700), slow elimination rates ( > 25  days)  proportion  and to  bioconcentrated  an  elimination  bioconcentrations  concentrations.  (2,3,6-TCP,  and  apparent  for  were  exceptions  which  species.  elimination.  pattern  which  The  2,4,5-TCP),  by all three leech  bioconcentration  without  tissue  exposure  trichlorophenois  both  final  seemed  in  the  same  relative  were  two  of  to  preferentially  be  Interspecies variability was Bioconcentration  the  different  varied  chlorinated  the  observed in  between  species,  phenolics,  while  was most rapid for H. stagnalis and slowest for E. punctata  Table 2.6:  Bioconcentration of chlorophenols by various aquatic organisms sampled from Canagagigue Creek, Ontario, Canada. Data from Metcalf et al. 1984.  Bioconcentration Factor Organism  2,4-DCP  2,4,6-TCP  2,3,4,6-TeCP  Leech  (Dina dubia)  173800  68727  122000  Leech  (Erpobdella punctata)  163733  100405  145750  Aquatic Worms  (Oligochaeta)  39200  9595  9250  Dragonfly Larvae  (Anisoptera)  -  784  34750  Caddisfly Larvae  (Pvcnopsvche SD.)  2533  2054  12750  Clams  (Ferrissia sp.)  -  270  3750  Snails  (Phvsa so.)  -  396  1666  Crayfish  (Orconectes DroDinauus)  16  52  250  Bull Frog Tadpole  (Ftana catesbeiana)  -  52  -  Rock Bass  (Ambloplites rupestris)  379  36  450  Aquatic  mussel  (Tessier et al. 1984) have  species,  been  and organic pollutants  a high bioconcentrating  developed  have  favoured  as  biomonitors  both  metal  (Kauss and Hamdy 1985), because they  capacity for certain of these pollutants  laboratory maintenance  of  and monitoring protocols.  In field  and have  well  comparisons  40 between the mussel species (Elliptio obscura), proved  complanata) and the leech species  (Nephelopsis  downstream of a B K M source in the Rainy River, Ontario, the leeches the  better  bioconcentrating chlorophenol  biomonitor  capacity  congeners  and  of  chlorophenols,  ability  (Metcalf  to  and  conducted by Hall and Jacob (1988)  Hayton  marmoratato  be effective  information  the  discharge  facilities,  into  differences  the  sporadic  the  on  their  relative  1989).  higher  proportion  Further  of  investigations,  showed N. obscura and a second leech species,  Percymoorensis on  indicate  based  of  Lower Fraser River.  in bioconcentration as well  in providing time integrated monitoring chlorophenols,  These  studies also  from  lumber  indicated  as hinting at differences  storage  interspecies  due to leech  size.  Laboratory testing of the above pair of leech species also revealed the importance of environmental positively Overall,  factors  correlated one  in biomonitoring. to  water  can conclude  biomonitoring  organisms  Leech bioconcentration  temperature  from the  and inversely  above  and sensitive  investigations  indicators  of  was  correlated  found to  that leeches  chlorophenol  to  be  water pH. make  good  contamination.  Based upon past success in using leeches as biomonitors of chlorophenols in both laboratory and field conditions, it seemed likely that leeches could be effective biomonitors of the other related classes of compounds, such as the chloroguaiacols, chlorovanillins  and chloroveratroles.  chlorinated  phenolic  obscura,  readily available  is  biomonitoring in the  While  several  candidates, current study  one  species  of  species  leeches in  are  good  particular, N.  region, encompassing  the Fraser  River Watershed, is  easy to maintain in the laboratory and has proven to be an  effective  under  biomonitor  both  though not tested as extensively  laboratory as N.  and field  obscura,  conditions.  also makes  P.  a good biomonitoring  candidate, since it is also available in the study region and has been used in  Fraser River  chlorophenol biomonitoring.  marmorata,  successfully  41  2.4. Leech Biology  Leeches environmental  are  generally  conditions.  hardy organisms,  able  to  tolerate  a wide  variety  N. obscura, of the family Erpobdellidae, is a common cold  water species of leech, inhabiting waters from the mid latitudes  of Canada south to  the northern United States and Rocky Mountain states (Linton et al. 1983). extensive  survey  obscura  is  ranging  from  of  leech  generally  of  species in Colorado (Herrmann 1970)  associated  oligotrophic  to  with  quiescent  eutrophic.  N.  lentic  obscura  revealed  waters has  An  of  been  that N.  productivities  found  in  waters  ranging from 0.5 °C to 24 °C and pH 6.0 - 9.8 (Herrmann 1970; Metcalf and Hayton 1989). N. obscura has been found in waters of total ash and organic solids contents of approximately 10 - 1000 mg/L and 10 - 300 mg/L respectively, and total alkalinities of 10 - 100 mg/L (Herrmann 1970). N. obscura is as  snails  feeding  a predator and scavenger,  (Gastropoda)  and  insect  on any dead organisms  larvae  hunting  aquatic  invertebrates,  (Chironomid, Coleopteran  it can locate (Anholt  1986).  Life  groups) history  such and  studies  conducted by Davies and Everett (1977) on the species N. obscura, in Alberta,  revealed  that this species lives from 12 to 19 months in the wild and at least two  different  generations are present which  hatch  in  the  in any one period. spring  and  are  The first consists of those individuals  sexually  mature  individuals may die off early in the summer. leeches,  which  following  in  summer or fall  sexually. weight.  hatch  the  summer;  these  the  next  spring; these  There is also a second generation of individuals  and sometimes carry over  The critical factor  by  are  another  not  mature  year before  triggering breeding by N. obscura  until  the  maturing  seems to be body  Further, the body weight at which any given leech species reaches maturity  seems to population  vary with the lives  in  environmental  (Davies  characteristics  and Everett  1977;  of  Peterson  the  particular habitat  1983).  Therefore,  that leech  generations may carry over to a third season if they did not reach an appropriate body  weight  concerning  to  trigger  identification  sexual  maturity  by  the  second  season.  of N. obscura refer to Appendix 1.  For information  42  P.  marmorata of the family Hirudinidae, is one of the widest ranging of North  American  leech  species,  being  United States (Sawyer 1972).  found  from  Alaska  through  to  the  P. marmorata has been found associated  mid northern with both lotic  and lentic habitats, but shows less tolerance for oligotrophic waters than N. obscura. P.  marmorataaad  has a narrower optimal temperature (5 - 25 °C) and pH (7.2 - 8.2)  range, but has wider tolerance for dissolved inorganic (20 - 2800 mg/L) and organic (50 - 1000 mg/L) solids content and total alkalinity (10 -150 mg/L) (Herrmann 1970). Like N. obscura, P. marmorata preys N.  obscura, as well  takes place  as being  somewhat  later  an opportunistic  than for  temperature, than leech weight.  on  N.  many  aquatic  scavenger  obscura,  being  invertebrates,  (Sawyer triggered  1989). more  concerning  Breeding by  water  Egg laying takes place in mid to late summer and  the young hatch in the same year and overwinter in the lake (Sawyer information  including  identification  of P.  1989).  marmorata refer to Appendix 1.  For  43  3. METHODOLOGY 3.1. E X P E R I M E N T A L ORGANISMS  Leeches  were collected  in August 1990  and June and September  1991,  littoral zone of Black Lake, located about 150 km east of Princeton, B . C . not  support  a  invertebrate  fish  population,  population.  due  Leeches  of  leech  in the  Typically 5 - 1 0  conditions,  external  [Haemopsis]  morphology,  (Appendix  1).  obscura  was  the  by  consisted out  Leeches  more  numerous  approximately of  a mixture  mature  10:1 of  obscura  and location was  of Biology, University of  of  in  the  the  different  a  rich  shoreline  Two species  were  tentatively  (TV. obscura) a n d  Davies, male  on  and  the  basis  female  of  gonopores  later confirmed as correct by Dr. R.  Calgary).  two  leech age  These Verrill  In general,  - 6.0 g) than did N. of  had  15 - 90 min.  '(P.marmorata)  identification  ranged to a larger size (0.5  marmorata  spawned  behaviour  Leech species  (Department  marmorata  texture,  but  traps were placed along the  Nephelopsis  marmorata  This lake did  10 crn^ wire mesh traps (5 mm  - 2.5 m and then retrieved after  laboratory as either  Percymoorensis  Lake  anoxic  appeared to be common in the catch from the lake.  identified  Davies  winter  were captured using  mesh) baited with calves liver. at depths ranging from 0.5  to  from the  leech  traps.  classes,  specimens  of  obscura (0.1 g - 2.0 g). species,  outnumbering  Leech populations ranging  in  from juveniles  P. N. P.  Black up  to  adults. L aquaria containing  aerated  native lake water (depth = 5 - 10 cm) and a sand bottom littoral zone habitat.  Leech  loading  were maintained in the  ranged from 20  to  50  leeches  laboratory, in 38  per aquarium.  Scattered  were provided for secure hiding places.  Water temperatures  The  free  leech  intervals. species  stock P.  were  was  fed  calves  marmoratawas kept  in  liver,  observed  separate  laboratory stocks of P.marmorata  aquaria.  of  chlorinated  to prey upon N. There  however, N.  was  no  rocks  and driftwood  ranged from 10 - 15 ° C . phenolics,  obscura, observable  at  seven  therefore, mortality  the in  day two the  obscura populations suffered 20 - 40%  44 mortality during the month after capture. sexually  Mortality occurred mostly in the larger,  mature adult leech population.  Black Lake is a small pothole lake, which is in an area isolated from any direct sources of B K M pollution, however its proximity to a highway was of concern with respect  to  potential  chlorinated  phenolic  contamination.  Laboratory  analysis  revealed that none of the test compounds were present in leech tissues.  3.2. LABORATORY STUDIES  Laboratory rate  and  turbidity, general  leech  assess  bioassays  the  leech  effects  weight  protocol  Conditions specific In keeping  of  and  followed  were  pollutant  interspecies  throughout  to each bioassay with  which had indicated  steady  reached after  about seven days,  present study.  The semi-static  to  measure  concentration, differences  the  on  pollutant depuration  water  temperature, pH,  bioconcentration.  laboratory bioassays  is  The  described  below.  accompany the results for each experiment.  earlier leech  that  conducted  monitoring studies by  state  Hall  tissue concentrations  a seven day exposure  of  & Jacob (1988), chlorophenols  period was  were  selected for the  bioassay protocol used by Hall & Jacob (1988), where  water was replaced completely once every 24 h, was followed.  This exposure protocol  is  are  a  compromise  constantly  between  decreasing  systems, which allow  pollutant  at  dichloroguaiacol  an  bioassays,  constant  which  and complex exposure  flow  characterized through  by  bioassay  concentration.  were spiked with a 100 pL mixture of the following test  appropriate  (4,5-DCG),  static  concentrations  for relatively  Laboratory bioassays compounds,  strictly  concentration,  3,4,5-trichloroguaiacol  using  a  methanol  (3,4,5-TCG),  carrier;  4,5-  4,5,6-trichloroguaiacol  (4,5,6-TCG) and tetrachloroguaiacol (TeCG), 5,6-dichlorovanillin (5,6-DCV) and 3,4,5trichloroveratrole methanol carrier.  (3,4,5-TCVer).  Control  Simultaneous exposure  bioassays  were  spiked  with  100  to all compounds was adopted over  pL of single  compound exposure, since field studies by Carey and Hart (1988) indicted that aquatic  45  organisms are exposed to a mixture of chlorinated phenolics in the Fraser River and indeed many other B K M receiving waters. were stirred vigorously with a glass rod.  Following spiking, all bioassay Leech bioassays  waters  took place in one or two  litre volumes of water, contained within one or two litre Mason Jars, which had been rinsed three times with hexane.  Since leeches are somewhat  try to climb out of bioassay jars,  amphibious, and will  plexiglass covers (3 mm thickness) with 9 air holes  (1.5 mm i.d.) drilled through the surface of the plastic, were used to cover jars. loading into bioassay obscura,  Leech  chambers ranged from about four to six grams of leeches (N.  n = 5; P marmorata, n = 2 - 3) per 1 L water.  Bioassay medium  water  - hard  analytical  was  made  formulation.  grade chemicals,  from  reconstituted  Bioassay  water  was  laboratory made  distilled  up using  water  the  following  supplied by the BDH Chemical company; NaHCO^ (110  mg/L), C a S 0 • 2 H 0 (60 mg/L), M g S 0 • 7 H 0 (120 mg/L) and KC1 (4 mg/L). 4  of  2  4  2  Water pH  of this formulation ranged from 7.7 - 7.9, but was adjusted to the desired pH for each bioassay by drop-wise addition of 2M HC1 and 5M KOH, which had been pre-extracted twice with hexane. a temperature  Temperature control was maintained by conducting bioassays  control chamber.  Vancouver City  well  water  was  in  provided by the  Department of Fisheries and Oceans, for the flow through experiments. Turbidity  bioassays  laboratory bioassays. in  the  suspended  stirring apparatus.  followed  a  slightly  different  protocol  from  other  In order to maintain the sediments in each bioassay chamber  form,  bioassay  water  was  continuously  stirred using  The leeches appeared to be irritated by the  action, producing more mucous  a magnetic  continuous stirring  than in previous unstirred bioassays.  Therefore, a  control bioassay was run without any stirring action, in order to determine the effect of  this  disturbance on leech  Turbidity experiments  bioconcentration. were designed  turbidities (18  - 147  seasonal  (Hall et al. 1991).  cycle  mg/L) encountered  fraction of < 63 um, which is  to mimic the composition  in the Middle Fraser River throughout one  Turbidity bioassays  thought  and range of  to represent  the  focused most  on the particle size significant  surface for organic pollutants (Voice et al. 1983: Schellenberg et al. 1984).  adsorptive Field  46 sampling indicated that the organic fraction of Middle Fraser River about 4 - 7% percent of total suspended sediments.  accounted  for  Therefore we selected a suspended  sediment composition of 95% inorganic to 5% organic content for bioassays. To model the character of Fraser River suspended sediment load more closely , an X-ray diffraction analysis of the < 63 pm fraction was carried out by Dr. Les Lavkulich, of the University of British Columbia Soil Science Department. diffraction it was possible  With X-ray  to identify and estimate the relative amounts of the clay  minerals present in sediment  samples.  The procedure is able to measure the spatial  orientation of atoms in a three dimensional crystal lattice, which is unique to each clay  mineral.  The  principle  clay  approximately 60% : 40% ratio. ratio  of  chlorite  sediments.  to  illite  components  were  chlorite  and  illite,  in  For the purposes of our bioassay we selected a 1:1  to  represent  the  inorganic  fraction  of  the  suspended  Both chlorite and illite were supplied, in gravel form, by the U.B.C. Soil  Sciences Department.  Clays were washed with distilled water and methanol  solvent  and then processed into a fine powder by pulverization in a ring grinder at the U.B.C. Geology  Department.  The  selection  turbidity bioassays  of an appropriate source of organic material for spiking into the is not straight forward.  The exact nature of the organic content  of  suspended sediments in the Fraser River downstream of Prince George is difficult  to  assess  Both  and probably varies  anthropogenic  and  with  natural  seasonal  sources  changes in environmental  account  for  suspended  conditions.  organic  particles.  Municipal and industrial discharges of biosolids and woody plant material, as well as plant material entering the system from surface runoff and stream bank erosion are examples plants  and  bioassays, crushed  of  outside animals  sources. are  Natural  important  we selected a single alder tree leaves  internal  of  aquatic  sources.  In  bacteria, planktonic  order  to  simplify  organic component for laboratory bioassays.  (Alnus  organic fraction of suspended  populations  rubra)  sediments.  (< 63 pm)  were  selected  Dried,  to represent  The leaves were collected  the  the  in green form  from trees located on the University of British Columbia Endowment Lands, dried at 104° C and ground into a fine powder, using a mortar and pestle.  47  Suspended as  carriers  inorganic  to  introduce  clay  introduced  to  sediment  solution bioassay  inorganic/organic sediment  spiking  sediments  to  of  g/L)  (0.05  jars  particle  l e v e l s o f 0.025  solutions  prior ratio  to of  g / L (4.5  leech  at 0.08  NTU)  g / L (9 - 15 N T U ) .  and untreated  the  chlorinated 95%  - 6.0  known  concentration  were  Appropriate  amounts  bioassays.  and  -  organic  phenolic  5%  were  nephlometric  (18 - 22 N T U ) , 0.15 g / L (34 - 39 N T U ) . run  of  solution  (0.01  spiking.  prepared of  g/L)  Bioassays  conducted  at  turbidity units  total  the were  with  an  suspended  - N T U ) , 0.08  g/L  A bioassay o f 100% inorganic clay content  was  C o n t r o l bioassays u s i n g clear bioassay water  turbid water 0.08 g / L (19 - .45 N T U ) were also  (0.5  -  1.0  conducted.  3.3. F I E L D S T U D I E S  3.3.1  Study  Area  The Columbia,  Fraser Canada,  three B K M s  BKMs  (aerated  lagoon);  all  the  first  the  Prince  for  two  of  the  major  C I O 2 substitution,  which  mills.  central  plateau  m o n i t o r i n g study,  because  River  clarification  and  Canfor new  and  (Figure  generally  results  m o n i t o r i n g area  o f its  3.1).  reduced  B K M sources covers  about  are a  52  Fraser  River  the  entry  of  a  major  tributary,  the  Nechako  and an area o f strong water m i x i n g at R e d R o c k C a n y o n , about Prince  form  km  upstream  of in of  of  the  this reach o f  the  at  stretch  Prince  George  30 k m d o w n stream  George. C o n t r o l s a m p l i n g took place at Shelley B . C . , about  single  initiatives,  the  present  Within  is  a  A O X formation  Fraser R i v e r , north from S h e l l e y , B . C . d o w n to Stoner Creek. there  share  in  Prince  treatment  policy  changes,  British  p r o x i m i t y to  effluent  government  in  of  A l l three  Intercontinental,  process  No  region  secondary  provincial  bleaching  of bleaching. The  in  Fraser  from  undergoing  George  the  mills,  pressure  c h l o r i n a t i o n stage  George  thi§  primary  Under  were  increasing  Prince  selected  practice  outfall.  mills  was  near  d i s c h a r g i n g d i r e c t l y into  George  effluent  River  6 k m upstream  o f the  of  48  Figure  3.1:  Fraser  River  biomonitoring  study  area.  49  Northwood B K M , where a Water Survey of Canada sampling station is located.  The site  is located near an abandoned sawmill facility, which could be a potential source of tri-, tetra- and pentachlorophenol, however there should not be any of the pulp mill specific  chlorinated  Upstream  of  potential  contributors  chlorophenol stations.  the  guaiacols,  Shelley  site  of  compounds,  vanillins are  several  chlorophenol  2,4,6-TCP  or  veratroles other  associated  active  sawmill  contamination.  For  and 2,3,4,6-TeCP were  with yards this  monitored  this  site.  which reason  at  the  are two field  One sampling station was set up at Shelley, on the east side of the River, in a  zone of moderate water flow. The downstream monitoring station downstream of Prince George.  was located at Stoner B.C., about 40 km  This site was chosen since it had been used for routine  monitoring conducted by the British Columbia Ministry of the Environment.  Since  the site is about 10 km below a strong mixing zone (Red Rock Canyon), it was hoped that water from the Nechako and Fraser Rivers along with the B K M effluent would be completely mixed upon reaching the Stoner station.  Sampling stations were set up on  both sides of the River, about 1.5 km downstream of the entry point of Stoner Creek to the Fraser. This distance  was beyond the reach of any back eddy generated by the  confluence on the Fraser River and Stoner Creek.  3.3.2. Study Periods  In  order  conditions,  to  evaluate  monitoring  trials  periods: summer (July 8-15, 26, 1992). on  earlier  the  leeches were  under  conducted  a  wide during  three  of  environmental  different  seasonal  1991), fall (October 17-24, 1991) and winter (February 19-  Monitoring was carried out during each trial for seven day periods, based findings,  indicating  that  leeches  attained  concentrations of chlorophenols after seven day exposures Jacob 1988).  variety  steady  state  tissue  at 4 and 12 °C (Hall and  Environmental parameters such as water temperature, pH and turbidity  were monitored at the beginning and end of each monitoring trial.  Water turbidity  50 samples were collected in 50 mL glass screw-cap tubes with a teflon liner and taken back  to  the  laboratory for  turbidity measurement  using  turbidometer (HACH Chemical Co., Ames Iowa, U.S.A.). nephlometric  3.3.3.  turbidity units  a HACH  Model  2100A  Turbidity was measured in  (NTU).  Sampling Procedures  Effluent  samples  Canfor/Intercontinental  were  collected  (200  mL/day)  and Northwood mill outfalls, by mill employees,  from  the  during  five  days of every week long monitoring trial and composited into one single five day sample from each of the two outfalls. used  for sample  collection  and storage.  One litre, solvent rinsed amber bottles were All samples  were  shipped back to  the  laboratory about eight days after the collection of the first sample, in ice packed coolers and stored in a dark cold room at 4 °C in the laboratory. effluent  samples  were  either  extracted immediately  Upon receipt,  or preserved with concentrated  H2SO4 (2 mL/L of effluent) for later analysis. Water samples were collected sampling  techniques.  using both grab sampling and automatic water  Automatic water  sampling was  carried  out  control site in July 1991 and at Stoner in both July and October 1991.  at  the  Shelley  One automatic  water sampler broke down before the October trial so grab sampling was used at the Shelley station from this point onward.  Below freezing air temperatures may impair  sampler electronics and mechanics as well as causing blockage due to frozen water, so  only  grab samples  were  collected  in February.  Winter  water  samples  were  collected on only four of seven days at both stations, namely February 19, 21, 24 and 26. The automatic water sampler, an ISCO Model 2900 (ISCO Inc., Lincoln, Nebraska, U.S.A.) consisted of a 10 m teflon lined sampling tube, which was attached to a battery (12 volt NiCad) operated programmable peristaltic pump. into 24, 500 mL plastic bottles.  The samples are deposited  Previous research by Jacob (1986) showed no  51  significant samples  difference  in  the  quantities  of  chlorinated  phenolics  between  water  stored in the plastic bottles or standard amber glass sampling jars.  The  automatic water sampler was programmed to take a 400 mL sample from a depth of about 0.5 m, every 8 h.  Therefore, over each seven day sampling run a total of 21  water samples where collected. purge cycle samples  was  programmed prior to and after  concentrated  sampling.  To minimize cross contamination between samples, a each sampling.  To preserve  the  H2SO4 (2 mL/L of water) was added to each bottle prior to  In the laboratory water samples were composited for analysis into seven  1200 mL water samples, representing one sample every 24 h period. Grab samples were collected from surface waters in solvent rinsed 1 L amber glass  bottles  and preserved  in concentrated  H2SO4 (2 mL/L of water).  All water  samples were shipped back to the laboratory in ice packed coolers and stored in a dark cold room at 4 °C. Suspended sediments alone in July 1991 February 1992.  were  sampled at only  downstream  Stoner test site  and at both Shelley control and Stoner test sites in October and  No suspended sediments were recovered from the October trial, due to  the disappearance of the sediment traps. B.C.  the  Suspended sediment traps, supplied by the  ministry of Environment, Prince George branch,  consisted  of  four  plexiglass  tubes, 30 cm long, with a 4 cm wide opening at one end, fixed in a plastic holder. trap was suspended at about a i m perpendicular to the flow  The  depth, from a float with the long axis of the tubes  of the water and the openings  facing the water surface.  The traps were then anchored to the river bottom with two approx. 15 kg concrete blocks.  Where possible the concrete blocks were also tied off to shore.  It should be  noted that the dynamics of this particular type of sediment trap change as the trap collects  sediment, resulting in a slightly biased sample.  As the sediment  collection  tubes become full, the force of the downward eddy, created at the mouth of the tube decreases,  resulting  in  the  fractions as the tube fills.  selective  sampling  of  progressively  heavier  sediment  At the bottom of the trap there tends to be a more  representative sample, while at the top there is a bias towards larger sediments. phenomenon becomes significant during medium and high flow periods on the  This  52 Fraser, when sediment loads are high and sediment after a seven day period.  capacity  One solution would be to remove sediments from the trap at  short intervals rather than at the end of possible, due to logistical  traps attain nearly full  the  sampling run, however  this was  not  difficulties.  For Fraser River biomonitoring, leeches  were housed  in cylindrical stainless  steel wire mesh (< 1.0 mm mesh size) cages, measuring 15 cm by 6 cm dia.  Leeches (n  = 10/cage) were suspended from anchored floats at depths from 0.5 - 1.0 m. leeches were placed near the east side of the Fraser at Shelley.  Control  Test leeches, at  Stoner, were stationed near both east and west sides of the River in July and October, to test for across  river differences  in bioconcentration.  only stationed at the east side of the River at Stoner.  In February leeches were N.  obscura was used for all  monitoring trials and P. marmoratav/as used as a measure of interspecies variability at the Stoner site during the July trial.  All leech samples were frozen and shipped on  ice to the laboratory about 24 h after the end of the bioassay. leeches were wrapped in solvent  rinsed aluminium foil  In the laboratory  and frozen at -15  °C, until  analysis.  3.4. ANALYTICAL PROCEDURES  3.4.1. Chemicals and Reagents  Hexane chromatography phenolics  and  methanol  organic  solvents  used  in  preparation  (GC) standards, spiking standards and for extraction  solvents  were all of  from BDH Chemical Co.  gas  chromatographic pesticide  Reagents used in  grade  of  gas  of chlorinated and purchased  extraction and derivitization (K2CO3, acetic  anhydride, K O H and HC1) were analytical grade and purchased from BDH Chemical Co. All  reagent  remaining  solutions organic  were pre-extracted twice with hexane in order to remove any contaminants.  Compounds selected for laboratory and field assay included 4,5-  53  dichloroguaiacol  (4,5-DCG),  3,4,5-trichloroguaiacol (3,4,5-TCG), 4,5,6-trichloroguaiacol  (4,5,6-TCG) and tetrachloroguaiacol (TeCG), 5,6-dichlorovanillin (5,6-DCV) and 3,4,5trichloroveratrole trichlorophenol  (3,4,5-TCVer).  In  addition two  chlorophenol compounds,  (2,4,6-TCP) and 2,3,4,6-tetrachlorophenol  for analysis in field investigations.  Analytical  (2,3,4,6-TeCP) were selected  standards, for laboratory analyses of  the chlorinated guaiacols, vanillin and veratrole were obtained in 99%+ from  Helix Biotech in Richmond,  tribromophenol)  B.C.  2,4,6-  pure form,  Chlorophenols, surrogate standard (2,4,6-  and internal standard (2,6-dibromophenol)  were  ordered from  the  Aldrich Chemical Co. (Milwaukee, Wisconsin, U.S.A.). Stock solutions of chlorinated phenolics were prepared for use as GC reference standards ranging  and from  spiking  solutions.  Individual stock  solutions  of  each compound,  1000 pg/mL to 3000 pg/mL, were prepared in methanol.  The moderate  polarity of methanol made it a good choice for preparation of the standards, since it allowed for easy dissolution of the slightly polar chlorinated phenolics and acted as a good intermediary solvent  for the introduction of organic analytes  extractions and bioassays.  From individual stock solutions,  to water based  mixed spiking standards  containing all the test compounds were prepared at concentrations of 10 ug/mL and 1.0 ug/mL.  All stock  solutions  amber screw cap vials at -15 °C.  and spiking standards were  stored in  teflon-sealed  Standard solutions were never used for more than 6  months.  3.4.2. Extraction Procedures  3.4.2.1. General Rational  Pulp mill effluent, following  the  sample phase.  same  water, sediments and leech extractions were all carried out  general  method  with  certain  modifications  specific  to  each  Since chlorinated phenolics are in the completely ionized form at pH >  12, the samples were first extracted into a basic solution of hCjCOg.  The neutral  54  veratrole  remained  Due phenolics  to are  their not  chromatograms  and  peak  anhydride.  be  to  are  formation  of  1992).  the  of  before  capture  detector  chlorinated  to  compounds  such  used was  organic  GC  analysis  is  al.  Bleached  highly  1992)  phenolic  Effluent  produces  the  acetyl  reactive  groups.  chlorinated oxidation  diazomethane  of  choice  solvent with  the  hexane  electron  sensitive  to  allows  for  which  is  are  it  is  capture  concentrated  the  possible  of  lowest  importance  the  Morales  et  al.  case,  high  to  the  common 1989). the  distinguish be  a highly  acetates an  were  appropriate The  electron  detection  when  by  since  to  (ECD).  with  may  chlorinated  (Kennedy  to  is  in  phenolic  compounds  which  another  known  detection  phenolic  unaffected  our  using  resulting  impossible  chlorinated  and  the  is  in  shape  procedure  1985,  analysis  diazomethane  acetylation,  al.  gas  phenolic  this  since  veratroles  peak  form,  solutions,  et  1992).  chlorinated  However,  (Starck  al.  acetylation,  phenolate  alkaline  chlorinated  addition,  the  phenolics,  in  method  asymmetric  extract  chlorinated  ( M o r a l e s et  reveal  3,4,5-TCVer, making  In  compounds,  electron affinities  limits  dealing  with  for very  concentrations.  Sample  kraft  and  more  using  the  underivitized  d e r i v i t i z e d by  to  chlorinated  not  Following  were  used  the  as  in  often  compounds  Methylation,  compounds.  environmental  3.4.2.2.  susceptible  quinoid  two  volume  et.  of  respective  carcinogen.  (Morales  classes  3,4,5-TCG  with  medium, to  pressure,  c a p i l l a r y G C analysis  Samples  h i g h l y reactive  all  reaction  these  extracted  by  by  methylation  methylation  potent  compounds  vapour  compounds  separation.  the  reaction.  However,  low to  phenolic  alkaline  especially  derivitization  between  pure  for  Neutral  acetylation  step.  and  The  attack  suitable  catechols  this  polarity  poor  ionizes  susceptible  low  of  t a i l i n g and  compounds,  not  high  in  p a r t i c u l a r l y amenable  GC  acetic  unchanged  mill  Preparation  effluent  a m o d i f i e d form o f the procedure  (BKME)  weekly  recommended  composite  samples  by Starck et al. (1985).  were  extracted  This  55  method was selected because of its general high recovery of chlorinated phenolics (> 80 %) and relatively low variability (< 30%). Effluents were mixed to resuspend any settled solids prior to extraction.  A 50  mL volume was measured into a 100 mL graduated cylinder and decanted into a 250 mL separatory funnel.  Residual solids were backwashed from the graduated cylinder  with 20 - 30 mL of effluent from the separatory funnel. 2,4,6-TBP surrogate and mixed by hand.  Samples were spiked with  Effluent pH, measured using E M Scientific pH  0 - 14 pH indicator paper, generally ranged from pH 6 - 7 prior to extraction. 5M K C 0  of the samples was adjusted to pH 8, using 5 M KOH solution. effluent)  was added to each sample to make a final  2  3  The pH  (1 mL/50 mL  K2CO3 concentration of 0.1 M .  Effluents were acetylated by addition of acetic anhydride (1.0  mL/50 mL effluent)  to  the separatory funnel, followed by vigorous manual shaking and venting of gas for 60 sec.  Samples were  phenolics  were  then allowed to stand for 15 min.  then extracted  by the  vigorous manual shaking for 3 min.  addition of  Acetylated chlorinated  10 mL of  hexane,  followed  by  Vigorous extraction generally led to emulsion of  hexane, which was broken up by the addition of 10 drops of methanol, followed by centrifugation covered  at about  with teflon  3000 rpm. for  caps.  15  The hexane  min., in conical  extracts  were  then  glass  centrifuge  transfered into  tubes 15 mL  graduated test tubes and concentrated under a gentle stream of nitrogen gas, in a 30 35 °C water bath.  Extracts were stored in the dark at -15 °C, prior to GC analysis.  3.4.2.3. Water Sample; Preparation  Water effluent resuspend  samples  extraction,  were but  any sediments  at  extracted  using  a procedure  a larger  scale.  Samples  on the bottom of  the  sample  water samples were extracted, unless other wise indicated.  similar were  to  mixed  bottles.  that  used  vigorously  for to  Whole, unfiltered  Water samples (1 - 1.2 L)  were spiked with surrogate adjusted to pH > 12 with 5M K O H solution and extracted with 100 mL of hexane to remove base neutral compounds.  This basic extraction  56 served  to  extract;  clean  this  up  the  solvent  samples,  fraction  allowing  also  for  contained  greater the  concentration  neutral  compound  of  the  final  3,4,5-TCVer.  Waters were then neutralized to pH 7 using 2M HC1, and extracted in 2.0 L separatory funnels,  by the  sequential problems water.  same  hexane were  (100  method per effluent mL) extractions  encountered  with  samples,  with the  were carried out.  either  Fraser River  To break the minor emulsions encountered,  that  No significant  water  1.0  exception  three  emulsion  or laboratory bioassay  mL of methanol was added  drop-wise to the sample in a 500 mL Ehrlenmyer flask, while swirling the extract. Base  neutral and acetylated  acid hexane  fractions  were  filtered  #4 Whatman Filter Paper, into 500 mL round bottom flasks.  separately  through  Hexane fractions were  concentrated by roto-evaporation at 40 °C, transferred to 15 mL graduated test tubes, further concentrated under nitrogen gas and stored in the dark at -15° C, for later GC analysis.  3.4.2.1. Sediment Sample Preparation  Frozen (-15  °C) sediment samples were thawed, homogenized by mixing with a  solvent rinsed metal spatula and weighed into 500 mL teflon screw cap bottles.  Sub-  samples  were  were dried at 104 °C for percent moisture determination.  rotary extracted (10 rpm) in 250 mL of 0.1M r v j C O g for 15 h.  Sediments  It was found that 3,4,5-  TCVer was not recovered in this procedure, so no sediment results for this compound are  reported.  h ^ C O g extracts,  containing  ionized  chlorinated  phenolics,  were  centrifuged at about 3000 rpm. for 15 min., in conical glass centrifuge tubes covered with teflon caps, to separate suspended sediments extracts  were  decanted  into  stored as per water samples.  2.0  L separatory  from the aqueous fraction. funnels,  extracted,  K COg 2  concentrated and  57 3.4.2.4. Leech Sample Preparation  Leeches used in the monitoring of chlorophenols by past researchers (Metcalf et al. 1984; Jacob and Hall 1988) have been extracted using a procedure involving the following steps; acid digestion of leech tissue in concentrated HC1, sequential extraction,  back-extraction into  chlorophenol  derivatives.  chlorinated guaiacols 50%).  O.IM hC^COg, acetylation  However,  early  attempts  to  and hexane apply  and chlorinated vanillins resulted in low  this  extraction  method  analyte  hexane  to  of the  recovery  (<  It was necessary to come up with a modified method of chlorinated phenolic  extraction.  Two different  extraction methods  need for a back-extraction from hexane polytron  extraction  were  developed,  both eliminating the  to K2CO3; 1) the acid digest method, 2) the  method.  1) The acid digest method: frozen (-15  °C) leeches were thawed, blotted dry with a  paper towel and weighed to the nearest 0.001  g.  Leeches were placed into 40 mL  amber screw-cap tubes with teflon lined caps, spiked with 2,4,6-TBP surrogate and digested for 2.5 hours in concentrated HC1 (1 mL/0.2 g leech).  Digests were decanted  into a 250 mL separatory funnel and neutralized to pH 7 with 5M KOH (approx. 7 mL). Samples where then made up to a volume of 50 mL with pH 7 laboratory distilled water.  1 mL of 5M K2CO3 per 50 mL of sample was added.  Samples were derivitized by  addition of 1.2 mL acetic anhydride, followed by manual shaking for 90 sec.  Samples  were allowed to stand for 15 min. after which they were extracted with 10 mL of hexane for 3 min., by vigorous manual shaking.  Samples generally emulsified  hexane extraction and had to be centrifuged as per effluent  after  samples.  2) The polytron method: frozen (-15 °C) leeches were thawed, blotted dry with a paper towel and weighed to the nearest. 0.001 g.  Leeches were placed into 40 mL test tubes  with 2.0 mL of 0.1M K2CO3 surrogate spiked with 2,4,6-TBP.  Leeches were then pre-  extracted for 30 seconds, using the grinding action of a Brinkmann Model PT 10/35 polytron, fitted with a Model PT 10-TS probe.  The polytron is supposed to break up  58 tissue at the cellular level, releasing cell contents into the K2CO3 extraction  medium.  Another 18 mL of 0.1M K2CO3 was added to the test tube and the sample was extracted for  another 90 sec.  separatory funnel.  at medium , speed.  The extract was decanted into  a 250 mL  The test tube was then rinsed with 30 mL of 0.1M K C 0 , 2  was added to the separatory funnel.  3  which  The resulting 50 mL volume of 0.1M K2CO3 leech  extract was acetylated and extracted as per the above acid digest method.  3.4.2.5.  Reference  Fresh  Standard Preparation  acetylated  prepared from  reference  10 pg/ml mixed stock  samples from a given bioassay. mixed  stock  standards,  solutions  of  solutions,  hexane  chlorinated  extracted acetic  phenolics  15 min.  for GC analysis  -  0.1 pg/ml  were  of each series of  and  surrogate  into  a separatory  Standards were acetylated by adding 1.0 mL  anhydride to the separatory funnel, followed by vigorous  manual shaking and venting of gas for 60 sec. for  0.01  Standards were prepared by spiking 0.1 - 1.0 pg from  funnel containing 50 mL of 0.1M K2CO3. of  ranging from  Samples were then allowed to stand  Acetylated chlorinated phenolics were then extracted by the addition of  10 mL of hexane, followed by vigorous manual shaking for 3 min.  Hexane extracts  were collected into 15 mL screw-cap tubes and stored in the dark, at -15 °C.  3.4.3.  Instrumental  Acetylated  analysis  chlorinated phenolic  analysed by capillary gas  and veratrole  compounds  were  quantitatively  chromatography on a Hewlett Packard model 5880A G C ,  equipped with an electron capture detector (temperature 310 °C) and a model 7672A automatic sampler and a splitless injection port (temperature 250 °C). gas  (60  cm/second  - 70 flow  cm/second rate)  flow  were  rate)  used.  and nitrogen detector The following  successful in separating all test compounds:  oven  Helium carrier  make-up gas temperature  (30  - 35  program was  75 °C (3 min.) to 120 °C at 15 °C/min.;  59 120 °C (0.1 min.) to 200 °C at 3 °C/min.; 200 °C (5 min.) to 220 °C at 5 °C/min.; 220 °C (0.1 min.) to 265 °C at 20 °C/min. for 4 min. column  (i.d.  Brockville, well  as  apparent  0.32  mm),  by  Chromatographic Specialties  Ontario, provided satisfactory  a number of detection  chromatographic  other  of  separation of all eight  chlorinated phenolic  trace  column is  their presence, especially for  supplied  A J & W Scientific 30 metre DB-5 capillary  amount  not  of  generally  compounds  organic accepted  in unknown field samples.  strong  of  test compounds as (Figure  compounds as  Company  3.2).  The  a  single  using  enough  evidence  of  The current method of choice  confirmation of trace organic contaminants is mass spectrometry (MS), however  we were unable to make use of this method, since the levels of contaminants we were dealing with were available to us.  generally  far below  the limits of detection  of the  MS system  Therefore, we chose to confirm our findings on a second GC capillary  column of greater stationary phase polarity; a J & W Scientific 30 metre DB-1701 (i.d. 0.32 mm) also supplied by Chromatographic Specialties Company (Figure 3.3). were  calculated  dibromophenol  according to  the  response  factor  of  each  compound to  Results the  2,6-  internal standard (Appendix 2).  3.4.4. Quality Control  Most glassware used in this research was kept aside for the exclusive  use of  this project, in order to prevent undue contact with outside sources of contamination. Before glassware of unknown origin was used it was fired in a muffle furnace at 530 °C  for 60 minutes to remove volatile organic compounds.  glassware  and  methanol  rinse  equipment  was  and  hexane  three  first  rinsed  rinsings.  in  distilled  Glassware  Prior to extractions all water, rinsings  analysed for chlorinated phenolic contamination by GC-ECD. have been shown to be susceptible  followed were  by  one  periodically  Chlorinated  phenolics  to photodegradation (Tratnyek and Holgne 1991),  therefore all sample extracts were stored in the dark at -15 °C and GC analysis was carried out using amber coloured GC vials.  60  Figure 3.2: Separation of chlorinated phenolic compounds on a 30 m D B - 5 capillary column.  1)  2,4,6-Trichlorophenol  2)  2,6-Dibromophenol  (internal  9) standard)  10)  2,4,6-Tribromophenol(surrogate 4,5,6-Trichloroguaiacol  3) 4,5-Dichloroguaiacol  11) 5 , 6 - D i c h l o r o v a n i l l i n  4)  2,3,5,6-Tetrachlorophenol  12)  Pentachlorophenol  5)  2,3,4,6-Tetrachlorophenol  13)  3,4,5-Trichlorocatechol  6)  3,4,5-Trichloroveratrole  14)  Tetrachloroguaiacol  7)  2,3,4,5-Tetrachlorophenol  15)  Tetrachlorocatechol  8)  3,4,5-Trichloroguaiacol  standard)  61 Figure 3.3: Separation of chlorinated phenolic compounds on a 30 m DB-1701 capillary column.  1)  2,4,6-Trichlorophenol  2)  2,6-Dibromophenol  9)  (internal  standard)  3,4,5-Trichloroguaiacol  10)  4,5,6-Trichloroguaiacol  3)  2,3,5,6-Tetrachlorophenol  11)  Pentachlorophenol  4)  2,3,4,6-Tetrachlorophenol  12)  5,6-Dichlorovanillin  5) 4,5-Dichloroguaiacol  13)  Tetrachloroguaiacol  6)  3,4,5-Trichloroveratrole  14)  3,4,5-Trichlorocatechol  7)  2,3,4,5-Tetrachlorophenol  15)  Tetrachlorocatechol  8)  2,4,6-Tribromophenol  (surrogate  standard)  62  Background levels  of contamination  and chlorinated phenolic  monitored through extraction and analysis matrices.  The exception  was  recovery  where  of both blank control and spiked  bleached pulp mill  effluent,  where  sample  no blank  effluent  matrix was available, however recovery could be gauged by spiking of a subsample of effluent  of  phenolics  known  contamination  levels in the sample  control of water samples level used was  Matrix  spikes  For sediments  Fraser  River  of  leech  of  concentration  extractions  was  hexane  the  known chlorinated  after  extracted  provincial park, sediments  concentration where also examined. control  subtraction  from the total  at Bromley Rock  of  and  spiking.  Quality  was monitored using laboratory distilled water; the spiking  1.0 ug/L.  Similkameen River  level  of  British  known  Sediment spiking level carried out  by  beach  analysis  sand  Columbia  from was  chlorinated was of  0.1  the  used.  phenolic  ug/g.  Quality  untreated laboratory  leeches and spiked leech tissue samples; spiking level was 1.0 ug/g.  into  To  further  assess  each  sample  prior  sample  extraction to  extraction.  provided a warning signal  derivitization and extraction. chemically, closely in any sample chlorinated  performance,  surrogate  Monitoring  or internal check  of to  compounds  surrogate  were  recovery  assess the  spiked  in each  success of  the  The surrogate of choice should be a compound which is  related to the target compounds, not likely to be already present  and does not interfere  phenolic  analysis  phenolic of intermediate  is  the  in the  analysis.  compound  substitution level  2,4,6-tribromophenol,  (Morales et al. 1992).  level for each sample depended upon the expected generally ranged from 0.25  An accepted  surrogate for a  halogenated  Surrogate spiking  final volume of each sample, but  - 1.0 ug/L for water and effluent and 0.25 - 1.0 ug/g  for  sediments and leeches. To assess and correct for the injection and overall GC-ECD internal standard was added to each sample prior to GC analysis. standard should have the same qualifications  performance an A good internal  as outlined for a surrogate compound.  On the basis of method development research by Xie (1983), 2,6-dibromophenol was selected as an internal standard. necessary  Since this compound is a polar phenolic, it was  to pre-derivitize a batch of 2,6-dibromophenol (1.0 ug/L).  Variability  63  between batches of internal standard was not measured, but as long as the same batch was used for any single set of samples and standards there was no overall effect on the  final  result.  However  qualitative  examination  did  not  reveal  any variation  greater than approximately 20% between batches of internal standard. The method detection limit (MDL) was estimated for each sample matrix (Table 3.1).  The range in M D L was dependent upon a number of factors including; sample  weight or volume, sensitivity  of  concentration  the  analyte  calculate detection  to  of  the  extracts,  background level  electron' capture  detector.  of  interference  It was  limits for each individual compound of interest  according to the extraction and GC running conditions used.  and  necessary  to  and modify them  Effluent MDLs ranged  from 0.1 ug/L for tri- and tetra-chlorinated compounds to 0.5 pg/L for di-chlorinated compounds. analysed  Water MDLs  greater  volumes  were about ten times lower than for effluents,  since  of  effluents.  water  and  they  where  Sediment and leech MDLs ranged from 10 and 20 pg/kg  cleaner for  than  the  dichlorinated  we  compounds,  down to 0.2 and 2.0 pg/kg for tri- and tetra-chlorinated compounds in sediments (dry weight) and leeches (wet  weight) respectively.  the M D L was the cleanliness samples  contained  compounds,  many  which  of the organic solvent extracts.  organic  precluded  Generally, the main factor limiting  interferences.  analysis  of  Leech  highly  Effluent and sediment  samples  concentrated  contained  samples.  lipid  Column  chromatography, using silica gel or florisil was not used to cleanup extracts, because of poor recovery of target compounds. method of choice lack  of  access  concentrations be  Gel permeation chromatography (GPC) is the  for lipid removal, however to  GPC equipment  bioassayed  the large number of samples  precluded  its  in this study, interference  of minor importance in laboratory and field  Nephelopsis  obscura.  species Percymoorensis  Greater  lipid  marmorata .  interference More  such as tri- and tetra-chlorinated phenolics Therefore, the less chlorine substituted were limited by their ECD sensitivity.  use.  However,  at  and the  the  water  from lipid compounds proved to bioassays was  highly  using  the  encountered  chlorine  leech in  substituted  are more responsive  the  species leech  molecules  the E C D detector.  dichlorinated compounds (4,5-DCG,  5,6-DCV)  64  Table 3.1: Method detection limit ranges for the analysis of chlorinated phenolics in effluent, water, suspended sediments and leeches. Detection limits based on sample dry weight for suspended sediments and sample wet weight for leeches.  Compound  Effluent  Water  (ug/L) 4,5-DCG  Suspended  Leeches  Leeches  Sediments  (N. obscura}  (P. marmorata)  (u-g/kg)  (ng/kg)  (ng/kg)  0.5  0.02 - 0.05  8.0 - 12.0  3.0 - 5.0  15 - 30  3,4,5-TCG  0.1  0.002 - 0.005  0.3 - 1.0  1.0 - 3.0  15 - 30  4,5,6-TCG  0.1  0.002 - 0.005  0.2 - 1.0  1.0 - 3.0  5.0 - 15  TeCG  0.1  0.002 - 0.005  0.3 - 1.0  1.0 - 3.0  5.0 - 15  3,4,5-TCVer  0.2  0.002 - 0.005  NA  1.0 - 3.0  5.0 - 15  5,6-DCV  0.5  0.01 - 0.05  1.0 - 5.0  20 - 50  100 - 200  2,4,6-TCP *  0.2  0.005 - 0.01  0.5 - 2.0  1.0 - 3.0  5.0 - 15  2,3,4,6-TeCP *  0.2  0.005 - 0.01  0.5 - 2.0  1.0 - 3.0  5.0 -15  * Analysed in samples from Fraser River only. N/A: Not analysed in suspended sediments.  65  4. RESULTS AND DISCUSSION 4.1. L A B O R A T O R Y S T U D I E S  4.1.1. B i o c o n c e n t r a t i o n  a sa F u n c t i o n  of  Contaminant  Concentration  Experiments were carried out in April 1991, in order to determine the effect of the water concentration of chlorinated phenolics on leech bioconcentration.  Since  pollutant  tissue  concentration  bioconcentrations parameter of  is  (Barron  one  of  the  major  1990), it is necessary  determinants  to quantitate the  in order to model chlorinated phenolic bioconcentration.  the Fraser River, water concentrations  of  final effect  of this  In the context  of chlorinated phenolics vary widely on  both temporal and spatial scales as a result of proximity to effluent point sources, changing  pulp  mill  process  Therefore,  an  chlorinated  phenolics on leech  assessment  field monitoring data. phenolics would water  of  the  effect  and  seasonal  variations  of  variance  in  bioconcentration  is important  with  reached  high  enough  levels  to  concentration  of  interpretation  of  either  saturate  until uptake  kinetics. from an organism is also of  importance in assessing the sensitivity of a biomonitor. result in decreased  levels of contaminants. linear  flow.  resulting in altered uptake and  The elimination rate of a target contaminant  will  to  water  increasing chlorinated phenolic concentration,  mechanisms and storage sites or cause toxic effects elimination  water  in  It was hypothesised that the bioconcentration of chlorinated  increase  concentrations  technology  relationships  bioconcentrations  and reduced  Rapid clearance from tissues sensitivity to low ambient  Earlier research conducted by Metcalf et al. (1988) reported between  leech  bioconcentration  and  elimination  prolonged half lives exceeding 30 days for tri- and tetrachlorophenols.  time  with  From this  research it was hypothesized that leeches would show similar slow elimination rates for  the  bioassay  chloroguaiacols, with periods.  relatively little loss of compound  after  seven  day  66  (TV. obscura), which had been collected  Leeches 1990,  were  exposed  concentrations temperature  ranging from 0.1  of  12.5  concentrations (Carey  semi-statically  1988)  greater (Hall  than  7.5.  1.0. ug/L is  and Jacob 1988)  carry out experiments  seven  to  five  chlorinated  The environmental  questionable,  to be generally less than 1.0  conditions,  since  (Schreier et al. 1991)  at levels lower than 0.1  under laboratory  days,  phenolic  - 10 ug/L (n = 5 leeches per test group) at a water  °C and a pH of  phenolics concentrations  that  for  from Black Lake in August  ug/L.  of test  Fraser River  studies  have shown chlorinated It would be desirable to  ug/L, however,  minimum leech  relevance  pilot studies indicted  bioconcentration  detection  limits  would be reached after seven days exposure to water concentrations in the area of 0.1 pg/L.  Mean leech weights, between test groups ranged from 1.18 - 1.45 g. Elimination of chlorinated phenolics  were followed  over a 28 day period, by  transferring a subsample of leeches to clean water after seven days exposure to a 1.0 ug/L concentration.  Water was replaced every 24 hours and samples of leeches (n =  5), were taken at time 0, 72, 168, and 672 h after termination of seven day exposure. Mean leech weights, between sample groups ranged from 0.63 in  leech  weight between sample  significant  differences  groups  in the  - 1.36 g.  The variation  elimination experiment  in bioconcentration due to leech  weight.  Therefore, it  necessary to normalize elimination data to a leech weight of 1.0 g.  A  strong  contaminant  linear  level  organism (Phillips strong  linear  is  of  1978).  relationship  between  in the  leech  bioconcentration  selection  Laboratory bioassays between  was  No mortalities  or depuration periods.  relationship importance  in  Tables detailing  normalization calculations are given in Appendix 3 (Table A2 and A3). were recorded during either exposure  resulted  of  an effective  were successful  bioconcentration  of  and  ambient  biomonitoring  in demonstrating a chlorinated  phenolic  compounds and water concentration (0.1 - 10 ug/L), when transformed to a double log scale (Y = aX ) (Table 4.1; Figure 4.1). b  equation Y = aX*\ varied from 0.77 bioconcentration  behaviour.  Slopes of regression lines, indicated by b in the  - 1.0, indicating possible differences in uptake and  67  o 05 O  CM CO 05 O  (35 O  10 05 O  05  CO O  co CO  as  CO CO  , . D) CD  _cz  i2.  X  A  II >-  cr  o CO CO  CM O r^- I— o> co CO LO  +1 o  LO  «= £ CD O. > >_ C D CD co CO i5  TJ c CO  "cO O o  LO  O c\i co XI  I  O  co  +1  +1  tf-  r-o  tfT-  +1  CO 00  CO  +1  CO CO CD  CM  c  CO r-CM 5 +1  +1  +1  CM 00  <35 CM LO CM 00 CO CM  o  00  o  00  +1 to •tf  CD  5  CO Q. „  (0  JZ  •o  M  ®  —  co  CO  i-  1  +1 o  CO "55 o  •tf tf-  +1  c  O CD  .E  05  tf•tf  CC k_ •*—'  Q) O  §  CO CM LO CM  o  2  O CO » D X) CD C 2  "5  +1 o  •tf  c  SI'S  "  +1 o  T—  LO tf-  II  3  o  CO  O)  +1 00  II  > >  i  +1 00  CO  l~~  CO  CM  +1  tftfT —  +1 u, a>  05  co co o d -H -H C\J  o  x: O O _o|  ~5> i  LO  co »-  •fe  +1  o  CD E  ^  g s o  9  xi  +1  co  r» +1  II  +1 •H  LO IO CM  -H  II  Q  (f)  -H  c  s  o  ^ b o -H o CO q xII  cd II  -H  O) CD  Oi  5  D)  X  S  ° s P CO CD  ?  "O c o  tf-  2  *-  x: CO  ™ O -H CO CO  II II  as  c  .2  , _  at a  m  ci -H CO OJ  cor em  ex  II  >-  CD  C O "5 o CO Q. CD  X  CM LO CO CM  SD  CD  •tf  X  so  o  .—'  o_  X  x  CO CO  ort  CO  on  £5 c CD O T3 c CD  SD  CO c o  r^ co 05 r*. CD ci CL Q. CL CL CL CL X X CD X CD CD C CD D CD  Q.  E o O  (5 CD (5 O O O Q  • LO CO LO tf-" LO" tf-" co"  n " N  N  W  «  . E Etf> CO II ~ c C D co g  a. g- E gP E co E  N  O  W  o  -S  n co co co co co co E •  +-+-<£»  n co >•  68  Figure 4.1:  The effect of chlorinated phenolic exposure concentration on leech (N. obscura) bioconcentration of 3,4,5-TCG and 3,4,5-TCVer.  10000 T  0.10 Log Water Concentration  + = Standard deviation 3,4,5-TCG; x = Standard deviation 3,4,5-TCVer  1.00 (ug/L)  69 The observed relationship could be described by the equation:  Eqn. 1  Bioconcentration  (u.g/kg) = a(Water Concentration (ug/L))b  used by Ellgehausen et al. (1980) to describe the bioconcentration of organochlorine pesticides  by algae (Scenedesmus acutus), a daphnid (Daphnia  (Ictdlurus static  melas)at equilibrium water concentrations.  bioassay  Similar  linear  was  a good  relationships  steady  were  chlorophenol  for  catfish  This indicates that our semi-  approximation of found  magna) and  state  water  concentrations.  bioconcentrations  in  salmonids inhabiting the Fraser River (Carey et al. 1988; Servizi et al. 1988). Leeches  were  relatively  laboratory bioassays.  efficient  bioconcentrators  of  chloroguaiacols  in  Table 4.2 shows the percentage of 4,5,6-TCG found in leeches  after seven days exposure.  Table 4.2:  The average proportion of 4,5,6-TCG found in leeches after seven days of semistatic  Nominal Water Concentration (Ug/L)  exposure at various concentrations.  Total Amount Spiked 7 days(u.g)*  Total Amount Detected in Leeches (n = 5) dig)  Proportion of 4,5,6-TCG in Leeches (%)  0.1  0.7  0.222  31.8  0.5  3.5  1.00  28.6  1.0  7.0  2.30  33.0  5.0  35.0  5.89  16.8  10.0  70.0  13.2  18.9  Water was changed and freshly spiked at 24 h. intervals.  Note  that  at  bioconcentrated  the  two  a smaller  highest  water  percentage  of  concentrations  (5  &  10  compound, indicating the  ug/L),  leeches  approach of a  saturation in uptake rate. Since the leeches bioconcentrated such a high proportion of compound in the  70  water, it is difficult to argue that water concentrations were at a steady state, unless most  of  the  uptake  took  place  relatively  quickly, reaching  nearly  steady  state  bioconcentrations within 24 - 48 h (1 - 2 water changes), with relatively little uptake over the following  120  - 144 hours.  In this case water concentrations  and leech  bioconcentrations would have been relatively stable for most of the bioassay period. The relatively low since  bioconcentration of  3,4,5-TCVer in leeches was puzzling,  the bioconcentration potential of this compound has been shown to be great.  Bioconcentration factors of 3,4,5-TCVer in the zebra fish (B. rerio) were on the order of 3100 (Neilson et al. 1984), an average of 75 times the values observed for leeches in our bioassays.  It is possible that the concentrations of  3,4,5-TCVer covered in our  experiments caused a toxic reaction, resulting in inhibited uptake of this compound, however this seems unlikely for two reasons: firstly, leeches have been shown to be resistant  to high concentrations of chlorinated phenolics (Metcalf et al. 1984)  and Jacob 1988)  (Hall  and secondly sub lethal threshold concentrations for 3,4,5-TCVer to  fish embryos and larvae (Brachydanio  rerio)weie found, in one study (Neilson et al.  1984), to be 450 ug/L, 45 times the highest exposure concentration tested in this study. However,  statements  regarding the  applicability of  toxic  threshold  concentrations  between species (i.e. fish vs. leech) have to be taken with caution, since toxicity can depend on many species specific physiological characteristics (Barron et al. 1990). The (pg/kg)  bioconcentration  factor,  defined  as  the  steady  state  bioconcentration  divided by the water concentration (ug/L) can be derived by rearrangement  equation  1, yielding.  Eqn. 2  a = Bioconcentration (ug/kg)/Water Concentration (ug/L)'  3  where a, the y intercept, is equal to the BCF (Ellgehausen et al. 1980).  When the BCF  was related back to water concentration there appeared to be very little change over the  range  of  water  concentrations  tested  (Table  4.3).  Leeches  retained similar  relative proportions of compounds in their tissues at each water concentration.  71  7—  cr co CC  in  ci  su-  es  0  +1  + 1  +1  c o  '4—•  CO  •4—'  §  c o o o  co.  CM CM  in  0  0  cn in  +1  +1  CO co  m  -,—  CO  CO CM  +1  +1  +1  T-  CM CO  00  CM  5  + 1  + 1  m  03  o  LL  +1  CO CM  CM CM iri  0  CD  in  o  co  ,  CO CO in  CO  CM  o CO  CD  +1  o  CM GO  O  o  +1  CM 00  C D i_  CM CO  CM  00  +1 w  •*r  CO  o  CL  o  CO in  0  +1 o  CO co +1  CO  •si-  o CO  ^-  +1  +1  +1  CO  CM  CM CO  00  00  9>  CM  6) o>  o m  +1 o m  O CM  O  +1  +1  o m co  o m O •sf +1  o in  CM ' R  00 N  Q " 0 a CO O O £ CO CO g CO co 1  M  CM  -H  +1 5  'co 50  5 6  co S> CO  co  T3  c o Q.  E o O  (3  0  Q  O I— •  1  in  in co"  CD O  HCD in  CO O  CD >  o hin  CO  E E  £  1 1 . - u)  ..  ._  N N 10 N if m mN W WJ '51 5 1 w11 "j§_ 1 1 0) 11aJ_ ffi e • = < = n Ii C Q . Q> . =CD Q, CO E E N§ CO E CO E M  c  m  co"  aj  CO  00 (0  CO CO .  CO  CO CO  4- (&>  O  X-  72  Elimination Figure  4.2.  The  kinetics or a l l o w not  be  of data  were  an accurate  too  phenolics  over  sparse  either  estimate  c a l c u l a t e d , i f i t is assumed  equals  half  indicate  the  that  measured  days)  for  the  life  in  calculated  half  species  the  of  leech  leeches lives  for  aquatic  4,5,6-TCG  and T e C G  (Mytilus  Figure  4.2  leeches  (T  tissue  leeches by  other of  is  T  > 28  consistent  d i - , t r i - , and  contrast to  that  obscura)  accurate  would  to be under  determination  require  longer  half  Metcalf  et  /  could  2  be  half  h),  while  T  (>28  1 / 2  al.  to  o f less  24  alburnus.  lives  depuration  of  times  range rates  by  than  auratus)and  half  three  elimination  other h  for  TheTj/  one  species  24 and 72 h respectively. of  of  (1988) in  eliminated rapidly lives  T\/2  lives  stagnalis)  to the s l o w  Carassius  was reported  (672  tetra-chlorophenols  species  edulis)  1  i n F i g u r e 4.2  with  Helobdella  reported  gairdneri,  While T  days  in  elimination  The relatively l o n g  species Alburnus  (Salmo  outlined  of  data presented  researchers.  In sharp  nature  leeches.  m  at  punctata,  a l . (1980)  the  are  concentration o f c o m p o u n d at  approached  Erpobdella  et  )  1 / 2  period  i n the b r a c k i s h water fish  indicates  (N.  describe  c h l o r i n a t e d p h e n o l i c s appear  for P C P i n two fish mollusc  in  than 40 days.  Renberg  day  at T < 3 days (72 h).  variety  dubia,  leeches,  organisms.  a  28  T = 0 h, then  is  reported of  (Dina  25 days to greater  reported  life  is reached  a  o f h a l f life  that the  chloroguaiacols  chlorophenols  to  concentration at  chloroguaiacol half  chloroveratrole h a l f  from  chlorinated  2  of  Data in  chloroguaiacols in  and  more  sampling  periods. Elimination There  was  higher  tissue  simply  have  model,  an  between  apparent levels  been  between  concentration model,  curves  where  due  to  the drop at  and  yields  a  an  tissue  h  distribution  an  initial  rapid  e l i m i n a t i o n rate phase  (Butte  1991).  indicate  aquatic  and  biological  line  all  levels  variation.  straight  two  by  in  168  and  characterised  chloroguaiacols  experimental  water  time)  uptake,  water  sharp  observed  the  vs.  for  after  that  the  only  organism,  a  of  compartments, phase  h,  sharp  character.  however  In  a  plot three  the  elimination a  tissue  compartment  compound  by  may  elimination (log  the  followed  the  decrease  compartment  semilog  relationship.  similar  72  initial  For a two  elimination  elimination  displayed  takes  curve  longer,  L i n e a r regression o f data i n F i g u r e 4.2,  place is slow  yielded  73  Figure 4.2: The elimination of chlorinated phenolics from leeches (N. obscura) over a four week period, after seven days exposure to a concentration of 1.0 ng/L at a water temperature of 12.5 °C and water pH 7.5. Sampling times were at time 0, 72, 168 and 672 h. Horizontal dashed lines represent the concentration of compound at T 1/2, expressed as half the mean tissue concentration at sampling time 0 hours. All tissue concentrations are normalized for a leech weight of 1.0 g.f  zi.  c q  «  2 c  100  CD O  c o o  CD CO CO  300  200  1 00  400  500  600  900  800  700  Elimination time (hours)  1000  B  CD  .  —  TeCG  Bp  c o  -*—>  CO  3,4,5-TCVer  -t— 1  c CD o c  O O  + 10  CD  +  3  CO CO  1—1—1—\  |  1  100  1  1  1 | 1 200  1  1 1 I 1 1 1 ' 1 ' 300 400  ' 1 1 | 1 1 1 1 llT'T'ft | 1 1 1 1 1 1 1 1 1 500  600  700  800  900  Elimination time (hours)  + = Standard deviation. t Leech sample weights: Sample time 0 h, 1.23 ± 0.44 g; Sample time 72 h, 1.36 ± 0.23 g; Sample time 168 h, 1.17 ± 0.19 g; Sample time 672 h, 0.63 ± 0.15 g.  74  weak  correlations  TCG, r  that  characterized phase.  2  elimination  by  an  initial  showed  likely  that  compartment linear  a linear  kinetics  rapid  correlation  elimination 168  curve  h,  from  possible  the  that  for  leech  phase  possible  in  directly  a  in  in  compounds. due  form  to  an  more  eliminated  (e.g.  bioconcentrations  of  by  and  have  O-methylation  species.  It  a  a  stronger  is  phenol  metabolised  (e.g.  biliary  excretion).  A  after  T  between chemically  group,  enzyme  plausible  relatively  The  rate  mechanisms  the  two  time.  depuration  easily  the  al.  yielded  compound  of  et  follows  elimination  this  release  Metcalf  also  elimination  3 , 4 , 5 - T C V e r is  by  the  model  slow  three leech  a rapid  Since  indicating  prolonged  for  would  = 0.37; 3,4,5-  2  compartment  leeches  concentration  characterized  a  r  = 0.59,  2  reported  model  periods  differences  chloroguaiacol  by  leeches  depuration  tissue  4,5-DCG,  three  followed in  sampling  3 , 4 , 5 - T C v e r was  is  or  low  log  time:  non-linear  chloroguaiacols  more  chloroguaiacols,  it  a  chlorophenols  of  that  between  and  conjugation) the  and  for  fit  release  with  = 0.32; 3 , 4 , 5 - T C V e r , r  2  two compartment  indicating  chloroveratrole distinct  for  elimination  model  concentration  = 0.68; T e C G , r  E l i m i n a t i o n curves  (1988),  =  tissue  = 0.41; 4 , 5 , 6 - T C G , r  2  possibility  is  between  it  is  mediated explanation  rapid  elimination  of 3,4,5-TCVer. Metcalf extremely  et  slow  chloroguaiacols the  Fraser  since  the  threshold  of  rates  that  slow  elimination  ambient  with  the  chlorinated  i n leeches.  rate  to  in  to  periodic  high  body  between slow  phenolic However,  the high  of  chlorophenols  to  elimination  of  biomonitors  on  of  use  as  sensitivity  of  increases.  biomonitor  Therefore,  the  is  lowered.  Low  pulses  contaminant,  since  of  longer  after  bioconcentration  elimination  of  the  in  water  since  chlorinated  may  incident.  and  chlorinated  concentrations ambient  a  biomonitor  burdens  leech  rate  the  for  rate  routine  tissues by  BCFs  slow  their  increases  detectable  exposure  leech  apparent  retained  contaminant  relationship  high  o f relevance  correspondingly  coupled  burdens  is  The  contaminant  highlight  retains  attributed  rates.  obscura  water  linear  concentration,  body  N.  of  The  from  by  amount level  1988)  elimination  A  organism  suggest  (1984,  River.  elimination the  al.  be  phenolic  water phenolics estimated  75  concentrations regression influences, which model.  vary  are  often  relationship, such with  as  below estimates  water  season  and  the  lower  limit  not  match  may  temperature, location,  pH,  must  of  measured and  be  those  covered  by  the  levels.  In  addition,  suspended  assessed  to  sediment create  a  laboratory other  partitioning,  more  complete  76  4.1.2. Bioconcentration as a Function of Leech Weight Experiments were carried out in April 1991, in order to determine the effect of leech  weight on bioconcentration of chlorinated phenolics.  Animal  size and weight  are recognized determinants of final tissue bioconcentrations of some aquatic species (Barron  1990).  bioconcentration weight  related  Earlier of  tetra-  investigations and  pentachlorophenol  bioconcentration  matter in this current study.  (Hall  differences,  and by  Jacob  1988)  leeches,  found  indicating  the  need  concerning  to  evidence  of  pursue  the  In the context of routine biomonitoring programs, it is  essential to quantify this effect, both to determine if there is an optimal leech size for biomonitoring and to aid in the interpretation of monitoring data from leech groups  of variable size.  It was  hypothesised  that the bioconcentration would be  related to the surface to volume ratio of the leech, weight. would  which is inversely related to  It was anticipated that smaller leeches, with a high surface to volume ratio, attain  higher  Leeches 1990,  weight  (N.  bioconcentrations.  obscura), which had been collected from Black Lake in August  were exposed  semi-statically  for seven days to a 1.0  pg/L  concentration  chlorinated phenolics at water a temperature of 12.5 °C and a pH of 7.6. segregated  into  four  different  weight  groups  individual leech weights ranged from 0.148  =  - 1.77 g.  rate and life cycle (Davies and Everett 1977) range of leech  (n  5  leeches  per  of  Leeches were group); mean  Based on the expected growth  of the leech species N. obscura, this  weights covered individuals born during two  separate  seasons, late  summer of 1989 and spring and early summer of 1990.  Leeches under 0.5 g (n = 10)  lacked a clitellum, which is an indicator of sexual  maturity. Therefore, there may  have  been  age  related  differences  in physiology  between  the  larger  and smaller  leeches. Results  describing  the  effect  of  chlorinated phenolics are given in Table 4.4. bioconcentration of the chloroguaiacols 0.94).  leech  weight  on  bioconcentration  of  A strong inverse relationship between  and leech  weight  was  observed  (r = 0.90 2  The best linear fit was obtained by plotting the variables on a log log scale, in  77  CO 0)  0)  o  o  o CD  0)  6  ci  o  CO CM  -t-  c g  co ZJ  cr UJ  c o  CO  co  CL X CD  X  X  (0 II  >  CD  rr  CO O CO d -H CM  LO T  II c  CNJ  +1  +1  co CO  LO co  ll  CJ>  CM  3  Q.  c o  CD  CM O CM  O CO  O CM  CO  CO  +1 +1 +1 +1  ZJ  2  CM CO  CM CO  LO CM CO  CJ)  CO CO  CJ) CO  o  CJ)  +1  +1 +l  LO  CM  JZ DJ CD  c 5 CD c o O  CD 3 CO CO  JZ  o  CD CD  n=4  O  *  O CL  e  o O  CM CO •tf  CM CJ)  co O)  CO CO  +1  +1 +1  O) CO CO  o  T—  co  o  CO LO  •tf  o CM  o  O)  00  o  +1  •* o  CM CO +1 CM  LO  co  T—  CO  1—  -H  -H  CO CO  CM CO  78 which the smallest extremely efficient  leeches, represented  by the steepest portion of the curve, were  bioconcentrators of chloroguaiacols.  Larger leeches (1.2  showed less change in bioconcentration with equal changes immediately  suggest  that  a trade off  between very small leeches,  in leech  size  selection  which are highly efficient  and larger leeches which are less sensitive,  in weight.  - 1.8  g)  These data  must be  considered  and sensitive bioindicators  but also show less variability with size.  As indicated in Figure 4:3, 3,4,5-TCVer displayed a different behaviour; there did not appear to be any correlation between leech TCVer.  weight  and bioconcentration of  3,4,5-  The slopes, b, of the regression equations listed in Table 4.4 indicate that the  magnitude of apparent weight effect differed between the chloroguaiacols.  3,4,5-TCG,  b = - 0.83, showed the greatest effect, while 4,5,6-TCG, b = - 0.54, displayed less of a weight  related  behaviours  difference,  between  the  Bioconcentration process where  differential  bioconcentration  chloroguaiacols.  models  for lipophilic organic compounds  often  describe  the  as a simple partitioning process between the water and organic lipid phase, bioconcentration  coefficient  is  dependent  upon the  log  of  the  across  respiratory surfaces,  blood flow  rates, into the bioconcentration process (Barron 1990).  A  on  contaminant  by  ambient  plausible  bioconcentration  distribution.  explanation  and  Physiological  environmental  leech  concerning leech physiology. a greater rate of oxygen Mooreobdella  Many  for  weight  rates  and depuration  For example, in the case of  poorly perfused peripheral tissue compartments, blood flow  influenced  water partition  also incorporate rate limiting physiologically based determinants such  ventilatory volume  influence  octanol  and the lipid content of the organism (Barron 1990; Connell 1991).  recent models as  implying compound specific  can exert a rate limiting  processes,  in  turn,  may  be  conditions. the  observed  may  be  inverse  inferred  from  relationship current  between knowledge  In general, small leeches have been observed to show consumption.  Oxygen consumption in the  leech  species  microstoma ranged from 1.7 pL/mg in small individuals (20 mg) down  to 0.3 uL/mg in large individuals (250 mg) (Sawyer 1989).  Davies et al. (1987) found  that survival of N. obscura, under anoxic conditions, was greater for larger leeches,  79 implying  greater  Figure 4.3:  oxygen  requirements  for  smaller  sized  individuals.  Bioconcentration of 3,4,5-TCG and 3,4,5-TCVer by leeches (N. obscura) of differing weights.  1000  3,4,5-TCG =  O  3,4,5-TCVer =  <>  cn C O  s= CD O C O  100  +  O  CO CO  CD  o  +  10 0.1  1.0 Log  + = Standard deviation 3,4,5-TCG;  Weight (g)  x = Standard deviation 3,4,5-TCVer  —i  1  1  1—t-  10.0  80  Since the primary uptake site for lipophilic organic chemicals in aquatic organisms is  the  respiratory surface  smaller leeches promotes  (Connell  1991),  a greater  greater total exposure  oxygen  consumption  and bioconcentration.  rate in  In a similar  fashion, rainbow trout, show increased ventilatory volume (Barron 1990)  and cardiac  output (Barron et al. 1987a) with elevations in water temperature, which is linked to increased bioconcentration of PCBs (Veith et al. 1979). There is with age.  evidence  that depurative metabolic  function in leeches may change  Leech carbohydrate and lipid metabolism as well  detoxification  reactions  take  place  in  the  homologue, dispersed throughout the body. to increase with age (Sawyer 1989).  betroiydal  as xenobiotic (DDT)  tissue,  a  primitive  liver  Betroiydal tissue content has been found  While no attempt was made to age leeches in this  experiment, life history studies of N. obscura, conducted by Davies and Everett (1976) indicate  that there  were  significant  lightest leech weight groups. and  probably born  5 - 7  age  differences  The two heaviest months  before  lower  bioconcentrations Bioconcentration  observed by  comprehensive organic  to  the  smaller leeches  total  investigations  compounds  in the  mass of of  heaviest  two  lightest  weight  leech  groups.  capacity could have contributed to the  may be  by a greater surface  tissue.  factors  guppy (Poecilia  further enhanced by  Saarikoski et  affecting  the  reticulata).  al.  uptake  a greater  area of respiratory (1986) carried rate  of  order uptake and elimination kinetics  and steady  out  ionizable  Equations relating volume  and surface area to bioconcentration were developed, based on assumptions  rate constant,  and  in larger leeches.  surface to volume ratio; more specifically, sites relative  two  weight groups were sexually mature  the  Therefore, increased metabolic detoxification  between the  of first  state tissue concentrations.  The  k, of absorption of several chlorinated phenolic and carboxylic acids  was inversely related to the size of the fish.  81 Saarikoski et al. (1986) used Eqn. 3 to calculate the rate constant of absorption:  Eqn. 3  k = C /(C a  where  f  W  * t) = N / ( V * C * t) f  f  w  k is the rate constant of absorption, C is the concentration in the fish a  f  exposure  to  water  concentration Q, for time interval t.  In the second half of the  equation N represents the total amount found in the tissues and V f  weight) the fish.  after  f  is the volume (=  Eqn. 4 describes the effective permeability of the outer epithelium  to organic compounds:  Eqn. 4  P = N / ( A * C * t) f  where  P is  epithelium.  the  f  effective  w  permeability  and A  f  is  the  surface  area of  the  outer  Permeability is related to surface area, while the uptake rate constant is  related to volume or weight.  However, both P and k remain a  proportional to  each  other as long as the surface area to volume ratio remains constant (Eqn. 5).  Eqn. 5  P/k =V / A f  or  f  k =P*A /V a  f  f  According to Eqn. 5, if the surface area to volume ratio increases, increase  in the  uptake  rate  constant,  there will be an  k , and the concentration in the fish. a  It is  likely that a similar mechanism could be operating in the case of the leeches, where there is an increase in the ratio of surface respiratory sites to tissue volume ratio, resulting  in the  phenolic The  inverse  relationship  between leech  size  and chlorinated  bioconcentration. effect  strong inverse TCG  observed  of  leech  relationship  weight was  on bioconcentration  observed  was  compound  for the chlorinated guaiacols,  specific. with  A  3,4,5-  showing the greatest effect, while no clear trend was observed for 3,4,5-TCVer.  Available evidence  suggests that the effect may have been due to a combination of  factors, both physiological and physical.  Increased respiratory rate and decreased  82 depurative  enzyme  function  greater  surface  area  weight  leech  groups  function this  and  this  in  size and  goal  between  in leech  differences  the  Lesser  greater  that  1.0  current  be  weight g.  between and  and  related In  the  and  possible  biomass  contributing to  were  leech  factors.  size  variability  in N. best  beyond  leech  the  be  use  a laboratory  of  situ b i o m o n i t o r i n g . however,  bioconcentration between  variability following  the  leeches should  be  experiments,  were kept to a m i n i m u m (± 10 - 30 %).  not  exponential  indicates at  the  bred  that  variations  feasible  any  where  scope  of  leech  i n leech  related  intraspecies stock to  of  achieve  relationship  weight  end  of  different  leech  inverse  higher  observed  to  low  enzyme  from  solution  It was  the  The  the  age/size  obscura  1977),  the  with  Measuring  were  of  factors.  associated  Everett  study,  minimized  total  populations  would in  leeches  lower  i n relation  for routine  weight  should  scale.  test groups  age  and  complexity  (Davies  bioconcentration  younger  possible  demand the  locations  ratio  other  relationships  geographical  uniform  are  Given  physiological  smaller,  volume  respiratory  research.  variability  to  in  the  related weight  weights  weight  are  between  83  4.1.3. B i o c o n c e n t r a t i o n  as  Experiments were  a Function  W a t e r pH  of  carried out in September  1991,  in order to determine the  effect of water pH on bioconcentration of chlorinated phenolics by leeches. chlorinated phenolics percent  are weak  ionization of  these  acids, individual  compounds.  Since  pKa and water pH determine  In the  context  of  the  routine biomonitoring  programs, it is essential  to quantify this effect for monitoring sites of variable water  pH  interpretation of monitoring data from  and to aid in the  water chemistry.  regions  of differing  For the purpose of chlorinated phenolic monitoring on the Fraser  River, which ranges in pH from 7.5 - 8.5, the effect of water pH may be of lesser significance  for those compounds with a pKa greater than one unit above or below  this pH range.  However, certain compounds with a pKa falling in this range may  show substantial shifts in percent dissociation.  For example, 4,5,6-TCG (pKa = 7.4)  shifts from 56% to 93% ionization over the above pH range of 7.5 - 8.5. It  was  hypothesized  that  leech  bioconcentration  would  decrease  with  increasing water pH in a relationship roughly parallel to the dissociation curve of the individual compound, making it possible the  to estimate leech bioconcentration from  water concentration of compound, corrected for the percent ionization.  trichloroveratrole  is  non-ionizable  it  was  hypothesized  that  Since,  bioconcentration would  not change with water pH. Leeches  (N. obscura), which had been collected from Black Lake in July 1991,  were exposed semi-statically for seven days to a 0.1 ug/L concentration phenolics at a water temperature of 12 °C. selected  to  environments.  cover  the  Leeches  pH were  range  of  segregated  of chlorinated  Three experimental pHs (5.1, 7.1, 9.0) were most  naturally  into three  occurring receiving  test groups (n =  10)  water  and one  control group (n = 3); mean individual leech weights .ranged from 0.606 - 0.714  g  between test groups. Results  showing  the  effect  of  water  pH  on  percent  bioconcentration of chlorinated phenolics are given in Table 4.5.  ionization  A weak inverse  and  84  r-CD  CO •tf  CO CO  6  6  O  O  O.  O-  o  O  CO  cn  in  6  ci b  c » >< (0  Q.  LU ^ c —'  T-  .9 co C D  rr CO  >> co •o  ,2 ~ l o o  c  01  >  d  < Z  oi  cn  CO  LO  CD CO  CD  4—'  ro  c  „ cn JZ  ,9 O  ro i—  r; o co xi  ^  Cci  +1 +1  o>|  co  00 CO  CO CO  CM  ^  O  Si CO CD  x: CJ CD CD  8 ^| C O  CO CO  1  cn  ®o  oi +1 o>  Q.CD  1 ro  CD :  CO  tf +1 o  co co  co  +l  +1 o  CO  CO  *—  c CD D. oE  CO  CJ +1 LO O  IE®  °ro § 5  •;=  CO  CO  ^  i:  ,2 - I  8 ^  CO  §=*•  co +1  CJT-  5° <D  O  £  CN  LO  CO •tf  +i  tf+i  o  co  CM  CO  CJ  O  +1  ,—  Cvi  +1 CO  C  c-S o ro  £^  CO  >_ O  O-  co cj  5  Q _ <D  o  tf  CO  N  S§  ^ ro oo  t5 <D  1  q CD  D O 1 « CO , •H  in  o  o  E '5  tf Tat E «  CD  o  g> ^  CD Q .  x: X LO •tf 0) XI  3 O a.  E o o  C9  O Q in •tf  O hin  •tf co"  O H co cu .,_ >-  t t:  17  85 linear was  relationship observed  sample  pH  increasing the an  2  differences  strongest water  (r  linear  between =  0.36 in  -  0.67).  mean  leech  correlation  (Figure  4.4).  bioconcentration  veratrole  remains  i n d i c a t i o n that  pH  bioconcentration  weight  did  observed  The  non-dissociable  at  higher  alter  the  Correction of  was  unchanged may  of  with the  for  a  chloroguaiacols  bioconcentration  not  significantly  semi  l o g plot  3,4,5-TCVer  water  pH.  water  p H , the  membrane  Since  increase  permeability  or  of  a  chemical in  for results.  trend  pH  between The vs.  towards  character  bioconcentration  uptake  Figure 4.4: The effect of water p H on the bioconcentration of 3,4,5-TCG and  + = Standard deviation 3,4,5-TCG; x = Standard deviation 3,4,5-TCVer.  water  bioconcentration  leeches.  3,4,5-TCVer by leeches (N. obscura).  data  alter  showed the  and  of is  physiology of  86 Both the fish toxicity  and bioconcentration of chlorophenols have been  shown  to decrease with increasing water pH as a result of dissociation of the neutral phenol to the phenolate ion form (Saarikoski and Viluksela 1981, inverse  relationship between leech  the ionizable chloroguaiacols,  however  the pattern of the effect did not follow  chlorinated guaiacols,  with  Figure 4.5 plots the dissociation an overlay of the  each compound at the three experimental pHs. fraction  is  generally  As hypothesized, an  bioconcentration and water pH was observed  dissociation curve for the compounds tested. for the ionizable  1982).  insignificant  for the  curves  bioconcentration of  The change in the percent ionized  at pHs greater  than ±1 unit of the pKa.  changes in the bioavailable fraction take place at pHs near the pKa.  Major  For example, the  percent dissociation of 3,4,5-TCG (pKa = 8.0) ranges from < 1 - 9.1% over the range of pH 5 - 7. if  the  Above pH 9.0, 3,4,5-TCG remains > 90% ionized.  major bioavailable  form  is  the  non-ionized  This behaviour indicates that  species,  then  the  relationship  between bioconcentration and water pH in Figure 4.5 should be a mirror image of the dissociation curve of that compound.  According to this theory, the bioconcentration  of 4,5-DCG should change little from pH 5.1 to 7.1, since there is only a 1.2% change in percent ionization.  From pH 7.1  to 9.0,  we should see  a dramatic decrease in the  bioconcentration of 4,5-DCG, since the percent ionization increases from 1.2% to 50%. In  the  case  of  3,4,5-TCG  bioconcentration between pH 7.1 much greater ease.  we  should  - 9.0,  have  observed  a  sharp  decrease  in  had the unionized form been absorbed with  In a leech bioconcentration study by Hall and Jacob (Figure 4.6)  a similar phenomenon was observed for 2,4,6-TCP and PCP (pH 7.5 - 9.0).  In general,  the slopes of the bioconcentration vs. pH curves were much less steep than would be predicted if the non-ionized species were the only form absorbed. In studies using guppies (Poecilia  reticulata) Saarikoski et al. (1986) observed  that bioconcentration of ionizable chlorophenols pH was > 1 unit below the pKa.  was independent  of pH, as long as  However, as pH approached the pKa of the compound,  bioconcentration in guppies decreased, but at a rate less than predicted if only the non-ionized fraction were bioavailable.  The water  soluble  phenolate  ion has  shown to be bioconcentrated at a significant rate in fish (Saarikoski et al. 1986).  been  87  (6>|/6Tl)uo!iBJiueouoo enssn  (6>)/Brl)uonBJ)uaouoo enssy.  •o  CD  co  o  w c CO  3  T3 03  9  3  CO  O T CJ) CL LO  ° co  X CL  CD Q. N _ C CO  O  x: o  •2  CO  .2 c  _  „_ CD  o o E CD ° . 2 CL " co X CD  O)  — CD CD CD  §> S =S- c: x: o ~~  O CD i_ x: <D —  C O  <0 "55 "K eg co  uoj)ezjuo| %  UO!)BZjUO| %  (6>|/6Tl)uo!iBjjueouoo anssii  (6>|/6Tl)uonBJ}ueouoo enssy.  <= > c  8 » .2 c £  .2 x  co  co i  § §  O CO o CD .2 0)  I '« =5> co X  c — c CO "  II  CD CD C D)  N  O CD  X CL  c in £  •S . CD x re 2^ o.  o  o  co  £ Q  cS o oco  CO  «CJ c*o —2 LO CD 3  CO  uo|jBZ!UO| %  UO!JEZ|UO| %  co 00  c  (6>|/6rl)uo!}BJjueouoo e n s s i i  (6>|/6TI)UO!IBJ}U80UOO  onssn  co c\i "—  ai •D c  CL  o c x: o "CO o •«—. CO cCD o CD E CJ CD  c- •Q_  the wa ter CO the thr ee ex|  II  o> "cn ratiion  zj  Hall an  o E o  tz CO  pa  "5  IO  Q. CO X3 < _j  zi.  o  o ion  ina ted  CL  Hs  oni ze  he  CO  O •a O o c CO C L CO ~3 w E in TJ o c TD cf  ^0  CD o x: c CO o o o !Q o  -»-^  lee  sa  ue  cz CO c c  c CD  cO o o C O o  g  ok  an  •o II  c g  0  uouezjuoi %  (6>)/6rl)uouBj}uaouoo enssu.  (6>)/6Tl)uo!iBj}uaouoo enssy.  U0!}BZ|U0| %  U0!JBZ|U0| %  0  o  cz  c o o CD i U a> T 3 o CO o c o CO  £  uo!JBZ!UO| %  tf-  o> CD c ro CO x :  JC  CL  he:  c C O CO 0 CO CO CD CO _c  tted  -1c o CO 'o  0  dxi  o>- co o  OS  wat  BZj  o tfCD CM k_ i— c CD x:< O o TD X  OS  T> o co o "o 00 CL  b  CO _ l  CD 0 i_ ZJ CD  89  Further support for the  bioavailability of ionized  chlorinated phenolics  in situ monitoring studies on the Fraser River (pH 7.5 - 8.5),  comes from  where measurable levels  of TeCG have been detected in fish tissues, even though TeCG is 97 - > 99% ionized (Dwernychuk 1991; Schreier et al. 1991). Relationships appear  to  be  between  complex.  bioconcentration  vs.  The  pH curves  non-ionized  form of  the  form,  ionized  chlorinated  the  in the  the  pool of bioavailable compound. induced and  interaction  both  shallow  slopes  study  (Figure 4.5)  present  guaiacols  latter  bioconcentration  are  chemical  from  water  pH  by  the  exhibited  suggest that  the  preferentially  to  bioconcentrated  species does represent  a  significant  The complexity of the results could be due to a pH  physical  chemical  dissociation  of  chlorinated  phenolics  into the mechanism of uptake of ionized chlorinated phenolics  sediment  compounds  partitioning  takes place  Schwarzenbach fixed  organic  studies.  mostly  1985).  bioconcentration,  on  Sediment the  adsorption  organic  fraction  +  content as well  is  inversely  related  being positively  to  showed  that  sediment  anionic form occur. phase neutral  partitioning ion  pairs  pentachlorophenol increased.  sorption  lipophilic  (Schellenberg  water  et  is  (e.g. was  the  the  case  +  observed  as  for +  of  chlorophenolate  PCP"»K ).  1984;  sediments  et al. 1984).  both  the  neutral  Schellenberg et al.  chlorophenol  Westall (1985) proposed that the major mechanism of of  organic al.  to suspended  pH, as  comes  correlated to the ionic concentration ( K ,  2+  (1984)  of  Partitioning of chlorinated phenolics  L i , Ca ) of the water compartment (Schellenberg  the  and  alterations in leech physiology. Insight  of  of  relatively  chlorinated  however  phenolic  ion  Increased ionic  was  sorption  octanol  strength  of  the  the  sediment  electrostatically  partitioning  (KC1) of  and  of  tetra-  and  water  phase  was  It is quite likely that partitioning of chloroguaiacol ion pairs accounts for  apparent  bioconcentration  of  ionized  compound  observed  in  both  laboratory  leeches and Fraser River fish. Bioconcentration  of  non-ionizable  lipophilic  organic  compounds  is  generally  independent of pH as long as physiological function is not altered over the pH range tested.  Saarikoski et al. (1986) found no relationship between water pH and  90 bioconcentration  of  neutral  pentachloroanisole)  in  bioconcentration  3,4,5-TCVer  of  guppies  organic '(P.  compounds  reticulata).  showed  (tetrachloroveratrole,  The  a positive  observation  relationship  that  with  leech  water pH,  indicates that leech physiology may have also been affected over the range of water pH tested. A  comprehensive  leech  survey  conducted  by  Herrmann  (1970) in Colorado,  revealed that N. obscura is found in waters ranging from pH 6.3 - 9.8.  The absence of  the species from waters of pH < 6.3 may not be due to water pH alone, since other factors and  such  as  presence  concerning  conductivity,  dissolved  of competing leech  the  effect  of  water  inorganic/organic  content,  food  species are also important variables. pH on  leech  respiratory physiology  abundance  No literature was  found,  however the lowest experimental pH of 5.1 may have been below the tolerance level for  the species. Evidence for a physiological component of the effect of water pH on leech  bioconcentration can be inferred from studies of the effects of low water pH on fish physiology.  Effects of low water pH (pH < 4)  on fish include: acid-base imbalance,  ionoregulatory dysfunction and decreased cellular oxygen delivery (Spry et al. 1981). Increased  H  +  concentrations  buffers, such as HC0 ", 3  affect  blood  acid-base  yielding H 0 and C 0 . 2  balance  by  titration  of  blood  Metabolic acidosis also leads to altered  2  charges on enzymatic proteins, leading to altered cellular function (Spry et al. 1981). Ionoregulatory dysfunction, which occurs in fish  at the gill,  water pH < 6.0. This effect is dominant in waters of low C a manifested  concentrations,  2 +  by excessive loss of N a , Cl" and K ions (Spry et al. 1981). +  water also decreases uptake of 0 across  has been recorded at  2  +  at the gill by two mechanisms.  the gill is reduced by copious mucous secretions,  (Spry et al. 1981).  +  2  is  In fish, acid  Firstly, 0  2  diffusion  induced by acid exposure  Secondly, the affinity of hemoglobin for 0  blood levels of C 0 and H (Bohr  and  2  is reduced by elevated  effect).  The leech species N. obscurais, a faculative anaerobe (Sawyer 1989), capable of using  both glycogen  and amino  (Reddy and Davies 1993).  acids  as  energy  sources  under anoxic  conditions  If the assumption is made that leeches suffer acid-base  91  imbalance  from  hemoglobin  responds  to  plausible  that  then  it  is  glycolytic anoxic  exposure  pathway  at  metabolism  to  increased there  the  are  increased blood  may  lowest  C 0  have  test  succinate  water  and  concentrations  and  2  been  p H of alanine  H  a  with  +  of  decreased  compensatory  5.1.  Since  i n N.  obscura  H  the  leech  affinity  to  to l o w  energy  shift  major  and  +  end  0 , 2  products  of  (Reddy and D a v i e s 1993),  this hypothesis c o u l d be tested by metabolite quantification over the p H range 5 - 9. A exposed  phenomenon  to l o w p H water  3,4,5-TCVer numerous as  similar  low  at  pH  secretions  by  pH N.  obscura  by  pH  may  decreased  permeability third  barrier  adsorptive  guaiacols,  such  that  effect the  response  to  inverse  also  to al.  (1990)  by at  relationship  positive  reported  for  1980).  H o w e v e r , this  chemicals  neutral  when  different  organic  promelas)  showed  values.  chlorinated  these  correlations organic  between  compounds  two  relationship data  are  compounds  generally  examined (log  Kow  variations o f greater  A direct relationship between phenolics  varies  from  is  difficult about  1%  sites;  compound  bioconcentration  by  two  1)  uptake  mucous protecting  of  low as  a  2 ) acting  as  a  the the  bioassay  chlorinated  and  water  pH  and 7.1. and  log  organisms much  K o w have  (Ellgehausen  v a r i a t i o n between  fathead  minnows  o f magnitude  been et  al.  different  B i o c o n c e n t r a t i o n factors  orders  to  acting  from  between  1-7)  such  response  the  closely.  than  in  altered  shows  stimuli,  excess  mechanisms:  respiratory  aquatic  from  effectively  secretions  bioconcentration in  reported  conditions,  removing  have  fish  mucous,  i r r i t a t i n g external  Mucous  uptake  in  l o w e r e d leech bioconcentrations o f  hyperoxic  effectively  could  et  3,4,5-TCVer  blocking  observed  m u c o p o l y s a c c h a r i d e based  diffusion.  of  the  secretions  to be o f l o w e r magnitude between water p H 5.1  Strong  ionization  uptake  a  Singhal  oxygen  mucous  for the  in  response  compartment, an  appeared  cells,  physically  Such  increased  secrete  1989).  in  reducing  system.  Kow  Leeches  (Sawyer  organisms  the  account  mucous  the  have  could  5.1.  subcutaneous water  to  of  45  (Pimephales for  similar  log  b i o c o n c e n t r a t i o n and l o g K o w o f i o n i z a b l e  to  establish  to  greater  at than  neutral 90%  water for  the  pH,  since  compounds  percent tested.  H o w e v e r at p H 5.1 it is possible to compare bioconcentration to l o g K o w , since a l l the chloroguaiacols are i o n i z e d less than 1%, except T e C G (11.1%).  F i g u r e 4.7 plots the  92 observed positive  relationship correlation  Figure 4.7:  between between  the  log  leech  of  the  bioconcentration  bioconcentration  and  log  and K o w  log was  Kow.  N o  clear  observed.  Relationship between leech bioconcentration a n d log K o w of chlorinated phenolics at p H 5.1.  100 CD  c O 03  C CD  o c o o  g  CQ CD  o  1 = 4,5-DCG; 2 = 4,5,6-TCG; 3 = 3,4,5-TCG; 4 = TeCG; 5 = 3,4,5-TCVer.  Bioconcentrations observed  o fall  for 4,5-DCG,  probably  lower  4,5,6-TCG  than  bioconcentrations  were  considering  rapidity  the  species  N.  organic  material  the  obscura ( S e c t i o n may  compounds  alter  and  expected  4.1.1.; the  any  which  similar,  3,4,5-TCG. due  lowest'of with  were  although  Leech bioconcentration  t o partial compound  relationship  4.2).  5.1,  which  t o b eeliminated Since  between  trend  was  of TeCG  was  ionization.  a t p H  i t appears  Figure  a positive  both  dissolved  bioconcentration  3,4,5-TCVer may  be  from  the  and and  log  expected leech  particulate Kow,  93 through  competitive  lipophilic  adsorption  trichloroveratrole  (Jaffe  1991),  mucous  may  have  also  bioconcentrations.  Note that at pH 9.0,  3,4,5-TCVer  bioconcentrated  differences  was in  bioconcentration  adsorption  of  contributed  the  to  highly reduced  where the chloroguaiacols are > 50% ionized,  to  the  between  greatest  level.  di- tri- and  The  relatively  tetra-chlorinated  small  guaiacols  is  consistent with the results of Saarikoski et al. (1986), who reported that the rate of bioconcentration  in  guppies  (P.  reticulata) increases  linearly  with  lipophilicity  until log Kow reaches a value of about 4.0, at which point there is little increase in bioconcentration with increasing lipophilicity. 5.35  in leech bioconcentration experiments.  permeability  barrier  to  unstirred water layers guppy bioassays water  was  of  was  the  was  ions  affected  not  or ion  by stirring.  pairs  by  Bioconcentration of  follows  which  a different  ionizable  bioconcentration compound.  to the leech species N. trend in  Regression  a result  1966).  of  Initial  When bioassay of PCP  not  The phenolate  bioconcentration that  mechanism. and log  uptake  directly ion form  was  In leech pH Kow may have  The effect of water  inversely  related  to  the  related  to  of  also contributed significantly  to  of physiological disturbance  obscura, induced by low pH, which was manifested  between  the  non-dissociable  bioconcentration  weak and in reality are probably not linear.  water  concentration  There was evidence  of  of  in Section 4.1.5.  chloroguaiacols  was  bioconcentration  relationships  suggests  water been stirred continuously.  the bioavailable contaminant pool.  opposite  as  stirrer, bioconcentration  Furthermore, the  stirring,  stirring on leech bioconcentration is investigated  undissociated  exists  environments.  a stronger relationship between bioconcentration  however,  attributed to the  (Dainty and House  a magnetic  affected  been found, had the bioassay  pH,  which  to  Uptake of less lipophilic compounds, such as phenol and butyric  PCP anion  bioassays  stirred using  significantly  chlorophenolate  compounds,  at the respiratory surface  continuously  not  lipophilic  This phenomenon was  were conducted in unstirred bioassay  increased by 75%. acid,  highly  Log Kow values ranged from 3.41  and  compound,  water  pH  by an  3,4,5-TCVer.  were  generally  Therefore, the relationships derived are  only useful as a guide to aid in the interpretation of monitoring data.  94  4.1.4. Bioconcentration as a Function of Water Temperature  Experiments were carried out in January 1992 to determine the effect of water temperature  on  temperature  is  bioconcentration recognized  as  of  an  chlorinated  important  phenolics  determinant  organic compounds by aquatic organisms (Barron  1990).  situ biomonitoring in the Fraser River, it is essential  by of  leeches.  Water  bioconcentration  of  Within the context of  in  to quantify this effect,  since  seasonal fluctuations in water temperature range from 1 - 20 °C (Hall & Jacob 1988). It  was  hypothesized  that  leech  bioconcentration would  linear increase with increasing water temperature.  show  a continuous,  Leeches (N. obscura), which had  been collected from Black Lake in July 1991, were exposed semi-statically for seven days to a 0.1 pg/L concentration of chlorinated phenolics  at water pH 7.0.  experimental water temperatures between 4 - 20 °C, were selected the seasonal  temperature range recorded on the Fraser River.  Three  covering most of  N. obscura is a cold  water stenotherm, preferring cooler lentic waters of Canada and the Northern United States (Sawyer  1972).  Herrmann (1970) found N.  summer and early fall temperature from 0.5  obscura in  - 24 °C.  waters  ranging in  Leeches used in the current  study, originate from Black Lake, B.C. which undergoes a similar annual temperature variation.  From this information it is assumed that leeches in the current study were  not subject to temperatures out of their tolerance range. into  three  test groups  (n =  10/group)  and one  Leeches were  control group (n =  segregated  3)  at each  temperature; mean individual leech weights ranged from 0.540 - 0.635 g between test groups.  i  x  Table 4.6 shows leech bioconcentrations at water temperatures of 4.4, 20.0  °C.  Contrary  temperature (Student's  was  t-Test,  to the hypothesis,  not  observed.  p = 0.05)  lower  a steady  Leech at 4.4  increase  in bioconcentration with  bioconcentrations °C than at  11.8  were  11.8  No significant change in bioconcentration was observed for 4,5-DCG. bioconcentration decreased  significantly  °C for the  tetrachloroguaiacols, however bioconcentrations levelled off between  trichloroveratrole  11.8 and  initially and then levelled  tri- and  and 20 °C. Unexpectedly, off  95  CO CO  t-  X  X  co co  CM  co  13\ "* O) CO  5  g 15  CD  CO CD  CO  cr LU c o  c 0 o CD CD -a  X  >* t .Q o  1  s  o co  CD  tr  C L x  CD  CO O  cn o_  O  co CD cn  C L X CD  x  cL  CO  CD  —  CL  * *  in  CO  II  >-  CO  CO  II  II >-  >  CM | II  >  -a CD  ns o ° x: o  X a. CD  M-  ° «  15 ®  c  CL  c o O a)  CO CO  •tf  E  CD  in  CM  +1  +1 1—  +i CJ) in  +1  SI  — I CO •tf  XI CO  +1 •tf CO  CO  CO  •tf  I-  CD  15  CO  1-  +1  +1  +i  +1  CO  o> CO  GO CO  CO CO  CD O  CD O •  co  o a_  -o C CD > CD  <o  T3 C Z)  o a.  E o O  CM  r-  d  X CD CO >. CO  CD  CO  O 1^  1—  ,—  CO CO  E2  CD  +1 o  3  CO CO  CD  <D  '•^ CO CD O  o  +1 in  in  o  c o  c o  CO  +1 CM  o CM +1 o in  ^) o  id  8  CO CO  O)  It £  1—  CD O Q LO •tf  \— I  m co"  CO_  in" •tf"  l« S S  I  96  between  the  highest  induced  alteration  temperatures. in  the  These  balance  data  between  are  indicative  uptake  and  of  predictive  bioconcentration 4.8):  1)  4.4  to  and  assume  11.8  best  after  change  line  two  measured there  option assumes that the  by is  no  a l l three no effect  since  it  leads  to  effect  methods  of  of  where  net  change  over  the  data  points.  points  the  Neither  is  data  the to  on  (Figure between  two  2)  range,  model  of  F o r the  changes  between  temperature  kinetics  temperature  bioconcentration  temperature starts at about °C.  water  i n bioconcentration,  entire  temperature  12 - 20 ° C .  interpreting  a l i n e j o i n i n g data  c o u l d start at any point above 4.4 data,  the  relationship,  bioconcentration between  of  possible  bi-phasic  which  in  drawn  are  a  ° C , as  temperatures, steady  there  modelling  a  elimination  compounds between 4.4 and 11.8 ° C , w h i c h stabilized between about purpose  of  lowest  assume  modelled  perfect.  by  The  deviating  a  first  12 ° C , w h i l e i n reality, i t  The second option is a poor description o f  relationships  a  significantly  from  linearity  (r  2  the  = 0 -  0.33). From leech  information  biology  contributed  it  to  is  the  states  water  temperature,  the  likely  observed  (1989)  movements,  that  there  that  an  are  of  water  increase  measured  w h i l e L i n d m a n (1935)  metabolic  rate  of  ventilatory  and  11.8  through  concerning  there  effect  is  as  temperature range  °C)  published  N.  obscura  metabolic a  by  rate  physical  Leeches  did  bioassays,  activity appear  which  to are  and  show likely  on  at  had  be  relatively  swimming  behaviour  by  cellular in  the  increased  elevated  leeches  sites  increase  (T as  =  4.4  showed  2  and  20.0  uptake  bioconcentration. Research by Veith  et  al. (1979)  has  relevance  to the  results  -  requirements.  11.8 0  in  similarly  ° C leeches O2  over  Increased  bioconcentration  low  have  swimming  a general  A t 4.4  on  Sawyer  at  and  uptake  respiratory  to  could  temperature.  activity  correlated  which  bioconcentration.  oxygen  water  uptake  temperature  undulatory  and B a r r o n (1990).  greater to  factors  ventilatory  increased  water  et a l . (1983) observed  of  probably  of  of  e x p l a i n increased  hypothesized for fish, by P h i l l i p s (1978) little  leech  increasing  activity could  greater  number  increasing  Linton with  effect  temperature  in  observed  10 - 17 ° C .  a  the  o f the  current  °C and  97  Figure 4.8:  Alternative interpretations of the effect of water temperature on the bioconcentration of 3 , 4 , 5 - T C G and 3,4,5-TCVer by leeches (N. obscura).  1) Bi-phasic model  100  3,4,5-TCG = O  ut  3,4,5-TCVer = O c o c  CD O  c o O  10  cu at o 10  +  15  Water Temperature (° C)  2)  Linear model; y = a(b)exp.x  + = Standard Deviation 3,4,5-TCG;  x= Standard Deviation 3,4,5-TCVer  20  98  leech  research, since  it  shows  that bioconcentration of  not necessarily increase constantly with temperature. fish °C). fish  bioconcentrations  organic  contaminants  does  Veith et al. (1979) found that  of PCBs generally increased with water temperature (5  - 25  However the nature and magnitude of the effect was variable depending upon species  at  test temperature.  bioconcentrations  showed  no  observed.  For example,  increase  between  Fathead minnow  rainbow trout  5 - 1 0  increase  was  followed  a pattern similar to that observed for leech  (Salmo  °C, after  (Pimephales  gairdneri)  which  a steady  promelas) bioconcentration bioconcentrations  of tri- and  tetrachloroguaiacol, with sharp increase between 5 - 10 °C, followed by very little change at 10, 15 and 20 °C. describe that  the effect  there  are  temperature,  These data are also consistent with a multiphasic model to  of temperature upon bioconcentration as well  species  which  specific,  affect  competing  physiological  as with the idea  factors  influenced  by  bioconcentration.  Bioconcentration can be seen as a function of the rate of uptake vs. the rate of elimination. xenobiotic  Barron uptake  temperature.  (1990)  and  details  elimination  Potential  rate  the  importance  rates  and  the  of  physiological  enhancing  limiting , physiological  and  processes in  effect  of  biochemical  increased factors  controlling uptake and elimination include: ventilatory rate and volume, blood flow, membrane permeability, lipid metabolism (Barron 1990). enhanced  at  elevated  ventilatory volume. rate  constants  of  composition, rate  of  enzymatic  For example, fish uptake of organic contaminants can be  water  temperatures  through  Sijm (1991) reported increases ethanol  biotransformation and  and 4-amino  over the temperature range  10-35  increased  output  and  in both uptake and elimination  antipyrine in goldfish  °C.  cardiac  (Carassius  auratus)  One plausible interpretation of the  bioconcentration data for the tri- and tetrachloroguaiacol (Table 4.6)  leech  is that the rate  of uptake was enhanced to greater extent between 4.4 and 11.8 °C, while the rate of elimination  took  decrease in the  on  greater  importance  bioconcentration  of  increased rate of elimination specific  at  temperatures  3,4,5-TCVer  > 11.8 °C.  could be  to this compound.  explained  The anomalous by  a greatly  In fact, this explanation is  supported by data in Section 4.1.1.(Figure 4.2), which show that leeches are able to  99  eliminate  3,4,5-TCVer much more rapidly than the chloroguaiacols.  The and  current leech data are not consistent with experiments  Jacob  (1988),  bioconcentrations  who  reported  an  arithmetic  increase  leech  of chlorophenols over the temperature range 4 - 2 2  experiments  uptake was dominant over elimination,  data  reflect  may  in  conducted by Hall  a  shift  towards  increased  chlorinated phenolics tested in this study.  (N.  obscura)  °C.  In these  The discrepancy in the current  elimination  rates  specific  to  the  Metcalf et al. (1988) reported half lives of  chlorophenols in leeches of 1 - > 2 months, while those for the chloroguaiacols are estimated  to be < 1 month (Section  4.1.1.; Figure 4.2).  Also,  the relatively high  exposure concentration of 10 pg/L used by Hall and Jacob, while not acutely lethal to leeches,  may  leading  to  have  greater  had  a  sublethal  increases  in  effect  on  chlorophenol  bioconcentration  exposed to 0.1 pg/L chloroguaiacols  in the  than  elimination  observed  current study.  in  the  Therefore, the  temperature on bioconcentration could be concentration dependent.  function, leeches effect  of  In order to shed  light on the discrepancy between the two leech studies it may be useful to conduct a search  for  biotransformation  products.  Common  phenolics in aquatic species include sulfate compound (Kennedy 1989). of  conjugate  to parent  metabolites  of  and glucuronide conjugates  (1978)  reported  compound may indicate  that  temperature induced increase increased  of the parent  An assessment of the effect of temperature on the ratio whether  enhanced  elimination rate  accounts for the sharp levelling off of chloroguaiacol bioconcentrations Phillips  chlorinated  a  similar  technique  successfully  above  revealed  12 °C. that  the  in fish- bioconcentration of DDT was primarily due to  uptake.  These  data can also be explained,  theories  concerning  biota.  Water  interactions  with  the  partitioning of  temperature other  material (Schwarzenbach  influences  compartments, 1985).  in part, on the  organic  contaminants  compound such  as  basis of physiochemical  solubility  organic  Since the exposure  and  to  phases  (Phillips inorganic  concentration of 0.1  other than 1978)  and  particulate pg/L was  well below the solubility limit of the test compounds the first phenomenon should not have played a significant role.  There was however, an unquantified amount of  100 particulate  and colloidal  organic  material  in  the  from the secretion of mucopolysaccharide (Sawyer was  not  possible  slime.  to  reliably  Moreover, the  quantitate  effect  of  the  water  leech 1989)  bioassay  resulting  based slime by leeches.  concentration  temperature  system,  on  of  compound  slime  It  sorbed  to  was  not  secretion  measured, although visual inspection did not reveal any obvious  differences  between  test groups.  secreting  epithelial  A histological  search  for hypertrophy in mucous  cells may clarify the importance of this effect.  Increased water temperature should  have increased the rate of desorption of compound from organic particulates with a concomitant temperature  increase may  in  also  bioavailable  have  inhibiting bioconcentration. is  consistent  neutral  with  and  elimination  rate  relationship  partitioning  lipophilic.  and  observed  secretion for  increased  (Phillips  slime  1978).  production,  The sharp decrease in bioconcentration  increased  highly  induced  compound  organic  Together,  a  excess  leech  of  3,4,5-TCVer  bioconcentrations at T > 11.8 °C.  to  and  particulates,  temperature  the  slime  levelling  competitively  of  3,4,5-TCVer  since  induced  may  of  it  is  both  increase  explain  off  However,  the  in  inverse  chloroguaiacol  It is important to note that any such effect may be of  lesser importance in a field biomonitoring system, where water currents are likely to wash away excess slime. From  this  bioconcentration described  by  experiment  of a  all  is  test compounds  bi-phasic  tetrachloroguaiacol  it  concluded  except 4,5-DCG,  model.  There  bioconcentration  between  bioconcentrations remain constant.  that  is  An opposite  following  significant  4.4  and  temperature  a pattern best  increase  11.8  °C,  affects  at  in  tri-  which  and point  effect was observed for 3,4,5-TCVer.  The results can be explained as the competitive interaction of uptake and elimination processes, latter  with  the  becoming  Considering  the  former dominating over of  increasing  variability  in  the  importance the  effect  lowest temperature at  of  higher temperature  range  and the  water  temperatures.  on  organochlorine  bioconcentration described by Phillips (1978) and Veith et al. (1979) along with the differing results obtained by Hall and Jacob (1988), using the same species of leech as the current study, it appears that there are other possible interfering factors to  101 consider to the  such  temperature results  quantified  is  as: (e.g.  contaminant slime  difficult  description  biomonitoring  secretion),  to of  programs.  concentration,  elucidate,  the  effect  age,  specific  stage.  Though  life  for is  species  the  enough  purpose for  of  physiological a clear  routine  practical  reaction  explanation  biomonitoring,  application  to  of a  routine  102  4.5.  Bioconcentration as a Function of Suspended Sediment Load Experiments  water  turbidity  hypothesized phenolics solids,  on  that be  (Hansard)  have  been  and  through  organic  shown  to  vary,  determine  binding  the  to  context  of  phenolic  by  the  of was  chlorinated  of  suspended River,  the  bioavailability could  be  in  greater  effect It  of  particles  concentrations  seasonally,  the  phenolics.  bioconcentration  chlorinated  sediment  to  chlorinated  therefore  Within  upon  suspended  of  competitive  fraction.  sediments  since  1992, i n order  bioconcentration  inhibited  suspended  importance,  i n June  bioavailability  p a r t i c u l a r l y the of  carried out  leech  the  would  influence of  were  Fraser  the  than  Middle  600%  Fraser  (Hall  et  al.  1991). Leeches were exposed phenolics between by  at  (N  obscura),  which  had  water  introducing  accomplished  a  a  temperature  mechanism  through  jars,  In  litre  jars,  cylindrical  bioassay  were  water.  stirring  on  the  containing  stirring plates.  former jars  caught  semi-statically for seven days, to 0.1 of  12  test groups ranged from 0.364  bioassay  been  to  teflon  addition, one since  used. A  the  coated  a  in  magnetic  magnetic  bioassay  bioconcentration.  a  apparatus  No  was  set  August of  Mean  1991,  chlorinated  leech  suspension.  stirring  stir-bar,  did  not  L e e c h l o a d i n g to bioassay jars  control  7.1.  weights,  up  mortalities  to  placed were  function  remained assess  were  the  recorded  This  apparatus,  were  litre square sided bioassay jars stirring  in  B i o a s s a y protocol had to be m o d i f i e d  sediments  of  Lake  concentrations  a p H of  - 0.467 g.  use  Black  ug/L  and  maintain  continuous a  °C  at  in  on  was which  magnetic  replaced  by  two  reliably when  the  at 5 leeches effect during  of  per  litre  continuous  the  exposure  period. Based the  organic  represent  the  on evidence and  clay  greatest  (Voice  particles effective  et in  a l . 1983) ( S c h e l l e n b e r g et the  63  adsorptive  pm surface  suspended for  al. 1984) i n d i c a t i n g that sediment  chlorinated  fraction  phenolics,  decided to focus on the < 63 p m sediment fraction.  A n a l y s i s o f the < 63 p m  Fraser  B . C . , showed  organic  River  suspended  content,  measured  sediments as  the  from  percent  Stoner  v o l a t i l e fraction, r a n g i n g  a  5.4  it  fraction  relatively from  may  -  was of  constant 7.4%  103  in July 1991, 3.7 - 5.2% in February 1992, 4.2 - 4.8% in March 1992 (Duncan, personal communication)  3  .  Qualitative X-ray diffraction analysis revealed the dominant clay  species to be chlorite and illite in an approximate ratio of 60% to 40%. evidence  it  was  decided  to use  a suspended  sediment  mixture  Based on this  composed  of  95%  inorganic clay (50% chlorite : 50% illite) and 5% organic material.  Organic material  consisted  campus  of  dried,  crushed,  green  alder leaves,  picked  University of British Columbia, in Vancouver, B.C. turbidity  experiment  to  represent  the  seasonal  character of Fraser River sediments (Hall levels, representing  low,  from the  of  the  It was decided to design  the  range  of  et al. 1991).  medium and high flow  concentrations  Three suspended  and  sediment  periods on the Fraser River were  selected: 0.025 g/L ( 5.0 ± 0 . 5 NTUs), 0.08 g/L (19.0 ± 1.5 NTUs) and 0.15 g/L (35.0 + 3.0 NTUs).  Water turbidity (NTU) was  suspended  sediment  found to be linearly correlated (r  concentration (g/L).  A turbid (0.08  = 0.98) to  2  mg/L) bioassay,  with  100%  inorganic clay was included, in order to assess the effect of the organic content on bioconcentration. sediments  was  In run.  addition, Also  a stirred  included  bioassay  were  control  with  no  introduced  bioassays,  with  no  suspended chlorinated  phenolic exposure in clear water and turbid water (0.08 g/L). Results  describing the  upon bioconcentration, differences Table  between  4.7.  along  effects  with the  continuously  Comparing  the  of increasing effect  stirred  suspended  sediment  of removing the  organic  and unstirred bioassays  bioconcentrations  of  chlorinated  are  concentration content  and  described  phenolics  in  between  unstirred (Group 1) and continuously stirred (Group 2) bioassays, it can be seen that continuous  stirring of the  water  significantly  facilitated bioconcentration of the observed  for  continuously (Sawyer  3,4,5-TCVer. stirred  1989),  bioassay  than leeches  chlorinated guaiacols.  Qualitatively, jar in  (p = 0.01,  secreted the  it  was  much  two  The same effect  observed more  unstirred bioassay  tailed Student  that  slime, system.  a  was  not  in  the  leeches stress  t-Test)  indicator  However,  slime  production was difficult to quantitate reliably, since much of it remained stuck to the walls of the bioassay jars and tb the leeches themselves.  William Duncan, British Columbia Ministry of Environment, Lands and Parks, 1011 Fourth Avenue. Prince George, B C , V 2 L 3H9, 1993. 3  104  CD  CO CL Z)  o  o  -g x> ZI  oo o d o 'c  CO O) 1—  co +i T~ LO 1—  o c  CO  c o •a >  c>u '•c cu .CO •a CO  x o  CL  -  2  cu  LO  *  CO  •s  c  CD OO O C CD CO C D  ocC cc CO  jS CV)  >s  CO CO  Q. "D  fI v u  cu  <s  I  O  TJ  CO  — o  c O £= x: c o co " CD O —  •5 9  .ti o OJ O CD T- Z £ c> ° c  C O  2 c  o  °  CD b  TJ  3 o  c  T-  U>  CO  2 £  S E . . CD CD > cu ** X  O)  d•<fr  d  CO CO  CO r^  CO CD +1  CO  d  cd  CO  — 1  •tf  o>  •tf  CM  o  CM +1  +1  CL ZJ  O  O) CM CM  CO  CD  LO  XI  CO Q  Q  "9)  c  _  Zi  O  T  oo -So 8 »  T J CO CD £ : TJ C C CD CD O  °- s 38  +1  CO  H  CC73  O)  J£  | o c o CC 1—  CM +1  -g  ai  cE  a—  E +" <D  cu  co  o a C  zi  O  CD  CD  LO  •Jo  •tf  In zt  co o -° 2> C  i-  LO a>  CM  CD l< +1  +1  +l q  o>  CM CD  « «  * «  •tf  •tf  CO  Zi  o CD  O  — o  I o  •tf  CO +1 CJ)  ^  tf  ci  * * CO LO  * « LO CO  D H  o 2 CD CO CO  c+ld oai>  cri  +1 TJ  K_  CO <0  LO  CM LO CM  -g  CM Q.  CM  w  +1  +1  +1  +1  ci  CO CM  CM CO CM  CO  •tf  q  •tf  oo  CM  H  § <°-  CO  °  B 1 cu co • V  t  co  a:  5 ^5  T. cn  a. n  Q  2  CD  o 5 .  LO 00  LO CJ)  +l  +i  d  o o  •tf  d  cd  00  C3  d  T-  to  d  +1 LO CO  II  LO  o  oS  CO +1  CO  sc  «J cu E  c ZJ  o  CL  E o  o  (3  O  r— i  LO •tf"  co"  CD O  hCD LO" •tf"  -6 o CD o T3 O c o  c  ..  Q  Z  in  1  T-  S ^ to  11  --  5  o  d>  cn co  CM  d  r-i  O  II  O  o +1  &  §I  5  J3  CD  CD O CO  TJ  11  CO o  O  Io-o io 0 o  „ m >  II  >. •6  o 2  "O  '-«)'-(/)  T—  o  si  • D • • , — I D D ' Z — ( H  2  -8 "~  0 1  XI CO  c\i co  +l  CO  g) cu o cu I CO  _cu  +1  LO CM CM  co CL  T3  •tf  +i  CM  +l  i-  zs o  o  2- cu cu oo -3  CD  +i  CD  +1  CL  i_ CD CU t C0 fU *3 O  CJ CU  T— LO  O)  S  co  •2--D  2>  O  cd  CO  CO  -g ffl £ '«  C  oo  tf  tf  cu x : — g>  CO  o  f-  co S n » o  o  ii it D O  to  if) O  2 o  ^ II  a a  crj co r r cn g)  r „ cn ^  „  aj "  5 S S | | | « o  0  io c » ffl 5  0)0  c  A  '•5 U  6 £ cn cn  ll c cu N  «  N  (j  « 0)  N W  N W  N w  CL <ii  2  E E I E E E « W <0 rj fn nJ .5 co CO CO CO CO S>  «  ~ci  — Q>  — a>  ~  105  No  clear  concentration concentration the  Middle  with which of  no  trend on  range Fraser  contrary  observed  TCG  and  even  though  observed  leech of  0  -  0.15 For  sediment to  the  what  of  example, at  are  with the  the  0.025  veratrole  g/L  and  and  effect  of  suspended  F i g u r e 4.9 sediment the  ionizable  of  all  range  sediment shows  the  followed  was  0.04  0.08  0.12  Suspended Sediments (g/L) + = Standard Deviation 3,4,5-TCG; x = Standard Deviation 3,4,5-TCVer.  0.16  for less  concentrations, variable for  both  a similar  Figure 4.9: The effect of suspended sediment concentration on leech (N. obscura) bioconcentration of 3,4,5-TCGand 3,4,5-TCVer  0.00  the  reported  compounds.  100  solids  over  compounds  concentration  guaiacols  suspended  phenolics  seasonal  bioconcentration  increasing  polar  the  chlorinated  covers  was. hypothesized.  Both  guaiacols  examining  g/L, which  than  bioconcentration  3,4,5-TCVer.  when  bioconcentration  River.  suspended  was  was  nature 3,4,5trend  106 Available  evidence  bioconcentration explained  by  increasing  associated a  the  Some  with  combination  water  surrounding  suggests  flow  and  the  of  that  the  continuous  two  second,  observed  increase  stirring o f bioassay  processes: alterations  first, in  water  leech  current,  response  was  leech can be  response  to  micro-environment  leeches. species,  including the  erpobdellid, Mooreobdella  species in the same family as N. obscura, display a positive water  water  physiological  the  in  tending observed  to  swim  against  the  in all continuously  current  rheotaxic  (Sawyer  stirred bioassays,  unstirred bioassay tended to remain at rest.  microstoma, a response  1989). while  to  A similar  leeches in the  The natural habitat of the leeches used  in this bioassay is the littoral zone of a small still water lake; the artificially induced current could have resulted in an elevation in physiological activity due to stress of abnormal environmental Conditions. been  found  organisms and  to  be  (Barron  determining 1990), it is  Since ventilation factors  quite  in  uptake  plausible  volume of  and blood flow  contaminants  that increased  by  have  aquatic  swimming behaviour  stress led to increased ventilatory rates, resulting in an increase in the rate of  chlorinated  phenolic  uptake  Agitation of the exacerbated the effect.  through  subcutaneous  capillary nets.  water micro-layers surrounding the leeches skin could have It has been found (Dainty and House 1966) that there exists a  micro-zone of poorly mixed water layers at the membrane-water interface organisms,  which is  theorized to impede  the  bioconcentration  of aquatic  of lipophilic organic  molecules.  Saarikoski et al. (1986) compared the bioconcentration of PCP in guppies  (Poecilia  reticulata  environments.  in  both  magnetically  stirred  and  unstirred  bioassay  A 75 % increase in the rate of uptake was observed in the stirred  bioassay, which was attributed to enhanced penetration of PCP, due to the elimination of  the unstirred water  output  was  compounds,  not  thought  butyric  rates in both  micro-layer.  acid  to  be  of  and phenol,  In these experiments, major  importance,  were  stirred and unstirred bioassays.  bioconcentrations  of chlorinated phenolics  since  bioconcentrated These  the  at  findings  under field conditions  increase the  less  in cardiac lipophilic  virtually  the  same  indicate  that  leech  may vary  107  considerably,  depending  Overall, not  related  to  indicating  sediment  0.1%  content  phase,  with  dominant (pKa values  the  was  of  as  Since was  well  4.3;  Table  should  of  ambient  chlorinated pH  experimental  water  above  we  4.5),  have  that  been  would  and  to  the  the  finding  organic  consistent  been  than at  from  unit  7.1  and  aqueous  the  to  the  to  PCP  preferential  partitioning  of  negligible  as  be  the  compound p K a .  percent  particulate  the  and  organic  content  c h l o r o g u a i a c o l p K a values  partitioning  affecting  the  on  chlorophenol p K a  Sediment  above  that  being  6.94)  with  observed  one  the  found  phase  above  is  dependent  (pKa =  particulates. has  was  increasing content  (1985)  organic  2,4,5-TCP  is  with  is  evidence  c o m p o s i t i o n o f the  p H increased  constant  expect,  ionic  particulate  water  more  process  sediment  phenolics  available  reduced  compounds  hydrophobic  dominant  that  is  to  hydrophobic  phenolics no  contrary  phenolics  organic  p H was  direct  the  is  chlorinated  Schwarzenbach  This to  of  1984).  as  1984). form  is  partitioning of  decrease  al.  water  0.1%,  al.  p H and  sediment  unionized  fraction  the  the  the  et  et  water  to  bioconcentration  provided  partitioning  found  flow.  concentration  ionizable  Organic  (Schellenberg  ionized  long  of  water  chlorinated  (Schellenberg  o f sediments,  factor.  adsorption  leech  sediment  hydrophobic  = 4.75)  that  concentrations,  partitioning  organic  ambient  bioavailability of  particulate  than  the  finding  suspended  that  suspended greater  our  upon  organic  (section material,  bioavailability of 4 , 5 - D C G ,  3,4,5-  T C G , 4 , 5 , 6 - T C G and 3 , 4 , 5 - T C V e r i n our bioassay system, w h i l e T e C G ( p K a = 6.0) should show  significant Sorption  organic  exchange pairs  on  or  as exposed  by  ionized  surfaces  phenolics surfaces  surfaces  electrostatic  calcium,  to  suspended  fraction  and  at  of  a fixed  sediments. chlorinated  phenolics  p H is related  to  the  occurs ionic  (Schellenberg. et a l . 1984; v o n Oepen et a l . 1991).  charged  to particulate  binding  the  water  chlorinated  exchange  1993).  of  and i n o r g a n i c  of the ambient ionized  anion  to  particulates  (Xing  et  (Westall  1985).  attraction  magnesium,  al.  to  has  been  1993)  or  by  Phenolate  ion-pairing of  atoms  to  occur  electrostatic ions  p o s i t i v e l y charged  a l u m i n i u m or i r o n  T h e r m o d y n a m i c a l l y stable  shown  may  both  concentration Partitioning of  by  direct  attraction  ion-  of ion-  d i r e c t l y b i n d by i o n  inorganic  on c l a y surfaces  chlorophenolate  on  anions  surfaces  such  (Xing  et al.  with  K , +  108  Na , C a +  and M g  2 +  formation  2 +  showing  cations  present  a positive  in water  relationship  (Schellenberg et al. 1984; Westall 1985). organic content > 0.1%,  was  Schellenberg  strength  to  increase  et  (1984)  with  found  1985).  of  the  with ion-pair ambient  water  Partitioning of PCP anions  increasing  a strong  K concentration  (Westall  +  positive  adsorption of 2,3,4,6-TeCP and PCP anions suspended  ionic  demonstrated,  In the presence of suspended sediments of  (Schwarzenbach  shown al.  to  been  partitioning of the anion species to organic material is the  dominant transfer process octanol  has  relationship  between  into  1985). sediment  and the fraction of organic material in  sediments.  From the available evidence we would have expected to observe a decrease in leech  bioconcentration  increasing presented  due  suspended  to  sediment  in decreasing  reduced  chlorinated  concentrations  phenolic  due  to  the  bioavailability, following  with  phenomena,  order of importance:  1) Hydrophobic adsorption of undissociated  compounds to organic particulates.  2) Ion pair adsorption to both organic and inorganic clay surfaces. 3) Ion exchange of free phenolate ions to clay surfaces.  Observed results indicate that at suspended 0.15  g/L,  with  a  95:5  partitioning plays little organisms  such  relatively  water  chloroveratroles TCVer finding  as  ratio  of  inorganic  clay  role in bioconcentration  leeches.  soluble, should  bioconcentrations  This  ionizable display  of  to  organic  material,  of chlorinated phenolics  a possible  description of  chloroguaiacols,  a distinctly  followed  could be indicative  is  sediment levels ranging from 0 -  however,  different  the  the  by aquatic  behaviour  highly  the  lipophilic  However,  3,4,5-  the same trend as did the chloroguaiacols.  This  an overwhelming  effect of the introduced suspended sediments.  behaviour.  sediment  experimental  artifact,  masking the  It is plausible that such an effect could  be related to the profuse leech slime secretions observed in the continuously stirred bioassays.  Leeches  secrete  mucous  from  various  secretory  cells;  the  two  most  common being the pear shaped mucous cells located just below the integument and  109 the tubular mucous cells located deeper in the musculature (Sawyer secretion  serves  physiological  functions  such  as  osmoregulation  observed in the continuously  to stress from the constant suspended  sediments.  response  to  various  complex  with  as  The excess leech  may have been due  water current and skin irritation from the pounding of  Chemically different stimuli.  protein  stirred bioassays  Mucous  and excretion,  well as in species recognition and defence against physical danger. mucous secretion  1989).  content  In  general  in  the  types  of  leech  area  of  can include: glucose,  mucous  mucous 10%  may  is  secreted  in  a mucopolysaccharide  (Sawyer  1989).  monomers  and fucose.  Within the bioassay jars mucous was visibly present in large and small  significant  mucous  abundant  pool  significant the  of  effect  increasing  surface high  area  in  sediment  the  organic  on chlorinated phenolic  suspended  form  of  material  colloidal material. is  likely  binding, perhaps  sediments.  to  masking  load on bioconcentration.  with mucous particles were suspended  galactosamine  In addition, it was likely that there was a  area present  surface  glucosamine,  Prominent  carbohydrate  pieces both free and attached to leeches.  galactose,  be  have the  This had a  effects  of  Physically  associated  The close bonding of  suspended  sediments with mucous coupled with difficulties in collection and isolation of mucous made it unreliable to try to quantify the relative role of this artifact in chlorinated phenolic absorption. leeches  in  To control or reduce this effect it would be desirable to expose  a larger system under flow  through conditions,  where  waste products  could be removed and the amount of mucous relative to the bioassay would  be  insignificant.  The  effect  of  removing the  water is shown in Figure 4.10, under  identical  without  organic  bioconcentrations  concentrations material  as  the  pollutants.  organic  sediment  fraction  comparing bioconcentrations (0.08  (Group.  g/L) of 4  vs.  suspended  Group  6).  from  turbid  bioassay  between leeches  sediments, Increased  both  exposed  with and  chloroguaiacol  were observed with the absence of organic material. . These results  are consistent with the hypothesis act  water volume  dominant  adsorptive  that the organic fraction of suspended surface  in reducing the  sediments  bioavailability of  organic  However, an opposite, but not statistically significant (p = 0.05), effect was  110  observed  for  3,4,5-TCVer.  of  chloroguaiacols  were directly proportional to the pKa of these compounds (Figure 4.11).  4,5-DCG (pKa  = 9), which was  The  differences  in  bioconcentration  ionized to the least extent at water pH 7.1  also showed the greatest  increase in bioconcentration between turbid waters with and  Figure 4.10:  without introduced  The effect of the presence of organic material in suspended sediments on the bioconcentration of chlorinated phenolics. Asterisks indicate statistically significant differences (p = 0.01; Student's t-Test).  Numerical figures indicate  the ratio of bioconcentration in the bioassay with 100 % inorganic sediments to the bioconcentration in the bioassay with 95% inorganic : 5% organic material  ou 1.46  CD  40  1.42  c o 1.13 cz co  o c o O  0) 3 CO CO  I  20  1.68  0.87  10 +  4,5-DCG  •  3,4,5-TCG  95% Inorganic : 5% Organic Material  4,5,6-TCG  TeCG  3,4,5-TCVer  100% Inorganic Material  Ill  Figure 4.11: Relationship between chloroguaiacol pKa and the ratio of leech (N.obscura) bioconcentration in turbid (0.08 mg/L) bioassay water of 0 % organic particulate content versus 5 % organic particulate material.  2.0  to E  o 'c CO cn t_  1.8  o  g  'c CO O)  4,5-DCG  e 1.6 3,4,5-TCG  e  o  e  1.4 + c cu o c o o  g in  1.2 +  4,5,6-TCG  TeCG  CO  rr  —I— 7.0  1.0 4.0  5.0  6.0  8.0  10.0  9.0  pKa  organic material.  T e C G ( p K a = 6.0), w i t h the lowest p K a , showed the lowest increase i n  bioconcentration expected turbid  if  pKa  is  the  material  tend  more than  by the  to  two  be  present  highly  interactions  dominate  of  in  These  for the  p H , chlorinated  hydrophobic more  bioassays.  mechanism  partitioning  water to  hydrophobic  continued  the  dominant  A t fixed  controlled  Other  the  system  material. higher  between  the  transfer  phenolics  glass  are  exactly  chlorinated  hydrophobic of lower  undissociated  dissociated  chloroveratrole  of  unionized  interactions,  with  results  and to  such  as  binding  tri-  and  tetra-  surfaces  and  bioconcentration.  species  to  thus  tend  particulate  slime  be  in  a  organic  substitution  chlorinated  leech  would  phenolics  chlorine  form  what  to  and be  organic  phenolics. may  have  112  Experiments affecting as  seasonal  biomonitors  findings the  aimed  are  i n the  Fraser  questionable  who  mucous.  at the p H o f the Fraser  suspended to  during  sediment  mass,  the  basis  total  flow  played  colloidal  sediment Fraser  fraction River,  relatively  can  where  the  constant,  that bioassay  protocol  six  to  eight  laboratory  bioassay  flow  also  may  be  the  from has  d i d not  was  of  must  different  increase  taken  in  effect  any  this  material  made by  organic  in  Carey  actual  ratio  sediments  material  in  may  be  in  not  measured  bioconcentration  of field  issue  in  the  sediments  is  comparing  also as  in  biomonitoring  role  suspended  when  results  be  Laboratory  an  studies  in  water  would  the  suspended  variation  of  possible  material.  consideration  Therefore,  material  detail about the  Laboratory the  i n interpretation  the  organic  chloroguaiacol stirred.  of  suspended  into  on  organic  the  partitioning of  material  watersheds.  continuously  o f importance  organic  However,  moderate sediment  organic  While  used  chlorophenols  with  of  of  in  several  together w i t h  dissolved  leeches  effect.  of  fraction  go into  and  be  the  material  to  w i t h predictions  coefficients  associated  a profound  fold  input  taken  bioavailability.  factor  extraneous  when  solid  compounds o f any  consistent  presence  fraction  this  studies  the  that reduce  biomonitoring  by  but  macromolecules  revealed  are  period  percent  H o w e v e r , his predictions  experiments  the  suspended  evidence  C a r e y predicted  at its peak,  the  no  of  phenolic  partitioning  very l o w . by  of  T h e results  low  role  chlorinated  ( p H = 7.8).  was  the  showed  sediment  the  sediments  River  on  determined  chlorophenols  assessing  bioavailability of  form o f leech  (1988),  at  was  indicated indicted  observed  when  ambient  water  data.  113  4.2. FIELD STUDIES 4.2.1. Temporal Variations in Contaminant Concentrations in Effluent,  Water, Suspended Sediments and Leeches  In situ studies were conducted both to evaluate leeches as biomonitors under a wide  range of  comparative  seasonal  study  environmental  with  the  conditions  results  of  and to generate a data base for  laboratory  bioassays.  Field  monitoring  experiments were carried out in July & October 1991 and February 1992. took place on the Fraser River at Prince George B.C.  Monitoring  A test site was selected at Stoner  B.C., located about 40 km downstream of three BKMs, discharging secondary treated effluent into the Fraser River, from a total of two discharge points. selected at Shelley outfalls with  (Section  composite  B.C., about  10 km upstream of the most  3; Figure 3.1). sampling  of  A comprehensive treated  mill  sediments in addition to caged leeches.  phenolics  experiments  is positively  revealed  of  organic  bioconcentration. the  in  leech  3.3. bioconcentration  suspended  of  bioconcentration,  sediments  did  seem  Introduction of water current, by means  laboratory bioassay  suspended  of  chlorinated  - 10 ug/L) and  The quantity of suspended sediments did not  an important determinant  material  and  followed,  Negative correlations to water pH and leech weight  were observed in laboratory bioassays. be  water  correlated to contaminant concentration (0.1  water temperature (4 - 20 °C).  appear to  that  river  system resulted  in increased  although  to  the  competitively  of magnetic  presence inhibit  stirring, to  bioconcentration.  In the context of a Fraser River biomonitoring program, it was expected contaminant concentration, which is governed river  and  water  flow,  would  two  description of monitoring sites and  sampling procedures is given in Methodology, section Laboratory  northerly of the  sampling program was  effluent,  A full  A control site was  be  the.  that  primarily by B K M discharge into the  major  determinant  of  bioconcentration.  Temperatures in the Fraser vary from about 1 - 20 °C (Hall and Jacob 1988), however,  114  temperature 0.33)  -  bioconcentration  relationships  in  the  laboratory  and only affected bioconcentration at T < 12 °C.  in the Fraser River, ranging from 7.0  - 7.8  (Hall  were  weak  and Jacob 1988), and was  stock of uniform size were encountered, therefore  bioconcentration relationships were expected results.  Based  on  laboratory  bioconcentrations  could  correction  applied to  leech  factors  be  account  from for  not  Difficulties in leech weight -  to be of importance in the analysis of  relationships  predicted  2  Water pH is relatively constant  expected to be a major factor in determining leech bioconcentrations. obtaining a leech  (r = <  it  was  measured  hypothesized water  variation in water  that  concentrations, temperature,  leech with  pH and  weight. Exposure  conditions  during  monitoring trials  are  summarized in  Table  4.8.  Seasonal water flows ranged from high summer flow to low winter flow on the Fraser River. the  Water temperature decreased  lower  range  of  temperatures  October 1991 and February 1992. pH unit over the study.  progressively  over  the  study,  tested in laboratory bioassays  dropping below  (4.4  °C) between  Water pH was relatively constant, varying under 1  Turbidity remained high over the July and October study  periods, but dropped sharply during the winter low flow period. Mill  operations  were  normal  in  July  shutdowns occurred in all mills in October 1991. parameters are given in Table 4.9.  of  testing  decreased  higher through  concentrations would  be  2  the  it was  greatest  concentrations were  C10  Increasing chlorine dioxide (CIO2) substitution  levels,  From  the  predicted that effluent in  July  (Table 4.10)  more than double  1991.  matched  those of  combined with secondary effluent  periodic  and higher chlorine substituted  All three Prince George mills were in the process  substitution study.  however  Effluent flow and bleaching process  the formation of both A O X (Axegard 1986)  phenolics (Liebergott et al. 1990).  February,  A O X loading data were calculated from measured  effluent flow and AOX concentrations. reduces  and  consequently C10  2  A O X loading  substitution  concentrations  Effluent  trends  and A O X  of chlorinated phenolics in  these predictions; July  October and February.  levels  generally  chlorinated effluent  High  phenolic  concentrations  C 1 0 substitution 2  treatment did not eliminate chlorinated phenolics  115 from  effluents,  February.  except  in  the  Decreased  use  of  case  of  elemental  2,3,4,6-TeCP,  which  chlorine  in bleaching led  ( C y  became  non-detectable to  shifts  in  Table 4.8: Field conditions for the July 8-15, October 17-24, 1991 and February 19-26, 1992 at two monitoring stations on the Fraser River at Prince George, B . C .  Parameter  July 1991  October 1991  February 1992  Shelley  Stoner  Shelley  Stoner  Shelley  Stoner  1808  21 8 4  750  881  449  574  1 1  12  6  6  0  0.5  PH  7.8  7.8  6.9  6.9  7.3  7.3  Turbidity (NTUs)  26  23  17  41  4  6  River Flow (m3/sec)* Temperature ( °C)  * Water flow data provided by Water Survey of Canada, Environment Canada. Flow at Stoner station calculated as the sum of flows on the Fraser River at Shelley and the Nechako River at Isle Pierre.  Table 4.9: Pulp mill process conditions for the July 8-15, October 17-24, 1991 and February 19-26 1992 at Prince George, B . C .  Parameter  October 1991  July 1991  February 1992  Outfall A *  Outfall B "  Outfall A*  Outfall B**  Outfall A *  Outfall B "  166,025  163,513  125,522  140,475  137,458  137,475  70  70  90  81  90  NA  16.5  20  5.5  12  8.5  1 6  2739  3270  690  1686  1168  2200  Avg. Effluent Flowt (m3/day) Avg. Percent Chlorine Dioxide Substitution Effluent AOXt (mg/L) AOX Loading (kg/day)  NA = not available. * Outfall A = Canadian Forest Products Ltd. (Canfor) outfall. " Outfall B = Northwood Pulp and Paper Co. outfall. t AOX concentration and efliuent flow supplied by Bill Duncan, B.C. Ministry of the Environment, Lands and Parks, Prince George, B.C. branch.  116  Table 4.10:  Flow weighted average effluent concentrations of chlorinated phenolics discharged from Prince George bleached kraft mill outfalls during three seven day monitoring periodsf.  Compound  Concentration (pg/L) July 1991  October 1991  February 1992  4,5-DCG  1.0  0.5  1.4  3,4,5-TCG  17.3  6.0  5.7  4,5,6-TCG  2.8  1.2  1.6  TeCG  7.8  2.7  0.8  3,4,5-TCVR  2.5  1.6  2.9  5,6-DCV  2.8  0.6  5.6  2,4,6-TCP  5.2  1.2  1.5  2,3,4,6-TeCP  1.2  0.2  ND  Total  40.6  14.0  19.5  ND = none detected; refer to Table 3.1 for detection limits, t Results from five day composite samples: July 8-12 and October 17, 21-24, 1991, February 19-21, 24, 25, 1992.  chlorine substitution of chlorinated phenolics (Liebergott et al. 1990).  Levels of 4,5-  DCG and 5,6-DCV increased between July and February, while TeCG and 2,3,4,6-TeCP levels dropped sharply.  3,4,5-TCG, 4,5,6-TCG and 2,4,6-TCP all showed decreases with  increasing C 1 0 substitution. 2  all  effluent  samples,  3,4,5-TCG was the most prominent marker compound in  which represents  a shift  in dominance  from TeCG,  which the  was prominent compound from Prince George effluents of the late 1980's (Schreier et al. 1991). and  0  2  Future implementation of chlorine reducing technology, such as 100 % CIO2 delignification  monochlorinated  species.  may  lead  to  shifts  towards  dominance  of  di-  and  117  Despite h i g h CIO2 substitution, station  consistently  similar  proportions  of  effluents,  the  showed to  phenolic  were  detected  not  concentrations relative that  discharge the  of  concentration concentrations  to be  the  were  what  detected  to  the  chloroguaiacols  species  July  detected  can probably be attributed  and and  most  in in  Stoner  and  A s i n the  marker  effluent  samples,  unknown could the  by  a  of  explain  the  increase was  much  possible  indication  capillary  trend  in  both  such  in  February,  effluent  Fraser River at Stoner, B.C. No target compounds detected in water samples from  Concentration (ug/L) July 1991*  October 1991*  February 1992f  4,5-DCG  ND  ND  0.073 ± 0.009  3,4,5-TCG  0.052 ± 0.005  0.043 ± 0.005  0.067 ± 0.016  4,5,6-TCG  0.002 + 0.001  0.006 ± 0.003  0.015 ± 0.003  TeCG  0.024 + 0.001  0.010 + 0.002  0.012 ± 0.003  3,4,5-TCVer  ND  ND  0.024 ± 0.003  5,6-DCV  0.036 ± 0.004  0.071 + 0.021  0.467 + 0.018  2,4,6-TCP  ND  ND  0.017 ± 0.002  2,3,4,6-TeCP  ND  ND  ND  ND = none detected; refer to Table 3.1 for detection limits. •July and October 1991 results from analysis of daily composite samples, n = 7 ( 3 samples composited per day). tFebruary 1992 results from analysis of four grab water samples; Feb.19, 21, 24, 26.  of  which  d u r i n g this p e r i o d .  the control station located at Shelley, B.C.  as  Water  number  Table 4.11: Seven day mean water concentrations of. chlorinated phenolics detected in the  Compound  column  in the  high greater  observations.  observed  to less d i l u t i o n o f the l o w r i v e r flow  compounds  5,6-DCV,  decreasing  sharp  in  dual  source  case  Relatively  but  in  of B K M derived  station.  samples,  test  TeCG,  Chlorinated phenolic  control  concentration  the  (Table 4.11).  unresolvable  A  from  4,5,6-TCG  prominent  water  followed  water  samples  3,4,5-TCG,  River.  river,  October.  water  samples  Shelley  previously  A O X discharged  between  Fraser  interferences a  of  the  detected  was  analytical  of  levels  upstream  5,6-DCV to  to  River  i n effluent  appeared  Alternatively,  degradation  chemical  of  were  analysis.  those observed  at  proportion  there  measurable  3,4,5-TCG  chlorinated  Fraser  118  High February 4.12).  low  concentrations flow  of  chlorinated  period carried  phenolics  over to. the  present  suspended  in  water  sediment  from  samples  the  (Table  February suspended sediments concentrations of 3,4,5-TCG and TeCG increased  by 68 and 36 times respectively over July levels.  As in the case of water samples, 5,6-  DCV appeared in concentrations out of proportion to effluent levels.  Note that 3,4,5-  TCVer was not recovered from sediment spiked material by the analytical procedures followed and therefore, could not be monitored in suspended sediments. lipophilic sediment  veratrole phase,  family  using  using organic solvent  of  the  compounds  alkaline  may  buffer  not  be  extraction  easily  separable  employed.  The neutral from  Further  the  testing,  extraction failed to recover spiked compound, indicating that  veratroles may bind by a different mechanism to suspended  sediments.  Table 4.12: Suspended sediment concentrations of chlorinated phenolics and percent organic contant in the Fraser River; July 8-15, 1991 and February 19-26, 1992. Results reported on a dry weight basis.  Compound  Concentration (pg/kg) July 1991*  February 1992**  4,5-DCG  ND  95  3,4,5-TCG  0.91  62  4,5,6-TCG  ND  8.4  TeCG  0.36  13  3,4,5-TCVer  NR  NR  5,6-DCV  ND  1 76  2,4,6-TCP  ND  3.0  2,3,4,6-TeCP  ND  ND  Organic Content (%)tt  3,1  7.8  ND = none detected; refer to Table 3.1 tor detection limits. NR = not recovered in sediments by extraction procedure used. * July 1991 study; no control samples taken. ** February 1992 study; 2,4,6-TCP (0.63 ug/kg) found in control sample, tt Measured as loss on ignition.  119 Size fractionation of July suspended  sediment  material revealed a composition  of 4.3% coarse sand (> 250 u-m), 78.5% fine sand (> 63 pm < 250 pm) and 17.2% silt (< 63 um).  February suspended sediments were not size fractioned due to a lack of enough  material. July  Qualitative X-ray diffraction analysis was carried out on the silt fraction of  suspended  sediments to better  characterize  clay  components.  minerals were chlorite and illite in an approximately 3:2 ratio. quartz, feldspar  and horneblend components.  The percent  Prominent clay  Also prominent were  volatile  organic material,  as measured by the loss on ignition, was-3.1% in July 1991 and 7.8% in February 1992, which is consistent for the 1.6% and 6.0% organic fractions predicted by Carey (1988) for  spring freshet  and winter low  flow  periods  particulate content during the high flow low  carbon mineral components  on the Fraser.  A lower organic  period is related to the increased input of  eroded from bed sediments and river banks during  freshet periods (Carey 1988). Sediment is  dependent  Schellenberg  partitioning of  primarily et  al.  both  neutral  and ionizable  on the organic content  (1984)  found  a strong  of  hydrophobic  compounds  sediments (Schwarzenbach  positive  relationship  between  1985).  sediment  adsorption of 2,3,4,6-TeCP and PCP and the fraction of organic material in suspended sediments.  In the case of ionizable hydrophobic compounds, such as the chlorinated  phenolics,  sediment  concentration fraction  (Schellenberg  preferentially  although phase  partitioning  stable  (Westall  adsorbs  ion-pairs 1985;  et  al. to  (e.g.  is  also  1984;  dependent  von  suspended  Oepen et particulates  upon al.  water  pH  1991).  and  The unionized  (Schellenberg  et  al.  phenolate-K pair) can also partition to the  1984),  sediment  +  Xing et al. 1993).  ionic  Preferential partitioning of the unionized  fraction is consistent with the observation that the ratio of 4,5-DCG (pKa = 9.0)  to  3,4,5-TCG (pKa = 8.0), 4,5,6-TCG (pKa = 7.4) and TeCG is greater in sediment than in water  samples  partitioning  as  from February  1992  (i.e.  there  is  an increased  rate  of  sediment  pKa increases).  The increase February  1992  was  in suspended likely  sediment  associated  material in suspended sediments.  bound compound between July  with  the  increase  in  the  1991  percent  Using the following equations developed by  and  organic  120  Karickhoff  et al.(1979) to describe sediment partitioning of hydrophobic pollutants to  sediments:  Eqn. 6  Ksw = Koc* foe  Eqn. 7  Koc = Kow* 0.63  Where: Ksw = sediment water partition coefficient, coefficient,  foe = fraction of  coefficient,  increased  organic carbon and Kow =  4.13  and TeCG  they give  should be  considerably  less  a 2.5  than  fold  the  increase  measured  68  in  Ksw between  and  36  fold  July increase  concentrations of 3,4,5-TCG and TeCG.  Table 4.13:  Predicted sediment-water partition coefficients (Ksw) for 3,4,5-TCG and T e C G in the Fraser River at Stoner, BC. in July 1991 and February 1992.  Sampling Period  during the  an idea of the possible  in sediment partitioning behavior of chlorinated phenolics.  there  water partition  The equations were developed to describe the behavior  neutral organic compounds, however  differences  octanol  Ksw values are predicted for 3,4,5-TCG  February low flow Table 4.13. of  Koc = carbon normalized partition  Ksw  Fraction Organic Carbon (foe)  3,4,5-TCG  TeCG  Jul. 1991  0.031  550  2700  Feb. 1992  0.078  1380  6800  Kow 3,4,5-TCG = 4.45; Kow TeCG = 5.14.  seasonal  From  Table  and  February,  in  sediment  121  Sediment with al.  concentration  the  S c h e l l e n b e r g et  sediment  load  probably  concentrations. much  than  study,  July  above  1991  range  suspended 1992,  from about  concentrations  that f r o m 0.3  -  1.4%  sediment  partitioning  on  Laboratory  investigations  (section  concentration in  the  (e.g.  small  be  an  -  is  150  pool  is  trials  et  suspended sediment  appeared  to  were  permit  it  is  3,4,5-TCG  water  in  to  the  column.  4.7)  may  also  content). fraction  to size  macromolecules  traps used  July  non-available  seasonal  roughly  1991  sediment  have this  and  to  aquatic  levels  of  using  the  February  the  effect  the of  negligible.  c o n c l u s i o n , since  suspended  sediment  quantity  adsorbed  personal current  the  and  been  that  to  load, while  Therefore,  probably  i n the  the  estimated,  support  (Derksen  measured  If it is assumed  H o w e v e r , o f the is  not  report  corresponded  bioconcentration  defined  compound,  River  a l . (1991)  adsorbed  Table  organic  sediment  size,  pm) ( V o i c e  February  sample  15 - 150 m g / L .  bioconcentration  and  of  et  leech  c o l l e c t e d i n the  unquantitated  Hall  for  the  between  in  enough  concentrations,  4.1.5;  63  particle  qualitatively  Fraser  in  poorly  biosolids)  and  obtained  m g / L ; 5% a  the  carried  observed  column,  colloids to  was  (25  water  98.6%  (<  increases  not  monitoring  of 3,4,5-TCG  99.7  correlation  in  to be  sediment  to  tested.  sediments  1992  related  silt fraction  sediments  was  (1988)  remaining  no  there  is  i n size c o m p o s i t i o n o f the  suspended  Carey  sediment  fine  remaining  concentrations  suspended  -  the  c o u l d not be  February  compounds  the  both  and  in  for  sediments,  solids  however  with  February  July  suspended  range o f suspended The  organic  Differences  accounted  so this hypothesis  Total this  a l . 1984).  Although  finer  fractionation,  in  hydrophobic  greatest p a r t i t i o n i n g associated  1983;  be  of  carried  to  organic  communication) study . 4  This  organisms  to  remains such  as  leeches. Leeches compounds (BCF) and by  during  ranged  water leeches  bioconcentrated  profiles, at  three  f r o m 465  the  present i n leeches 4  all  tri-  Stoner were  monitoring  - 6000  the  site.  relatively  times  and  high  trials  water  (Table  concentrations.  tetrachloroguaiacols  In addition the  similar to levels detected  George Derksen, Environment C a n a d a , 1992.  4.14).  were  observed i n fish  chlorinated  phenolic  Bioconcentration A s expected detected  proportions  most  from  factors effluent  consistently  of chloroguaiacols  tissues by Rogers et a l .  122  co  r~  c o  CD  O  c  CL CD  — i  I—  CO >  CO  >_  3 0) 0)  CC CD  h-  X3 CD  c o  "a CL CO  CM  +1  +1  +1  o  •* CM  -H  CM  CO  CD O  o O  CO  T—  CD  S  CO CO  o  CL O  H cc  CO  3  co  •a  CO  .b: CO  -a  w  CD  ' CO  8  o  3  c\i  CD  CO  CO  co DC co LL  z  m i—  CM  CM O)  CO  OS  00 <  CO o i—  CO  t o  CL  co  o o  co  3.  ~  a.  a. co  2!.  Q. O ©  2P  CL  X  CO  ">  3  ID  CD U_  °  <D W O) LO  5  5  7  2  C D  \%  < < < <  r~ •* ^ CM a. O a  "O  CO  C CO  8 x:  C  I  CD .Q O  o O  fl) CM  cn  O co  c  CO  CD O  +1  c o O  o  CM  CD  O XI C O JB TJ  3  in  O) r 1  +i ^t"  6  d  +1  +1  CD  ^—  00  in  -  co co  J= OJ  C5  5  c  C O  '3 fi 3 t  Q. O  "8 £  Co CO  "co .  .- c °> ©  E  CO  o eo o o in  o >, O  CO  3  M—  o  "55  CO  c o >>  CD > CD CO CO  -3  CD i— O  o  c  tv.  CM  c o O  +1  +1  in +i  CO CM  CM  T—  CD O  00  U J  +1  c  o  CM  CD  3  CO CO  §9S CO CO  cn  5 CD  "O  c 3  O  a.  ca  » 5  «  |  E o O  a o a  in  a o in co"  (3  O H co m" •*"  CD >  O hin •<t co"  O hco t" CM"  ©  s -g  CO  »  t ©  CO  I  CO  co" CM"  S ©  8 2  u.  1 if 2  T-  T> O  c 11  O O CO  T3  z  »  M  •c •£ -c  3  I-a -=s oO) .£ 2o> o. ^ S  II  «CO £ m  c  O  I-  ft  S- »  $ CO S co  CO .©  D_  > O Q co in  II  -a  o 3  CD  5  XI  »  CO  to  tz  N  E Ss  -a o c  o o  eo  11  < < < <  CO CL  CO  £Z  II  in  g  3, CO  c  Q CO  a co  Q  c  .E  -O CD  to £" T3 oi Ji £ 2 B >. to f O  123 (1988a) and Dwernychuk et al. (1991). control  leeches.  contamination  of  No chlorinated guaiacols  However,  Shelley  tri-  tetrachlorophenols  and  leeches  were  successful  during  all  after water testing had revealed no detectable traces.  were detected in  in detecting  three  upstream  monitoring trials,  Note that measurable levels of  neither 4,5-DCG nor 5,6-DCV were bioconcentrated by leeches in any field trial, even though these compounds were detected in effluents, water  concentration  of  5,6-DCV  interpreted as either evidence bioavailable form or it is latter  explanation  is  coupled  with  at all times.  its  absence  The apparent high  from  leeches  can  be  that 5,6-DCV is transported in the Fraser in a non-  simply not bioconcentrated effectively  favoured  by  laboratory  studies  where  difficult to detect in leeches at water concentrations < 5 pg/L.  by leeches.  the  The  compound  was  The relatively low GC-  ECD response of 5,6-DCV also inhibits the study of this compound; detection limits (20 50  pg/kg)  in  leeches  range  tetrachloroguaiacols ( 1 - 3 5 pg/kg), however, organisms.  its  to  |xg/kg).  twenty  times  above  those  analytical  tri-  lower log Kow (3.41) make it less bioavailable to aquatic  biomonitors of either  5,6-DCV  or 4,5-DCG  indicate  that  at present  leeches  levels of  sensitivity.  One of the criteria of a good biomonitor is the ability to concentrate compounds  in  proportions reflective  bioconcentrated to  both  Leech  and  4,5-DCG has a relatively lower detection limit (3 -  Both in situ monitoring and laboratory bioassays  are not effective  for  of  ambient  tri- and tetrachloroguaiacols  measured  effluent  bioconcentrations  and Fraser  followed  levels  (Phillips  and 3,4,5-TCVer  River  decreasing  water  1978).  of  Leeches  in proportions similar  concentrations  proportions  target  TeCG  proportion of 4,5,6-TCG associated with increased C10 substitution 2  (Figure 4.12). and increasing  employed by  the  mills. Chlorinated  phenolics  were  detected  leeches throughout our monitoring study.  in  water,  suspended  sediments  and  The results indicate that the winter low  flow period on the Fraser River may be the critical exposure time for many native organisms, period.  since  the  highest  concentrations  of  compounds  were  found during  In support of our findings, monitoring of juvenile chinook salmon on the  this  124  Figure 4.12:  Relative concentrations of chlorinated phenolics in leeches and water samples from Stoner, B C . and effluents from two Prince George B K M outfalls, over three seasonal monitoring periods.  July 1991  October 1991  February 1992  0.08 ^  0.07  ~  0.06 +  B co  0.05  *" co o c o O  0.04  1_  0.02  CD CO  Water  0.03  0.01 0.00  n  July 1991  October 1991  February 1992  Effluent  zt CZ  o c CO  o c  o  o  cl  CD  _3  3= LU  n July 1991  October 1991  February 1992  125  Fraser  River  phenolics periods  by Rogers  were  i n the f a l l . Some  of  rate  increasing fish  when that  fish.  water  year  rates  (Kennedy  (Webb  on juvenile  and Brett phenolics  temperatures  and isolate  in  laboratory  water  concentration, to  water  at  concentration  appeared  i n field  strong  relationship  (Figure  4.13).  appeared  to  be  departure  from  burdens  than  o f chlorinated  during  medium  flow  (r  A t the  a  slight  linearity  i n the  until  o f higher  the  spring  are elevated.  winter  runoff  Evidence  as the Fraser  River  of C o n t a m i n a n t  decrease  body  period, suggests  is required,  over  decreasing  concentrations B C F , which  is  logarithmically  B C F (the  concentrations B C F with data  Table 4.3).  show  water  a relatively of 4,5,6-TCG  in  the  an i n d i c a t i o n  that  concentration  of  range o f  increasing  concentration  monitored  v s . water  ratio  the exposure  and f i e l d  B C F and water  C o n c e n t r a t i o n  increased in  a l l water  laboratory  o f the b i o c o n c e n t r a t i o n  i n m e t a b o l i s m and  periods.  over  towards  = 0.78) between  an increase  impacts  was observed  Together  water  changes  bioconcentration  concentration)  lower  felt  a Function  to be a trend  2  however  systems  only  energy  l o w (0.7 ° C ) temperatures,  dynamic  bioassays,  maintenance  nerka)  rates  as  salmon  Therefore,  exposure  the  (Oncorhynchus  metabolic  critical  studies.  raising  i n measurable  0.1 - 10 p g / L ; B C F was r e l a t i v e l y constant there  very  m a y not be  and fish  While  However,  period  sockeye  1973).  m o n i t o r i n g o f such  to identify  bioconcentration  body  and P C P ) are k n o w n to be uncouplers  1989),  m a y result  4 . 2 . 2 . In Situ B i o c o n c e n t r a t i o n  with  that  l o w flow  (TeCP  to be m i n i m a l  temperature  round  order  the w i n t e r  T h e effect  o f chlorinated  water  indicated  v  was found  growth  burdens  during  phosphorylation  upon  growth  a l . (1988a)  o f the chlorinated p h e n o l i c s  oxidative  demand  in  higher  et  field, there  there is a  126 relationships decreases  derived  below  in  the  bioconcentration  laboratory lowest  studies  level  predictions  tested will  (Figure in  4.14).  laboratory  increasingly  As  water  concentration  bioassays  (0.1  pg/L),  underestimate  field  bioconcentrations. ~  —•  •  i  Figure 4.13: Leech (N. obscura) bioconcentration factor (BCF) as a function of water concentration for 4,5,6-TCG detected in field studies (x) for July, October 1991 and February 1992 and laboratory studiest(O).  10000 Jul. 1991  o CO  1000  Oct. 1991  q  o 100 • 0.001  0.010  0.100  o -0 1 000  Log Water Concentration (u.g/L) t Laboratory leeches exposed lor seven days at T = 12.5 °C, pH = 7.5.  Figure 4.14:  Leech (N. obscura) bioconcentration as a function of water concentration for 4 , 5 , 6 - T C G detected in field studies (x) for July, October 1991 and February 1992 and laboratory studiesf (O).  10000  0.001  0.010  0.100  1.000  Log Water Concentration (p,g/L) t Laboratory leeches exposed tor seven days at T = 12.5 °C, pH = 7.5.  .  127  In  the  case o f 4 , 5 , 6 - T C G ,  4.14,  since  laboratory and  field  was observed  1992  were factors  (0.043  levels.  still  For (0.5  magnitude varied  markedly  example,  °C)  o f error  low  probably  water  resulted  is not  both  - 0.067  p g / L ) d i d not  H o w e v e r , higher  observed  unaccounted  than  (Figure  4.15).  for  laboratory  in  It  in  deviate  expected  in  appears  that  studies,  accurately  between  temperatures lower  at the laboratory temperature o f 12.5 ° C .  concentrations assayed  conditions  conditions.  February  the  depicted  field in  trials  October  and  1991  bioconcentrations  (6  °C) what  In the case o f 3 , 4 , 5 - T C G ,  water  from  the  lowest  situ b i o c o n c e n t r a t i o n s there  are  result  in in  situ higher  of  laboratory 3,4,5-TCG  environmental than  predicted  bioconcentrations.  Figure 4.15:  from  than  greatly  which  in Figure  Leech (JN. obscura) bioconcentration as a function of water concentration for 3,4,5-TCG detected in field studies (x) for July, October 1991 and February 1992 and laboratory studiest(O).  t Laboratory leeches exposed for seven days at T = 12.5 °C, pH = 7.5.  128  Insight expected  in  that l e e c h  into  the  bioconcentration effluent  photographs  effluent  of  km  data  of  1994).  completely  the  of  Stoner,  at l o w r i v e r f l o w .  both  of of  reduces  automatic  comprehensive  sides  Pulp  and  distinct  breadth mix  the  with  Fraser  was  the  and  Fraser  bulk  river  flow.  for  least  at  the  about  1 km 40  point.  occurs  Rivers  that  km  assumption  that  mixing  verify  river.  located  at  to  indicate  on  River  than  outfall  River  selected  effluent  Nechako  the  the  station,  Fraser  complete  of  with  greater  conducted  Company  monitoring  B K M outfalls  the  the  Paper  from  Stoner  water  probability  the  Fraser should  water  effluent  i n July show  no  for the  sampler  sampling  homogeneous  bioconcentrations  the  B a s e d upon concern  the l a c k o f a second  assumption  across  the  in  that  Indeed,  about 7  km  (Dwernychuk  and  A l s o the presence o f a strong m i x i n g zone at R e d R o c k C a n y o n , about  upstream  conduct  of  testing  instantaneously  that  confluence  from  not  mixed  indicate  resulting  equal  The  George  gained  variables  be  visibly  1994).  Prince  be  tracer  downstream Levy  the  would  sodium  Levy  do  be  Northwood  remains  and  downstream  the  unknown  can  should  streams of  plume  (Dwernychuk  effluents  hypothesized  situ b i o c o n c e n t r a t i o n s  BKM Aerial  the  on  incomplete  security  effluent  o f water  mixing,  sampling  even  equipment,  and l o g i s t i c a l problems it was decided  o n l y one  mixing  and  of  10  side  leech  October  significant  of  the  Fraser.  biomonitoring  1991.  It  was  differences  To  was  test  the  conducted  on  hypothesized  across  the  to  that  breadth  leech of  the  river. Statistically between July  monitoring  and  highest  October  on the  October.  The accounted equations are  stations  1991  differences  at  (Table  opposite 4.15).  east side o f the  July  compounds,  leeches  significant  of  being  s l i g h t l y greater  in  cross  channel by  relating more  differences  intersample leech  effective  leech  sides  of  bioconcentration  the  Fraser  weight  2,4,6-TCP west  side  did  in  both  of chloroguaiacols  were  trend  follow  in  leech leech  bioconcentration,  bioconcehtrators.  at  observed in  opposite  not  were Stoner  was observed  the  trend  of  in  other  leeches.  observed  differences to  River  L e e c h bioconcentrations  river i n J u l y , w h i l e the  bioconcentrations  for  in  H o w e v e r , the  bioconcentration weight. it  was lighter  cannot  From found west  be  regression that  side  lighter leeches  129  bioconcentrated less than the heavier east side leeches in July 1991.  October leech  sample groups did not differ enough in weight to cause any notable differences in bioconcentration.  Higher bioconcentrations  of chlorinated phenolics in leeches  on  one side of the river could be explained by incomplete effluent mixing resulting in cross channel differences  in water concentrations.  This theory cannot be  verified,  since water sampling was only conducted on one side of the river.  Table 4.15: Cross channel differences in tissue concentrations of chlorinated phenolics in leeches (NL obscura) exposed for seven day periods in the Fraser River at Stoner, B C . Results reported on a wet weight basis. Results in bold text indicate significant differences (two-tailed Student's t-Test; p = 0.05) between east and west side test groups.  Compound  Tissue Concentration (pg/kg) July 1991  October 1991  East side*  West side**  East sidef  West s i d e f t  4,5-DCG  ND  ND  ND  ND  3,4,5-TCG  49 ± 9  28 ± 7  16 + 4  20 ± 3  4,5,6-TCG  20 + 3  12 ± 2  3.7 + 1.8  5.8 + 1.5  TeCG  30 + 5  21 ± 5  4.2 ± 1.9  6.4 ± 1.9  3,4,5-TCVer  ND  ND  3.0 + 1.7  1.9 + 0.9  5,6-DCV  ND  ND  ND  ND  2,4,6-TCP  17 ± 3  ND  ND  2,3,4,6-TeCP  4.7 ± 0.7  9.8 + 1.6  11+2  19 +  8  3.4 + 0.6  ND = None detected; refer to Table 3.1 for dectection limits. * Sample size n = 19; mean weight ± SD = 1.18 + 0.14 g. ** Sample size n = 9; mean weight ± SD = 1.07 + 0.14 g. t Sample size n = 10; mean weight ± SD = 0.85 + 0.09 g. t t Sample size n = 10; mean weight ± SD = 0.872 + 0.15 g  Alternatively, the differences, could also be explained by micro-environmental differences one  between  monitoring stations,  leading  station  over the  other.  significantly  in either  temperature or pH, field  in water current.  to  While it is not likely  enhanced  bioconcentration  that sampling sites  notes indicated notable  at  differed  differences  No attempts were made to measure this variable, since it had not  130  been  originally  hypothesized  bioconcentration. and  other  substantial In leeches  after  effect  uptake  rate  chlorinated  on  1991  a  in  of  field  trials,  of  Investigations  into  (Saarikoski  al.  unstirred of  tri-  and  from  1986)  of  lipophilic  organic  poorly  molecules attributed  oxygen 1989),  to  and  to  laboratory actively,  East  when  the side  of  phenolics  this  have  had  water from  (Section  by  side,  leeches  were  ranged  water  west  investigations,  bioassays  guppies  bioconcentration  water  which  the  is  may  where  had  been  conducted  conducted  under  current)  leech  5.2  7.8  times  Table  4.7).  -  4.1.5; (Poecilia  differences  increased  through  exists  the  at  impede  1966).  of  rate o f uptake  o f surface  layers  House  agitation  xenobiotic  a disruption  by  uptake  the  obscura,  against  rates  stress, related  including  swim  could  from  reticulata)  between  by  75  %  stirred  with  the  the  The  water  diffusion  membrane-water  bioconcentration  enhancing  effect  micro-layers  ( S a a r i k o s k i et  "pores"  the  barriers  water  surrounding  a l . 1986).  scattered  of  of  Since  across  the  the  leeches  epithelium  could  result  in  a  flow  could  also  substantial  bioconcentration.  Enhanced  as N.  from  laboratory  PCP  flow  leech  phenolics.  P C P bioconcentration  ( D a i n t y and  up  family  of  affect  bioassays  water  area.  bioassay  mixed  organisms,  take  species,  that  presence  similar  bioassays;  e p i t h e l i u m , i n c r e a s i n g the  exacerbated  that  (i.e.  unstirred  revealed  of  aquatic  be  in  showed  laboratory  observed  from  tetrachlorinated  outer  increase  Evidence  would  current.  interface  (Sawyer  were  backwater  bioconcentration  micro-zone  can  flow  conditions  laboratory a  low  zone.  those  et  a  flow  stirring  than  of  theory  l o w e r bioconcentrations  high  bioconcentrations  current  the  stationed  continuous  A  from  the  in  presence  evidence  support  completion  and  is  current  which  were  greater  there  water  investigations,  July  stationed  However,  that  the  bioassays. w h i l e leeches  in  areas  of  stronger  p h y s i o l o g i c a l response  e r p o b d e l l i d , Mooreobdella display  a  current  (Sawyer  Leeches i n the  water  positive  in  A  continuously  unstirred  bioassay  water  microstoma,  rheotaxic  1989).  to  response similar stirred  tended  to  to  have  been  current.  Some  leech  a species  i n the  same  water  response  current, was  observed  bioassays  tended  remain  rest.  at  tending  to  in  swim  131  The natural habitat of the leeches used in this bioassay is the littoral zone of a small still  water lake; the artificially induced current could have resulted in an elevation  in physiological activity due to stress of abnormal environmental conditions. ventilation factors  in uptake of contaminants  plausible rates,  volume and blood flow  that increased  resulting  subcutaneous  have  flow  (Barron  of the determining 1990), it is  swimming behaviour and stress led to increased  in an increase  in the  rate of  chlorinated phenolic  quite  ventilatory  uptake through  capillary nets.  may  have  a  significant  combination a physiological response water  found to be one  by aquatic organisms  Both laboratory investigations water  been  Since  and field effect  observations on  leech  support the  bioconcentration  to increasing water flow  micro-environment surrounding the  leeches.  for the discrepancy between leech bioconcentrations  This  theory due  that to  a  and alterations in the  factor  alone  may  account  observed in the field and those  measured in laboratory bioassays (Figures 4.14 & 4.15).  132  4 . 2 . 3 . In Situ B i o c o n c e n t r a t i o n  In leech  weight  during field g,  order  the  documented  July  1991  laboratory  Inverse weight,  similar  could  laboratory  study.  those  (Table  be  were  4.16).  i n July tri-  chloroguaiacols,  although  coefficient  other  less  sensitivity  did  laboratory  apparent  for  and  to  bioconcentrations  TeCG.  laboratory  w e i g h t range  relationships,  in  The  the  form  laboratory  as  In  a  no  manifested  (Figure  since  by  may  the  laboratory.  of  predicted  field  reflect  conditions a(X)exp.b). 0.5 - 1.0 g.  bioconcentrations  were  not  possible,  v a r i e d m a r k e d l y i n terms o f water Marked  differences  are  only  studies  however of  their  lower  in  the  similar to  the  correlation showed  slopes,  was  than  especially  differences  between  less  than  half  laboratory  and  field  bioconcentration  over  D i r e c t c o m p a r i s o n , i n terms o f  since  for  significant  quantities  encompassed  decrease  concentration  apparent  under  regression  C o m p a r i s o n o f the percent  observed  similarly  true  g.  leech  no  difference  1.94  and  from  and  the  0.15  3,4,5-TCVer,  detectable  -  situ b i o c o n c e n t r a t i o n s  the weight range 0.5 - 1.0 g is p r o v i d e d i n Table 4.17. absolute  for  slope  The not  0.63  about  revealed  shallower  4.16).  slopes  of  were  behaved  in  investigated  from  A s expected  shallower  general,  i n the the  leeches.  showed  weight  studies,  weight  and  o f s m a l l leeches,  weight  investigations  leech  was  bioconcentration  tetrachlorophenols  decreased  relationships,  examined  the  and  compounds. weight,  a  phenolic  exposed  and  to  m o n i t o r i n g , since  field  2,4,6-TCP  test  leech  field  bioconcentration  relationship  i n mean  down  Laboratory  c o n f i r m e d by  structures,  than  for  W e i g h t  between  this  ranging  chlorinated  reported  L e e c h  a lack of availability  ranged  bioconcentration  detected  bioassays,  samples had  of  relationships  D u e to  between  between not  chemical  the  inverse  investigations  to  conditions  veratrole  field  the  in  field  relationships  relationship this  validate  a Function  i n v e s t i g a t i o n o n l y tested l e e c h  while  field  to  as  the  laboratory  and  (i.e. y intercept  TeCG,  over  the  field  a of y  weight  =  range  133  CO  I" g co 13  cr LU  co co  d  CO d  •  d  x X  co  tf d  o tf  CD d  X  LO CO d  d  c  c  CD  T-  tf  ^  CM  CO  CD  rr CO  >.  o  *  +1  tf  ^  CVJ +1 CM  LO +1 ,-  +i  tf +i  m  tf  r-  in CM  CQ  O)  CD o  ~c5)  55 to  c  o  CD  > be  -t—»  CD CO CO  c  CO  CD W W  ys ex  eg  o  CD CO i_ Z3 co CO CO X i  LO  o d  £  cn CD  IO  +i  9  i  CD  CD  CO  CO  c o  > CD  Q  O  C  C  Q  c  +^  CO  o  CD CJ  1  T3  +1 CO CM  4—'  Li. CD  O CL  CO  tf  d  TJ C  o CL  E o  o  CD O Q  CD  U  r, O  CD  >  o  huS tf"  co"  >  o  CL O  "S £  134  Figure 4.16: Bioconcentration of 3 , 4 , 5 - T C G (a) and T e C G (b) by leeches (N. obscura) of differing weights, exposed under both laboratory (water concentration, 1.0 ug/L; temperature = 12.5 °C; p H = 7.5; n = 19) and field conditions (water concentration = 0.052 pg/L; temperature = 12.0 ° C ; p H =7.8; n = 28).  a) 1000  3,4,5-TCG - LAB = O  "9)  3,4,5-TCG - FIELD =  •  JZ  O  CD  o  o O  100  t  CD ZJ  co co O  10 0.1  1.0  10.0  L o g L e e c h Weight (g)  : Standard Deviation 3,4,5-TCG - LAB; x = Standard Deviation 3,4,5-TCG - FIELD.  b) 1000  1 ^  +  C  o CD CJ  c o O  100 +  CD  co co  o  10 0.1  1.0 Log L e e c h Weight (g)  + = Standard Deviation TeCG - LAB; x = Standard Deviation TeCG - FIELD.  10.0  135  Table 4.17:  Predicted d e c r e a s e s in bioconcentration of chlorinated phenolics by leeches (N_. obscura) between 0.5 g and 1.0 g under both laboratory* a n d field conditions**.  Predicted bioconcentrations calculated from regression relationships  developed for laboratory bioassys (Table 4.5) a n d Fraser River field bioassys (Table 4.18).  Laboratory Study Compound  Predicted Bioconcentration  Field Study  (u.g/kg)  Decrease  Predicted Bioconcentration (u.g/kg)  Decrease  Leech Wt. = 0.5 g  Leech Wt. = 1.0 g  (%)  Leech Wt. = 0.5 g  Leech Wt. = 1.0 g  (%)  3,4,5-TCG  121  68.0  - 43.8  53.2  33  - 40.0  4,5,6-TCG  446  307  - 31.2  18.2  12  - 34.1  TeCG  333  202  - 39.3  27.5  20  - 27.3  * Semi-static seven day exposure; water concentration = 1.0 ug/L, temperature = 12.5 °C, pH = 7.5. " Seven day caged field exposure at Stoner BC. July 8 - 15, 1991; water concentrations: 3,4,5-TCG = 0.052 ug/L, 4,5,6-TCG = 0.002 ug/L, TeCG = 0.024 ug/L, temperature = 12.0 °C, pH = 7.8.  The combined effect of the following factors were argued to be responsible for the  enhanced  bioconcentration  bioassays: 1) greater rate of 0  2  observed  1991), 2) decreased  homologue),  small  leeches  observed  in laboratory  consumption (Davies et al. 1987; Sawyer 1989) leading  to an increased rate of xenobio'tic Connell  for  uptake at surface respiratory sites (Barron  content  of detoxifying  1990;  betroiydal tissue (primitive liver  leading to a lower rate of depuration (Sawyer  1989), 3) greater uptake  surface area to volume ratio (Saarikoski et al. 1986), 4) lower biomass to contaminant ratio for bioassays believe  containing the lighter leech weight groups.  There is no reason to  that the first three phenomena do not occur in field exposed  leeches.  The  fourth factor is an artifact of the laboratory bioassay protocol and is not a factor in the continuous flow conditions of the Fraser River.  In laboratory bioassays, leeches  were segregated into four weight groups (n = 5 per group) of mean weights, 0.148 0.303 g, 1.23 g and 1.77 g. 8.85  g/L of bioassay  water.  g,  This corresponds to biomass loadings ranging from 0.74 Therefore, there was more compound per unit biomass  available  to leeches in the  light  weight groups in effect,  exposure  concentration to lighter leech groups.  increasing  the  apparent  The magnitude of the the effect is  uncertain, however it may have been of little significance,  since water was  136 completely changed every 24 h and field exposures biomass  effect  could  account  for  the  bioconcentration per unit change in leech in situ bioassays in  showed similar relationships.  slightly weight  greater  rate  of  A  change  in  observed in laboratory relative to  (Figure 4.16), although the limited range of leech weight examined  the field make it difficult to compare the two studies.  4.2.4.  Interspecies  Criteria  for  biomonitoring  Differences  the  selection  laboratory  uncontaminated biomonitoring that all the  analysis,  stock.  between leech  appropriate  to both toxicity  this  criteria were  leech  species  should include: sensitivity  Nephelopsis  species, Percymoorensis 1991 field study.  an  maintenance  organism for above  Bioconcentration  of  of water contaminants  target compounds, with respect routine  in  in  for  routine  to the presence  of  and bioconcentration, suitability for the  obscura  laboratory was  early  and  selected pilot  availability  as  of  the  primary  investigations  indicted  study,  because  met.  For comparative purposes,  a second  leech  marmorata, was evaluated against N. obscura during the July  Refer to section 2.4 for a comparative description of leech biology  species.  No  clear  interspecies  trend was  observed.  Table  4.18,  indicates that N. obscura was a more effective bioconcentrator of 4,5,6-TCG, TeCG and 2,3,4,6-TeCP, while P.  marmorata attained  equal levels of 2,4,6-TCP. of  greater  concentrations  of  3,4,5-TCG  and  Laboratory bioassays, conducted at a water concentrations  1.0 pg/L, revealed a different pattern (Table 4.19), in which P. marmorataa.tta.ined  greater bioconcentrations under  of all chloroguaiacols.  laboratory conditions  cannot  be  explained  The dominance of P. by  differences  in  marmorata  leech  weight,  since the mean weight of N. obscura (1.23 g) was about half of that for P. marmorata (2.66  g).  maintained  Furthermore, leeches under  identical  used  conditions  condition prior to the experiments.  in both and  laboratory and  appeared  to  Therefore, differences  be  field in  studies  similar  were  physical  between laboratory and  137  field  results  uptake  can be e x p l a i n e d  and  depuration  There  are  differences content, flow  factors,  such  warmer  water  differences fathead  o f water  marmorata  in  environmentally rate  River,  induced  (1970)  was  to  have  tolerant  suspended  sediment  bioconcentration  by  clear  levels N.  a  conditions on  water  could  obscura.  have  lentic  of  (Connell  1991),  blood  rate  gill,  trout  uptake  to  that  for this  habitats.  about  (Barron  lentic  High  N.  by  1990).  interspecies between  the  gairdneri).  N.  than  P.  above,  respiratory phenolics  explanation  while  l e d to the t h e o r i z e d  F o r example,  obscura and P.  between  levels,  factors,  described  increased  o f Colorado leech  evenly  limiting  contaminants  of chlorinated  for N.  Certain  brought  (Salmo  more  by  1990).  et a l . (1979),  fashion,  ranges  sediment  lipid  may explain  by Veith  A basis  preferences  ratio,  conditions.  the  interspecies  volume  bioconcentrations  manifested  greater  affect  (Barron  physiology  similar  relatively  suspended  to  to  and r a i n b o w  and tolerance  distributed  for  rate  observed  obscura.  the habitat  of high  preference  N.  area  rate  increased  reaction, induced  exert  are i m p o r t a n t  bioconcentrations  species  be  flow  promelas)  In  to  by environmental  to  of PCBs,  stress  studied  flow  on respiratory  exposures.  and c o u l d  P. marmorata  distinct  may lead  o f the k n o w n habitat  Herrmann  and  blood  greater  b y the  environmental  metabolism  respiratory  volume  (Pimephales  field  and  and b l o o d  and  surface  o f depurative  term,  i n bioconcentration  thought  including:  i n the short  temperature  attained  metabolic  rate  temperature,  minnow  obscura  studies  factors  pattern,  as respiratory  ventilatory  effect  Fraser  biological  distribution  are i n f l u e n c e d ,  increased  The  many  bioconcentration  and  o f differing  physiology.  a c t i v i t y o f and m e c h a n i s m s  rate  which  in  b y the effect  species  i n the  comes  from  marmorata and f o u n d  and l o t i c obscura  and  habitats  showed  in  situ water  flow  stress  induced  increase  a  and in  138  Table 4.18: Bioconcentrations (ng/kg) of chlorinated phenolics in two leech species, Nephelopsis. obscura and percymoorensis  marmorata. exposed in the Fraser River  at Stoner, B.C.*, for a seven day period, July 8 - 15, 1991. Results reported on a wet weight basis.  Compound  Tissue Concentration (pg/kg) N. obscura f  P.  marmorataft  4,5-DCG  ND  ND  3,4,5-TCG  28+7  159 ± 63  4,5,6-TCG  12 ± 2  3.0 ± 1.0  TeCG  21 ± 5  5.0 ± 1.0  3,4,5-TCVer  ND  ND  5,6-DCV  ND  ND  2,4,6-TCP  19 ± 8  19 ± 6  2,3,4,6-TeCP  3.4 ± 0.6  ND  ND = None detected; refer to Table 3.1 for dectection limits. * Leeches exposed on the West side of the Fraser River, t Sample size n = 9; mean weight + SD = 1.07 + 0.14 g. f t Sample size n = 10; mean weight ± SD = 3.07 ± 0.52 g.  Table 4.19: Bioconcentration of chlorinated phenolics by two species of leeches, Nephelopsis Qb£Cjjia.and Percymoorensis marmorata. after seven days exposure to a water concentration of 1.0 ug/ L at a water temperature of 12.5 °C and water pH 7.5.  Compound  Tissue Concentration (pg/kg) N. obscuraf  P-  marmorataft  4,5-DCG  182 ± 44  311 ± 105  3,4,5-TCG  82 ± 18  303 ± 96  4,5,6-TCG  325 ± 27  354 ± 119  TeCG  219 ± 41  256 + 65  3,4,5-TCVer  45 ± 18  45 ± 8  t Sample size n = 5; mean weight ± SD = 1.23 ± 0.43 g. t t Sample size n = 4; mean weight + SD = 2.66 ± 0.49 g.  Both N. obscura and P. marmorata are phenolics  and have been useful in situ riverine  (Hall and Jacob 1988; Metcalf and Hayton 1989).  suitable  biomonitors  biomonitors N. obscura is  in  past  of  chlorinated  investigations  available  throughout  British Columbia as well as all the rest of Canada (Sawyer 1974), is easy to maintain in  139 laboratory and provides clean, easy to analyse extracts.  While P. marmorata may be  more suitable for Fraser River monitoring due to its riverine habitat preferences, N. obscura has shown  a greater  (Herrmann  1970; Sawyer  monitoring,  characteristic  tolerance  to lower  water  temperature  1974) and may be the better of  the  Fraser  River  during  choice  (0 - 20 °C)  for cold  spring,  fall  water  and winter  seasons.  4.2.5. E s t i m a t i o n L a b o r a t o r y  o f In Situ W a t e r  C o n t a m i n a n t  Bioconcentration  Concentrations  U s i n g  Relationships  Leech bioconcentration data provides a measure of the bioavailability of water contaminants. to in  However, it would be useful to relate leech tissue concentrations back  situ exposure  bioconcentration  (i.e.  derive  water  contaminant  concentration  from  data).  Laboratory environmental  levels  relationships  (temperature,  between  pH, water  bioconcentration, current)  water  and biotic  concentration and  (leech  weight)  factors  were used to calculate the original exposure concentration during July, October, 1991 and 5.  February 1992 field trials (Table 4.20). In  each  case  the  relationship  Sample calculations are given in appendix  between  leech  bioconcentration  and water  concentration (Section 4.11; Table 4.1) was rearranged to give an initial prediction of in situ water  concentration  Table 4.14).  Since this laboratory relationship was derived for a mean leech weight  of  1.17 g and water  from  measured  temperature  in situ bioconcentration  12.5 °C and pH 7.5, corrections  normalize  the prediction to the corresponding field conditions.  predicted  water  temperature,  concentration  pH and leech  took  weight.  (Section  into  account  The resulting  exposure value,  were made to  Thus the resulting concentration,  termed  predicted water concentration is given in column 4 of Table 4.20.  4.2.1;  the static  water water  140  E  c 0  k_ 3  8?  J o  = ^  co LU  X  CD a CO  3  CO  —  co  £ £  o  c  j= 3 2 ~ 2 ffl o  0  o  -a  CD  8I  c  I  0  CD  oo  c 5 -o i . CO CD g ^ c -B  co•  CD c  b  ci  3 C  CO O O  CM  b  d  in o q  o  c o  O O o  o  CD I - I-  o  3> i i C M -0 = I I 0II C" O O CM C D (5  LO  CO  CM  O  r-  CO  T-  O  T~  £T ^ " io ii  m  "5. -a-  CO  ffl  CO C O  2 >_  o  0  5  1 0  X  12 ^  o  Q-  0 i_  JZ  JD  aC$L Q.  -9 co £  c co  ~2 w . o _  31o co -  " D C  1  1  CC  0  ~c  T3  CD O  3  o o o  CD CO  o  c c  s^  E x: 0  0  a  co  c  0  o O  co  in o  CM  O  CO  o  o  o  CM  CO o  T—  o  6  0  o  c CO tr  n  T3 C 3  o  CL  E  o O  CD O H I  CD O  r— I  in in  co"  •>* co"  1I  m co"  c o  I  >  cu 00  §•  O  O)  fl)  C  o  « 5 E  n  CD O  CD  -r--  ci  2 .2> .2> oi ffl | |  cn 1^ CO  ffl TS £ 2  o CM  k_ 3 CO  CM  CO  CO -  5  k_  •*—'  O  O  H  0  o co  b 2  •g  £  -a  c  S  c -5 >. >, CO CD > CD CO  £Z ^  ffl  *£ • co eg •o -6  I  2  ,f  ! I § P  m  o o  U o  (5  CO  »  to OT * .2 co >" o  c 2 o ca pE S c co c  ^  =£. > "a  CO  CD O  OI  cn  as  ffl  "& =1 * 5 5!  6 2  0  o  0  ,' i "> C  O  6  T—  o p  o  00 CM O  00  CO 3  0  Q)  CO •4—1  O  o  c o  E  Q-  !  o 0  O) C  "a. E co co  X  9 o  •a  0-  co  OT OT  OT OT •0CM  CM OT OT CO CM  in  OT oo 3  ~3  O  O  XI 0  O O csi CO CD  ffl  ffl  CO  ffl  <B CL  E 0)  CO  CL  E ffl  fl) ffl ffl  III  141  Note  that  i n a l l cases  bioassay  overestimated  bioconcentration when  was  bioassays  provide  a  the  were  more  correction observed  in  method  estimate  provide  to  with  of  more  exposed  i n lotic  present  study habitats.  the  predictions only of  a single  water  accurate  environments. more  questionable  discovered To  conditions  a  bioconcentration  current),  was d i v i d e d b y  flow  for 3 , 4 , 5 - T C G the  concentration  a useful  between o f in  range  correction  factor  i n February 1992.  water  flow  water  current  bioassay  for predicting  estimation  o f exposure  situ w a t e r  T h e laboratory suitable  field  on  leech  water  since  current  correction  bioassay.  provided  estimates  The  except  contaminant  relationship  in  seven d a y  i n suspension.  under  (i.e. water  water  accidentally  sediments  increase  4.  of  was  concentration  stirring  levels,  for an unstirred  effect  but  retain  relative  column  are  The  to  o f water  situ measured  the  m a y be  stirred  magnetic in  the t w o estimates  characterization  water  examined,  was d e r i v e d from  together  may  formally  magnetically  o f the  Neither but  never  predictions  the  accuracy  factor  value.  i n the bioassays  underestimated  concentration  measured  equivalent  water  water  the  accurate  factor,  static  The  the p r e d i c t e d  and  was  accurate,  levels.  Further  bioconcentration  concentrations  from  leeches  relationships  derived  i n the  water  concentrations  in  lentic  142  5 . SUMMARY, RECOMMENDATIONS AND APPLICATIONS  Leeches T h e leech  were  TCG,  water -  contaminant  one to  leech  tissue  the  Environmental  but  as  as  the  secondary and  and  factors  biotic  guaiacol  In  also  primary test  field in  analysis not  and  the  apparent studies,  low  two  product,  compounds,  to poor  chlorophenol  the  effects  of  the  following  investigated:  concentration  (0.1  -  10 p g / L )  - Water temperature (4.4 - 20.0 ° C )  Biotic  sediment l o a d and c o m p o s i t i o n (0 - 0.15 g / L )  Factors: - L e e c h weight (0.15 - 1.80 g) - L e e c h species (N.  assessed  the  of  four 4,5,6-  3,4,5-TCVer.  An  chlorovanillins,  this  compound  bioconcentrations  - Water p H (5.1 - 9.0)  - Suspended  comparative  3,4,5-TCG,  the of  while  in  a n a l y t i c a l s e n s i t i v i t y by  Factors:  - Contaminant  for  bioconcentration  (4,5-DCG,  leech  compounds.  test o r g a n i s m ,  species,  quantification  achieved due  and  field  of  type  investigations  compounds  group  phenolic  compounds,  at  the  2,4,6-TCP  monitored.  laboratory, were  third  was  detection  assayed.  a  reliable  samples  capture  bioconcentration  used  laboratory  chlorinated  investigate  5,6-DCV,  and 2 , 3 , 4 , 6 - T e C P were In  chlorinated  c h l o r i n a t e d g u a i a c o l biotransformation  by  levels  of  of  was  used  environmental  made  electron  was set  derived  and  was  and  both  mill  TeCG)  represented  GC  of  kraft  attempt  integrated  biomonitors obscura  marmorata  An  importance bleach  as  species Nephelopsis  Percymoorensis purposes.  assessed  obscura,  P.  marmorata)  factors  on  leech  143  Three and  winter  under  field  m o n i t o r i n g trials, c o v e r i n g summer  (February  varying Under  1992) seasonal  field  conditions  phenolic  bioconcentrate  were  after  measurable  water  were  BKME  chlorine high  accurate  flow  body  primary  contaminant between  log  bioconcentration contaminant  (Table  levels  rate  of  laboratory  contaminant that  (estimated be  concentration.  T  a critical  leech  body  eliminated 1  /  2  ^28  factor burdens.  days)  of TeCG, Leech  leech  there  leeches  to  model  trend  m a y be  of  also  a  a  in  < 0.1 p g / L .  2,3,4,6-  from  chlorinated  Leeches  i n both  during  the  the  the  of  linearity (i.e.  than  concentration  at l o w rates  at  predicted  derived  function  guaiacols  the  B C F at l o w  uptake  contaminated  was  correlation  log  higher  a  that  winter  laboratory  from  in  flow.  linear  elevated  data  increase  In situ studies,  as  able  indicated  increased  departure  bioconcentration  o f ambient  also  strong  The resulting  Trichloroveratrole showed  also  day period.  an  by river  towards  i n water  seven  from  and  suggested  (Figure 4 . 2 ) , suggesting  i n the estimation  were  4.14).  biomonitoring  may occur  showed  o f leeches  burdens  a  resulting  concentration  describing  (Table  o f contaminants  bioconcentration  overestimates  Removal body  over  Leech  organisms  4.13).  o f t r i - and  2,4,6-TCP,  m o n i t o r i n g data  concentrations  (Figure  Leeches  are d i l u t e d the least  (< 0.06 p g / L )  lead  regression  leeches  that  at water  concentrations would  (Figure  H o w e v e r , a slight  suggested  lower  bioconcentrations  phenolics  4.12).  of  monitors  (3,4,5-TCVer,  water  contaminant  4.1).  concentrations  the  to evaluate  conditions  proportions  contaminants  the  contaminant water  (October 1991)  any trace o f the c o m p o u n d .  A l l contaminants  o f uptake)  seasonal  o f the r e l a t i v e  for aquatic  determinant  concentration.  the  to detect  i n bleaching.  water  effective  from 4 6 5 - 6000.  discharge  burdens  period, when  The  increased  i n mill  substitution  contaminant  low  fall  conducted  be  diverse  o f dichlorinated  indicators  the decrease  to  l e v e l s o f the compounds  and receiving  dioxide  under  ( B C F ) ranged  biomonitors  effluents  reflected  proved  analysis h a d failed  not effective  Leeches  leeches  compounds  L e e c h bioconcentration factors  TeCP),  were  1991),  in situ c o n d i t i o n s .  tetrachlorinated  to  conditions,  (July  from  of  water  water  revealed  relatively  slowly  that loss b y e l i m i n a t i o n w o u l d not water  concentrations  a d i s t i n c t l y different  from  seven day  depuration  144  profile,  with  constant,  rapid  Bioconcentration pH,  however,  compound  The  phenolate  contaminant species  N.  ionizable  bioconcentration  undissociated 4.5).  of  ion  There  obscura,  induced  bioconcentration  of  the  not  also  was  2  <7  relationships  evidence  of  days). inversely to  compound,  to  weak  the  disturbance  manifested  by  to  bioavailable to  the  of  a l l test  compounds  except  best described by a bi-phasic model (Figure 4.8).  was  significant  tri-  4.4  and  opposite  11.8  ° C , at  which  point  was  observed  for  effect  competitive  interaction  dominating  over  importance  at  the  affecting  seasonal  evidence  of  uniform  composition  any  effect  sediment  chlorinated  guaiacols  the  were  fraction  in  indicated  that bioassay  indicted  by  observed  when  ambient  chlorinated  the  form  the  six  of  to  phenolic  current  and  the  when  that  change  results  can  be  the  latter  of  suspended  There between  significantly. explained  processes,  with  the  becoming  leech  in  eight  may  of  as  An the  former  increasing  the  absence  of  of  secretions.  fold  increase  were be  effect in  on  The  in  leeches  showed  no  sediment  mixture  of  organic  organic  since  the  in  the  bioconcentrations  of  particulates.  input  of  that  sediment  River  organic  studies  results  as  also was  bioconcentration  Therefore,  environmental Fraser  However,  suspended  measured  stirred.  material).  material  chloroguaiacol  important finding  material  5%  Suspended  continuously an  :  organic  extraneous  a profound  bioconcentration.  inorganic  solid  to  suspended  bioavailability,  mucous  also  a  presence  unquantified,  bioassays  compounds  95%  the  reduced  by  role  using  diameter;  p r o t o c o l has  laboratory  water  4.7),  greater  complicated  material,  range  chlorophenolic  revealed  were  The  not  elimination  assessing  (< 63 p m  suspended  in  at  (Table  experiments  did  bioconcentration  4,5-  temperatures.  bioavailability of  Laboratory  results  and  temperature  water  aimed  bioconcentrations  uptake  lowest  higher  Experiments  of  tetrachloroguaiacol  3,4,5-TCVer.  in  3,4,5-TCVer.  bioconcentration  and  leech  a p o s i t i v e trend  (Table 4.6), i n a pattern in  of  (Table  DCG  increase  water  concentration  generally  significantly  was  related  the  were  physiological  by l o w p H , w h i c h  affected  /  related  contributed  non-dissociable  temperature  1  was  directly  regression form  T  chloroguaiacols  was  and  pool.  Water  e l i m i n a t i o n (estimated  variation  determinant exposed  of  145  leeches attained greater bioconcentrations contaminant supports from  concentrations  this the  theory  were  cross  water  Further  channel  bioconcentrations (Table 4.15). heterogeneous  significantly  (Figure 4.15).  apparent  of certain compounds, even lower  than  in  though water  laboratory  corroborating evidence  differences  in  bioassays  was  Fraser  obtained  River  leech  Strong river water mixing reduced the possibility of  contaminant  higher leech bioconcentrations  concentrations  at  the  Stoner  site.  However,  were related to positioning in areas of swifter  water  flow. The effect strong  inverse  of leech  weight  relationship  clear trend was  observed  was  on bioconcentration was  observed  for  for 3,4,5-TCVer  the  chlorinated guaiacols,  (Table 4.4).  found for field exposed leeches (Table 4.16).  compound specific. while  Similar relationships  Available evidence  A no were  suggested that the  effect may have been due to a combination of age and size dependent factors of both a physiological  and  depurative enzyme  physical  nature.  clear  between the  chlorinated  guaiacols  P.  than N.  similar levels of 3,4,5-TCVer.  attained  species  to  be  Differences weight  decreased  for  However, N. obscura may be better  was  tissue  observed  marmorata .  concentrations  of  Both species bioconcentrated  in tissue concentration of chloroguaiacols differences,  of  since  the  smaller species, N.  Under in situ conditions, N. obscura  of 4,5,6-TCG,  greater levels  suitable  greater  obscura (Table 4.19).  greater bioconcentrations  proved  in bioconcentration  obscura and Percymoorensis  attained the lower bioconcentrations.  marmorata bioconcentrated  and  area to volume ratio in smaller,  marmorata attained  could not be explained by leech obscura,  differences  species Nephelopsis  Under laboratory conditions  rate  contributing factors.  trend in interspecies  leech  respiratory  function and . a greater surface  younger leeches were possible No  Increased  TeCG  3,4,5-TCG  and 2,3,4,6-TeCP, while (Table 4.18).  P.  Both leech  biomonitoring chlorinated phenolic  compounds.  suited for monitoring in cool northern waters,  such as the Fraser River, since it shows a greater temperature tolerance range that P. marmorata. In addition, N. obscura provides cleaner tissue extracts phenolic analysis, allowing for lower limits of detection.  for chlorinated  146  The in  relative  importance  to  magnitude  relation  the  be seen i n Table 5.1. the  effects  changes in  on  in  each  of  levels.  range 5 - 9 .  of  these  classes  to  Water  pH  temperature the  appeared  range  and  occurring  in  which  biotic  the  factors  natural  is  investigated  environment  useful  affected  for  very  detecting  can  between  northern  sensitive  small  bioconcentration  to  changes  over  the  pH  system o f relatively stable p H ,  bioconcentration  affected many  were  w o u l d probably be m i n i m a l .  affect  by  Leeches  i n any single aquatic - 8.5),  to be  exhibited  compounds.  significantly  significantly  bioconcentration  of  concentrations,  In reality, the effect  found  environmental  changes  such as the Fraser R i v e r ( p H 7.5 not  the  T w o compounds, 3 , 4 , 5 - T C G and 3 , 4 , 5 - T C V e r are used to illustrate  contaminant  ambient  of  above  0 - 1 2  Temperature 12  °C,  however  ° C , w h i c h corresponds  aquatic  systems  from  fall  in  the  present  study,  an  important  was  to  the  through  to  effect  of  spring. Although  not  quantitatively  current  on  bioconcentration  water  biomonitoring  of  controlled,  some  flow  to  rate.  leeches  Leech  at  Attempts estimate  the to  use  weight  contaminant  after  exposure If  bioconcentration  with  while  bioconcentration, Fraser  a  concentrations  simulations,  River.  end  derived  concentrations.  water  systems,  extent,  smaller  water  phenolics  riverine  failed  investigated  by  such  as  be the  Fraser  conducting  all  was  a  powerful  determinant  of  the  weight  laboratory  the  correction  Fraser factor,  continuously  River  body  the  complex  to  burdens  leech may  similar  Use  was  of  important dynamic  to  chlorinated water  increase  applied,  Therefore,  of  sensitivity.  bioassays,  observed  be  water  overestimated  the  water,  and  of  maximum  4.20)  levels.  identifying  effect  semi-static  (Table  measured  in  duplicate  leech  in  bioconcentration.  from  bioassay  areas  promote  corresponding  stirred  underestimated successful  from  The  in  of  would  relationships,  factor  River.  monitoring  scale  concentrations in  to  may  the  in  predicted laboratory  determinants conditions  in  of the  147  SZ  2>  CO CO  LU  00 CO  UJ  CD  SI CJ CD CD  UJ  z  O c p co k_ "cz  -- I O CO •tf  UJ  0)  o c: o o o  LO LO  JQ  as  o  o  21  >.  x:  co TJ  0 CD  0  o  C  o co  >  c o  CD  CO  '  Ja 73  c  Q_ X 0  c o  c 0 o c o o g  LO 00  X Q_  CD  E c o  >  O w o  IT)  CD  ma  O  E n  CO  CD  o  i  c 0  co"  c  nd  ffe  o i-  CD CM  4—'  c  •tf  o  •tf  CD O hlO  c  o  CO  E o O  •tf  o  c o •2  1  •tf o  CO C ZI  CO  i n  CJ  "D  LO _0  i  o O  co  ^~  =^ «=! §> D) O 1  00  LO  CO CQ  C CD  o  CO  CQ  co  Ve  c  05  CVJ  CD  ed  CO  SO  AO 0 3  o  •tf  co  CO  o  CM CM  CO  1  CO  1  CD  c  o  o  t o  pe  o  CO -Q  LO  2 CD  o CL E o O  CD O H Lf) •tf  co"  CD >  O Hin •tf" co"  IJ  1I  I  » o a S -5 n •= <S IS -2  •3  M  148  Semi-static  laboratory  bioconcentration  data  is  that future  low level  conditions.  Further,  recommended  flow  through  relationship generate  at  bioassays  between  a  more  water  realistic  flow  regimes.  Direct  other  aquatic  organisms  between  leeches  biomonitoring program,  and  Evidence  contaminant (0.1  current  laboratory  could  be  it  individuals of  from  the  bioavailability  rate  model  and  leech  body  through  age  and  an i n d i c a t i o n o f effectiveness  reduction,  such  as  information  on  the  reduced relative  chlorine proportion  in  of  a  data  pg/L.  to  out  in  bioavailability  For  application of  a  to  differing  uptake  routine  the  order  from  It  under  quantitate  comparative  that  leeches  rate  laboratory  in  are  Leeches variety  useful  B K M derived  o f m i l l process  use.  0.1  carried  situ data  establish  changes  Leeches also gave  be  leech  to  studies leech  breeding  size.  indicates  burden  about  bioconcentration,  bioconcentration  to  accurate  accurately  evaluate  desirable  study  to  and  species.  standard  below  p g / L ) studies  necessary  flow  be  present and  is  aquatic  would  provide  concentrations  achieved  representative  not  - 0.001  it  application of  programs  utilizing  seasonal  water  did  changes  provided  of  for  contaminants. aimed at A O X  time  contaminants  gauging  integrated  in  the  Fraser  River. The  potential exists  involving changes  a  series  in  system.  distribution  Further, In  the  case  assessment  of  concluded  that  in  a  the  this of  a more  test and  proportion  strategy  could  Canadian  effects  "effluents  of  bleached  from  pulp  concentration  effects  on  the  (CEPA)  under  (Environment  Regulations, environmental  under risk  the  Canada  11(a)  with  information  contaminants  pulp  a  Act  program,  concerning  throughout  mill  using  federal  an  bleach  conditions  the  bleached  pulp  1992, mill  the  entering immediate  therefore  Canadian  May  on  is  having  Amendments in  government  effluent  E f f l u e n t s . . . . are of  situ b i o m o n i t o r i n g  spatial  entire  river  a p p l i e d i n a national m o n i t o r i n g p r o g r a m .  1991a).  Fisheries  associated  of  mills  under  environment.  Paragraph  be  in  generating  B K M effluents,  or  defined  comprehensive  stations,  quantity  harmful as  of  for  Pulp  were effluent  receiving the  and  enacted  risk  waters  environment  and  considered  Environmental to  sponsored  long-term to  be  toxic  Protection  Act"  Paper to  discharge  Effluent  reduce  the  149  (Environment  Canada  the  of  acute  and  the  need  for  area  spawning regulations  in  Monitoring  Program  program  1992).  chronic a  protection  mechanism  of  aquatic  ( E E M ) for  monitor  environment  (Environment  Canada  provided  a  guidance  Each  is  monitored  the  in  order  under  to  assess  and  1992). at of  a  determine:  the  Federal  effluents  paper  National  both  of  effluents  Canada, and  the  provides  a  on  amended  nation-wide aquatic  guidance  regional  regional  Effects  the  and  in  1991a),  Environmental  coordination  provincial  identified  Canada  effectiveness  Environment  are  administration  federal  offices.  for c o n d u c t i n g the E E M .  set  the  mill  were  (Environment  The  paper  pulp  is responsible  E E M program,  federal  whole  and  that leeches  good  chemical  technologies, phenolics,  be  of  both  site-specific  magnitude,  be  when  other  into  aquatic  detoxification  spatial  with  simple  conduct,  other  be f o l l o w e d  for  and  extent  and  core  and  variables  temporal  part the  are  changes  observed  leeches  between  benthic  fish  larva  impacts  .  could  be  used  up b y specific t o x i c i t y  for  as  routine  testing.  It  proved  they  would  of  impacts,  bioconcentration  indicators  o f trouble  be  make  chlorinated  analysis.  bioassays  to  reducing  community  leech  here  changes.  chlorine  levels  both  proposed  and  leech  situ  is  contaminant of  both  phenolic  bioconcentration  invertebrate  Since in  of  includes  conventional  leech  toxicity),  effects  S i n c e leeches  temporal  companies  chlorinated  tests.  measurable low  paper  testing  implementation  too  (eg.  the  contamination,  and  bioconcentrate  relationship  activity,  bioassay  phenolic  are  on  the  E E M program.  after  &  Specific  spatial  useful  pulp  focuses  including  toxicity  measuring  can  individual  1991b).  concentrations the  by  Monitoring  chlorinated  organisms  enzyme  correlated to  monitoring  they  water  research  Canada  of  out  components,  especially  since  carried  supervision.  an integral  indicators  may  be  specific  situ m o n i t o r s  EEM  further  and  could  in  Leeches  to  (Environment  environmental  sensitive  is  provincial  effluent  compounds  may  of  responsibility  E E M program,  effluent  gaps  effects. The  on  the  to  and  E E M Office  pulp and paper m i l l Under  in  National  of  knowledge  ecosystems.  pulp  effects  by  extensive  effects  the  and  to  However,  are  With effects fish  could  be  relatively  areas,  which  150  The E E M program represents relative  hardiness  leeches  potentially  potential  to track down and identify  integral  part of  of  leeches useful  federal  chlorinated  organic  assessment  and  a single  as _ well as  indicators  regulatory  their of  strong many  biomonitoring.  bioconcentrating priority  impact  Leeches  enforcement.  may  assessments play  a  of  role  ability  pollutants,  suspected pollutant sources.  and provincial  discharges.  as  application of leech  Leeches  with  The make the  could be an  projects  with  in  environmental  both  potential  151  6. REFERENCES  Addison, R.F., M.E. Zinck, and J.R. Leahy (1976).  Metabolism of single and combined  doses of 14C-aldrin and 3H-p,p'-DDT by Atlantic salmon (Salmo salar) fry. J. Fish Res. Bd. Can., 33: 2073 - 2076.  Allard, A.S., M. Remberger, T. Viktor and A.H. Neilson (1988). chloroguaiacols and chloroguaiacols.  Allison, R.W. and J.S. Gratzl (1987). prior to alkaline pulping.  Anholt, B. (1986).  Wat. Sci. Tech. 20, 131-141.  Oxidative pretreatment with hydrogen peroxide  J. Wood Chem. Tech.  7, 285-309.  Prey selection by the predatory leech Nephelopsis obscura'm  relation to three alternative models of foraging.  Axegard, P. (1986).  Environmental fate of  Effect of C I O 2 dioxide  substitution  Can. J. Zool. 64, 649-655.  on bleaching efficiency  and the  formation of organically bound chlorine: Part II. J. Pulp Pap. Sci. 12, J67-J71.  Axegard, P., U . Jansson and A. Teder (1984). towards chlorine dioxide.  Barron, M . G . (1990).  The E2 stage improves reactivity of pulp  J. Pulp Pap. Sci. 10, 1.  Bioconcentration. Environ. Sci. Technol. 24(11), 1612-1618.  Barron, M.G., B.D. Tarr and W.L. Hayton (1987a).  Temperature dependence of cardiac  output and regional blood flow in rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 31, 735-744  152  B a r r o n , M . G . , B . D . Tarr and W . L . H a y t o n (1987b). ethylhexyl Appl.  phthalate  Pharmacol.  88,  B . C . Environment (1992). Hazardous  (DEHP)  Temperature dependence o f d i - 2 -  pharmacokinetics  in rainbow  trout.  Toxicol.  305-312.  P u l p M i l l A O X R e d u c t i o n Plans.  Contaminants  Branch, B . C . Ministry  Industrial Waste  o f the  and  Environment,  Victoria  B.C.  B o u l e , P . , C . G u y o n and J. L e m a i r e (1982). photochemical Chemosphere  behaviour 11,  of  Phtochemistry  monochlorophenols  in  and environment dilute  IV -  aqueous  solution.  1179-1188.  B r a n n l a n d , R . , L . L i n d s t r o m and S. N o r d e n (1989).  Implementation  i n full  scale  - the  (R)  next step for P R E N O X X Pulping  Section,  In the Proceedings  1989 T a p p i  Conference;  195-120. ,  B r e z n y , R . , T . W . Joyce and B . Gonzales (1992). chloroaromatic  o f the  compounds  related  Biotransformation o f s o i l  to bleach  plant  effluents.  Wat.  Sci. Tech.  26,  397-405.  Butte,  W . (1991). of  Mathematical  description  xenobiotics i n a fish/water  contributions Weinheim  to the  Federal  C a r e y , J. H . (1988). Columbia.  o f uptake,  system.  accumulation  and  elimination  In B i o a c c u m u l a t i o n i n A q u a t i c  Systems;  assessment, R . N a g e l and R . L o s k i l l E d . , V C H p u b l i s h i n g Republic  Pathways  In Proceedings  of  Germany.  o f chlorophenols o f the  Canada  i n the Fraser  R i v e r Estuary,  British  - B r i t i s h C o l u m b i a W o r k s h o p on  Water  Q u a l i t y Guidelines and Objectives: Focus on the Fraser  1988; M a c D o n a l d , D . D .  Ed.;  Yukon  B.C.  Envionment  Canada,  Inland  Waters,  Pacific  and  Region,  Vancouver,  153  Carey, J.H. M.E. Fox, B.G.Brownlee, J.L. Metcalf and R.F. Platforn (1984). Disappearence kinetics of 2,4- and 3,4-dichlorophenol in a fluvial system. Can. J. Physiol, and Pharm. 62, 971-975.  Carey, J.H. and J.H. Hart (1988).  Sources of chlorophenolic compounds to the Fraser  River Estuary. Wat. Poll. Res. J. Can. 23, 55-68.  Carey, J.H. M.E. Fox and J.H. Hart (1988).  Identity and distribution of chlorophenols  in the North Arm of the Fraser River Estuary.  Celgar Pulp Company (1990).  Wat. Poll. Res. J. Can. 23, 55-68.  Proposed modernization of bleached softwood kraft mill,  Castlegar, British Columbia, Stage II Report, submitted in compliance with the British Columbia Major Project Review Process and Canadian Federal Environmental Assessment  Connell, D.W. (1991). groups.  and Review Process, July  1990.  Extrapolating test results on bioaccumulation between organism  I n Bioaccumulation i n Aquatic Systems: contributions to the  assessment, R. Nagel and R. Loskill Ed., V C H publishing Weinheim Federal Republic of Germany.  Dainty, J. and C R . House (1966).  "Unstirred layers" in frog skin. J. Physiol. 182, 66-78.  Davies, R.W.and R. P.Everett (1977). Nephelopsis  obscura\em\,  The life history, growth and age structure of  1872 (Hirudinoidea) in Alberta.  Can. J. Zool. 55,  620-627.  Davies, R.W., T. Yang and F.J. Wrona (1987).  Inter- and intraspecific differences in  the effects of anoxia on erpobdellid leeches using static and flow through systems.  Holarctic Ecol. 10, 149-153.  154 Dwernychuk, L. W., G.S. Bruce, B. Gorden and G.P. Thomas (1991). Thompson Rivers: A Comprehensive  Organochloririe Study  Fraser and 1990/91. Hatfield  Consultants Limited, West Vancouver, B.C.  Dwernychuk, L. W.and D. Levy (1994). monitoring  Upper Fraser River environmental  (EEM) pre-design reference document.  Hatfield  effects  Consultants  Limited, West Vancouver, B.C.  Earl, P.F. and D.W. Reeve (1989). pulp production.  Chlorinated organic matter in bleached chemical  Tappi 72, 183-186.  Ellgehausen H., J.A. Guth and H.O. Esser (1980). bioaccumulation  potential  Factors determining the  of pesticides in the individual compartments of  aquatic food chains. Ecotox. Environ. Saf. 4, 134-157.  Environment Canada, (1991a).  Canadian Environmental Protection  Act; Priority  Substances List Assessment Report No. 2, Effluents from Pulp Mills Using Bleaching.  Minister of Supply and Services Canada Cat. No. En 40-215/2e, ISBN  0-662-18734-2.  Environment Canada, (1991b). Environmental Effects and Study Design. Limited,  Technical Guidance Manual for Aquatic  Monitoring at Pulp and Paper Mills, Volume 1: Overview  Prepared for Environment Canada by BEAK  Consultants  Brampton, Ontario.  Environment Canada, (1992). Requirements.  Aquatic Environmental Effects  Monitoring  Minister of Supply and Services Canada Cat No. EPS l/RM/18.  155  Ernst, W. S. Weigelt, H. Rosenthal and P.D. Hansen (1991).  Testing bioconcentration of  organic chemicals in the common mussel (Mytilus edulis). In Bioaccumulation in Aquatic Systems: contributions to the assessment, R. Nagel and R. Loskill Ed., V C H publishing Weinheim Federal Republic of Germany.  Frederick, L . L .  (1975).  Comparitive uptake of a polychlorinated biphhenyl and  dieldrin by the white sucker (Catastomus commersoni). J. Fish Res. Bd. Can., 32: 1705 - 1709.  Gergov, M . , M. Priha, E Talka, O. Valttila, A Kangas and K Kukkonen (1988). Chlorinated organic compounds in effluent treatment at kraft mills.  Tappi J.  71, 175-184.  Gibson S.A. and J.M. Suflita (1990). trichlorophenoxyacetic  Anaerobic biodegradationof 2,4,5-  acid in samples from a methanogenic  aquifer:  stimulation by short chain organic acids and alcohols. Appl. Environ. Microbiol. 56, 1825-1832.  Grimvall, A., S. Jonsson, S. Karlsson, R. Savenhed and H. Boren (1991).  Organic  halogen in unpolluted waters and large bodies of water receiving bleach plant effluents.  Tappi 74, 197-203.  Hagbloom.M.M., J.H.A. Apajalhti and M.S. Salkinoja-Salonen (1988).  Degredation of  chlorinated phenolic compounds occuring in pulp mill effluents.  Wat. Sci.  Tech. 20, 205-208.  Hall, K. J. and C. Jacob (1988).  Bioconcentration of chlorophenols by leeches and  their use as in situ biological monitors.  Wat. Poll. Res. J. Can. 23, 69-87.  156  H a l l , K . J . , H . Schreier and S. J. B r o w n (1991). In W a t e r Fraser  i n Sustainable  River  University  Water quality i n the Fraser R i v e r basin.  D e v e l o p m e n t : E x p l o r i n g O u r C o m m o n Future  of British  methyl  pomaceus.  Columbia, Vancouver, B . C .  donor  Appl.  for  biosynthesis  Environ.  Microbiol.  H a w k e r , D . W . and D . W . C o n n e l l (1986). some aquatic organisms.  o f esters 55,  and  anisoles  Chloromethane, a in Phellinus  1981-1989.  B i o c o n c e n t r a t i o n o f l i p o p h i l i c compounds  E c o t o x . E n v i r o n . Saf.  11,  plant  effluents:  environmental  recent  impact: Part  developments  aimed  at  plant  effluents:  environmental  H e r r m a n n , S.J. (1970).  Isnard,  P.  and  water  S.  Lambert  partition  Jacob C . (1986).  aimed  at  Kraft  decreasing  mill  their  69-78.  1-37.  (1988).  coefficient  and  E s t i m a t i n g bioconcentration aqueous  p o l l u t i o n . M S c . thesis,  of British  their  distribution, and e c o l o g y o f C o l o r a d o H i r u d i n e a .  Use o f bioconcentration  chlorophenol University  developments  II. T a p p i J . 7 1 ,  Systematics,  A m . M i d l a n d Nat. 83,  mill  I. T a p p i J . 7 1 , 51-59.  recent  impact: Part  Kraft  decreasing  Heimburger, S . A . , D . S . B l e v i n s , J . H . B o s t w i c k and G . P . D o n n i n i (1988b). bleach  by  184-197.  Heimburger, S . A . , D . S . B l e v i n s , J . H . B o s t w i c k and G . P . D o n n i n i (1988a). bleach  the  B a s i n : Dorcey, A . H . ; G r i g g s , J. R . E d . ; Westwater Research Centre,  H a r p e r , D . B . , J . T . G . H a m i l t o n , J.T. K e n n e d y and K . J . M c N a l l y (1989). novel  in  solubility.  factors  Chemosphere  from 17,  capability o f leeches to evaluate Department  Columbia, Vancouver B . C .  of Civil  Engineering,  octanol21-34.  157  Jaffe, R. (1991). a review.  Fate of hydrophobic organic pollutants in the aquatic environment: Environ. Poll. 69, 237-257.  Jiminez, B.D., C P . Cirmo and J.F. McCarthy (1987).  Effects of feeding and temperature  on uptake, elimination and metabolism of benzo(a)pyrene in the bluegill sunfish  (Lepomis  macrochirus) Aquatic Toxicol. 10, 41-57.  Kauss, P.B. and Y.S. Hamdy (1985).  Biological monitoring of organochlorine  contaminants in the St. Clair and Detroit rivers using introduced clams, Elliptio  complanatus. J. Great Lakes Res.  Kennedy, C.J. (1989).  11: 247 - 263.  Toxicokinetic studies of chlorinated phenols and polycyclic  aromatic hydrocarbons in rainbow trout (Onchorhynchus  mykiss). PhD thesis,  Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.  Karickhoff, S.W., D S . Brown and T.A. Scott (1979). pollutants on natural sediments.  Kocurek, M.J. (1989).  Sorption of hydrophobic  Wat. Res. 13, 241-248.  Pulp and Paper Manufacture, third ed.. Vol. 5. Alkaline Pulping.  Joint textbook committee of the paper industry, Tappi, Atlanta, Georgia U.S.A. 30348-5113.  Kringstad, K. P., K. Lindstrom (1984).  Spent liquors from pulp bleaching.  Environ.  Sci. Technol. 1984, 18, 236A-249A.  Kukkonen, J. and A. Oikari (1991).  Bioavailability of organic pollutants in boreal  waters with varying levels of dissolved organic material.  Wat. Res. 25, 455-463.  158 Lee, S.K., D. Freitag, C. Steinberg, A. Kettrup and Y.H. Kim (1993).  Effects of dissolved  humic materials on acute toxicity of some organic chemicals to aqutic organisms.  Wat Res. 27, 199-204.  Liebergott, N., B. van Lierop, A. Nolin and T. Kovacs (1990).  A comparison of the order  of addition of chlorine and chlorine dioxide in the chlorination stage.  Tappi J.  73, 207-213.  Lindeman, V.F. (1935). leech  (Hirudo  The relation of temperature to respiratory regulation in the  medicinalis). Physiol. Zool. 8, 311-317.  Lindqvist, B., A. Marklund, L - A . Lindstrom and S. Norden (1986).  Nitrogen dioxide  preoxidation before oxygen delignification - a process for the future?  J. Pulp  Pap. Sci. 12, J161-J165.  Linton, L.R., R.W. Davies and F.J. Wrona (1983).  The effects of water temperature,  ionic content and total dissolved solids on Nephelopsis punctata(Hirudinoidea,  Mackay, D. (1982).  Erpobdellidae), 1 Mortality,  Correlation of bioconcentration factors.  obscuraand  Erpobdella  Holarctic. Ecol. 6: 64 - 68.  Environ. Sci. Technol. 16,  272-278.  McCubbin, N. (1983). environmental  The basic technology of the pulp and paper industry and its protection  practices.  Environmental Protection  Service,  Environment Canada, EPS report # 6-EP-83-1.  McCubbin, N. (1984). environmental  State of the art of the pulp and paper industry and its protection  practices.  Environmental Protection  Environment Canada, EPS report # 3-EP-84-2.  Service,  159  McCubbin, N., J.B. Sprague and N. Bonsor (1990).  Kraft mill "effluents in Ontario.  Pulp  Pap. Can. 91, T110-T113.  McKim, J.M. and P.K. Schmieder (1991).  Bioaccumulation does it reflect toxicity?  In  Bioaccumulation in Aquatic Systems: contributions to the assessment. R. Nagel and R. Loskill, Ed. V C H publishing Weinheim Federal Republic of Germany.  McLeay, D. and Associates (1987).  In Toxicity of Pulp and Paper Mill Effluent: A  Review 1987, EPS Report EPS4/PF/1.  Metcalf, J. L., M.E. Fox and J.H Carey (1984).  Aquatic leeches (Hirudinea) as  bioindicators of organic chemical contaminants Chemosphere 13,  in freshwater  ecosystems.  143-150.  Metcalf, J. L . , M.E. Fox and J.H. Carey (1988). screening tool for detecting  Freshwater leeches (Hirudinea) as a  organic contaminants  in the  environment.  Environ, Monitor. Assess. 11, 147-169.  Metcalf, J. L . and A. Hayton (1989). for chlorophenol pollution.  Comparison of leeches and mussels as biomonitors  J. Great Lakes Res. 15, 654-668.  Morales, A., D.A. Berkholz and S.E. Hrudy (1992). contaminants compounds.  NCASI (1989).  in water,  sediment,  Analysis of pulp mil effluent  and fish muscle-chlorophenols  and related  Wat. Environ. Res. 64, 669-681.  Pulping effluents in the aquatic environment, part I: a review of the  published literature.  NCASI Tech. Bull. # 572.  National Council of the Pulp and  Paper Industry Air and Stream Improvement, New York, N.Y., U.S.A.  160  N e e l y , W . B . , D . R . Branson and G . E . B l a u (1974). bioconcentration  potential  o f organic  Partition coefficient to measure  chemicals  to fish.  E n v . S c i T e c h n o l . 8,  1113-1115.  N e i l s o n , A . H . , A . A l l a r d , S. R e i l a n d , M . Remberger, A . T a r n h o l m , T . V i k t o r , and L . Landner,  (1984).  bacterial  O-methylation  their  T r i - and  bioconcentration  of  tetra-chloroveratrole, tri-  potential  metabolites  produced  and  tetra-chloroguaiacols:  an  and  their  reproduction.  effects  on  fish  by  assessment  of Can. J.  F i s h . Aquat. S c i . 4 1 , 1502-1512.  N e i l s o n , A . H . , A - S . A l l a r d , C . L i n d g r e n and M . Remberger chloroguaiacols, anaerobic  chloroveratroles  bacteria.  Appl.  and  Environ.  (1987).  chlorocatechols Micrbiol.  53,  by  Transformations  stable  consortia  of  of  2511-2519.  N e i l s o n , A . H . , H . B l a n c k , L . B r o l i n , L . Landner, P . Part, A . R o s e m a r i n and M.  Soderstrom (1989).  ed.),  In: C h e m i c a l s  Springer-Verlag,  of  Aquatic  A new perspective  c h l o r o l i g n i n degredation  T e c h n o l . 26,  the  chlorinated  (sorption/desorption)  phenolics.  rainbow  pulp trout  mill (Salmo  effluents  on  gairdneri).  the  respiration  t o x i c i t y o f a simulated  (Salmo  trutta  M.  lacustris).  Sublethal effects  and  the  Sci.  energy  of  metabolism  of  E c o t o x . E n v i r . Saf. 9, 378 - 384.  O i k a r i , A . , P. L i n d s t r o m - S e p p a and J . K u k k e n o n (1988). and  Environ.  on  556-560.  O i k a r i , A . , M . N i k i n m a a , S. L i n d g r e n and B . L o n n (1985). simulated  Environment (L.Landner  Berlin.  O ' C o n n e r , B . I . and R . H . V o s s (1992). question  in  bleached  pulp m i l l  S u b c h r o n i c metabolic  effluent  on j u v e n i l e lake  E c o t o x . E n v i r o n . Saf. 16, 202-218.  effects trout  161  O p p e r h u i z e n , A . and R . C . A . M . the  bioaccumulation  Environ. Poll.  51,  S t o k k e l (1988).  of  hydrophobic  Influence  organic  o f contaminated  micropollutants  chains.  of  on  fish.  165-177.  Paasivirta, J . , J. S a r k k a , T. L e s k i j a r v i , and A . R o o s (1980). enrichment  in  particles  chlorinated  Chemosphere  9,  phenolic  compounds  in  Transportation different  and  aquatic  food  441-456.  Paasivirta, J., K . H e i n o l a , T. H u m p p i , A . K a r l j a l a i n e n , K . K n u u t i n e n , K . M a n t y k o s k i , R . P a u k k u , T . P i i l o l a , K . S u r m a - A h o , J.Tarhanen, (1985).  P o l y c h l o r i n a t e d phenols,  environment.  Chemoshpere,  Peterson, D . L . (1983).  and  catechols  and H . V i h o n e n in  Erpobdellidae)  Freshwat. Invertebr.  P h i l l i p s , D . H . (1978).  the  469-491.  L i f e c y c l e and reproduction o f Nephelopsis  (Hirudinoidea,  organochlorine  14,  guaiacols  L . Welling  in  permanent  ponds  of  obscura  Verrill  northwestern  Minnesota.  B i o l . 2: 165 - 172.  Use o f b i o l o g i c a l indicator organisms to quantitate pollutants  in  aquatic  environments:  a  review.  Environ.  Poll.  16, 167-229.  Presley,  J . R . (1990).  Bleach  plant  faces  organic halides. P u l p . Pap! 64,  P r y k e , D . C . (1989).  230.  environmental  hurdle  in  adsorbable  252-255.  Substituting chlorine d i o x i d e for c h l o r i n e . T a p p i J. 72,  R e d d y , D . C . and R . W . D a v i e s (1993). obscura  new  M e t a b o l i c adaptations by the leech  during long-term a n o x i a and recovery.  147-155.  Nephelopsis  J Experiment. Z o o l .  265,  224-  162 Remberger, M . , A. Allard and A . H . Neilson (1986). chloroguaiacols,  chlorocatechols,  Biotransformations of  and chloroveratroles  in sediments.  Appl.  Environ. Microbiol. 51, 552-558.  Remberger, M . , P-A. Hynning and A.H. Neilson (1993).  Release of chlorocatechols  from a contaminated sediment. Environ. Sci Technol. 27, 1158-164.  Renberg, L . , O. Svanberg, B. Bengtsson and G. Sundstrom (1980). guaiacols alburnus, Nitocra  Chlorinated  and catechols bioaccumulation potential in bleaks (Alburnus Pisces) and reproductive and toxic effects on the harpacticiod spinipes (Crustacea).  Chemosphere 9, 143-150.  Rogers, I. H., J.A. Servizi and C D . Levings (1988a). chlorophenols  by juvenile  Bioconcentration of  chinook salmon (Oncorhynchus  tshawytscha)  overwintering in the Upper Fraser River: field and laboratory tests.  Water  Poll. Res. J. Can. 23, 100-113.  Rogers, I. H., I.K. Birtwell and G.M. Kruzynski (1988b). in eulachons  (Thaleichthys  pacificus)  Organic contaminant uptake  migrating through the Fraser River  Estuary. In: (D.D. MacDonald ed.) Proceedings of the Canada - B.C. workshop on water quality guidelines & objectives; focus on the Fraser, Nov. 16-17, 1988. Env. Can. Vancouver B.C.  Saarikoski, J. and M. Viluksela (1981). phenols to fish.  Influence of pH on the toxicity of substituted  Arch. Environ. Contam. Toxicol. 10, 747-753.  Saarikoski, J. and M . Viluksela (1982).  Relation between physiochemical properties  of phenols and their toxicity and accumulation in fish. 501-512.  Ecotox. Environ. Saf. 6,  163  Saarikoski, J., R. Lindstrom, M . Tyynela and M . Viluksela (1986). absorption of phenolics reticulata).  Factors affecting the  and carboxylic acids in the guppy (Poecilia  Ecotox. Environ. Saf, 11, 158-173.  Sawyer, R.T. (1972).  North American freashwater leeches, exclusive of the  Piscicolidae, with a key to all species.  Sawyer, R.T. (1989).  111. Biol. Monogr. No. 46.  Leech Biology and Behaviour Vol. 1. Oxford Science Publication,  Schellenberg, K., C. Leuenberger and R.P. Schwarzenbach, R. P. (1984). chlorinated phenols by natural sediments and aquifer materials.  Sorption of Environ. Sci.  Technol. 18, 652-657.  Schreier, H., S.J. Brown and K.J. Hall(1991). River Basin. In Water in Sustainable  The land water interface in the Fraser Development:Exploring Our Common  Future in the Fraser River Basin: Dorcey, A. H.; Griggs, J. R. Ed.; Westwater Research Centre, University of British Columbia, Vancouver, B.C.  Schwarzenbach, R. P. (1985).  Sorption behaviour of neutral and ionizable  hydrophobic organic compounds.  In Organic Micropollutants in the Aquatic  Environment: Bjorseth, A.; Angeletti, G. Ed.; D. Reidal Publishing Co. Boston Mass.U.S.A., 1985. p 168-177.  Servizi, J.A., R.W. Gordon and J.H. Carey (1988).  Bioconcentration of chlorophenols  by early life stages of Fraser River pink and chinook salmon (Oncorhynchus gorbuscha, 0.  tshawytscha). Wat. Poll. Res. J. Can. 23, 88-99.  164 Sijm, D.T.H.M. (1991).  Extrapolating laboratory results to environmental  In Bioaccumulation in Aquatic Systems: contributions  conditions.  to the assessment. R.  Nagel and R. Loskill, Ed. V C H publishing Weinheim Federal Republic of Germany.  Sinclair, W.F. (1990).  Controlling pollution from Canadian pulp and paper  manufacturers: a federal perspective.  Environment Canada, Minister of  Supply and Services, Ottawa, Canada. Cat. # En40-384/1990F.  Singhel, R.N., R.W. Davies and M . Kapoor (1990).  Undetectable levels of superoxide  dismutase activity in the leech Nephelopsis  obscuraunder hyperoxia.  Arch.  Hydrobiol. 119, 351-358.  Spry, D.J., C M . Wood and P.V. Hodson (1981).  The effects of acid on freshwater fish  with particular reference to the soft water lakes lakes on Ontario and the modifying effects of heavy metals.  Can. Tech. Rep. Fish. Aquat. Sci. No. 999.  Starck, B., P.O. Bethge, M . Gergov and E. Talka (1985). phenols in pulp mill effluents.  Stehly, G.R. and W.L. Hayton (1990). pentachlorophenol in goldfish.  Pap: Puu. 12, 745-749.  Effect of pH on the accumulation kinetics of Arch. Environ. Contam. Toxicol. 19, 464-470.  Suntio, L.R., W.Y. Shiu and D. Mackay (1988). chemicals present in pulp mill effluents.  Tana, J.J. (1988).  Determination of chlorinated  A review of the nature and properties of Chemosphere 17, 1249-1290.  Sublethal effects of chlorinated phenols and resin acids on rainbow  trout (Salmo gairdneri). Wat. Sci. Tech. 20, 77 - 85.  165  Tessier, A . , P . G . C . C a m p b e l l , J.C. A u c l a i r and M . B i s s o n (1984). partitioning of  the  o f trace  freshwater  metals i n sediments  m o l l u s c Elliptio  and  their  complanatain  Relationships  accumulation  between  i n the  tissues  a m i n i n g area. C a n . J. F i s h .  Aquat. S c i . 4 1 : 1463 - 1472.  Tratnyek, P . G . and J . H o l g n e (1991). environment: oxygen.  A QSAR  O x i d a t i o n o f substituted  analysis  o f rate constants for  E n v i r o n . S c i . T e c h n o l . 25,  T r i n h , D . T . and R . H . Crotogino (1987). fibers during washing.  factor  reaction  with  singlet  1596-1604.  The rate o f solute removal from kraft  pulp  J. P u l p Pap. Soc. 13, J126-J127.  V e i t h , G . D . , D . L . Defoe and B . V . Bergstedt (1979). bioconcentration  phenols i n the  o f chemicals i n fish.  M e a s u r i n g and estimating  the  J. F i s h . R e s . B o a r d C a n . 36(8),  1040-1048.  V o i c e , T . C . , C P . R i c e and W . J . Weber (1983). partitioning Technol.  of hydrophobic  17,  pollutants  Effect o f solids concentration on sorptive in  systems.  Environ. Sci.  513-518.  von Oepen, B . W . K o r d e l and W . K l e i n (1991). processes,  aquatic  measurements  OECD-Guideline  106.  and  Sorption o f nonpolar and polar to soils:  experience  Chemosphere  with  the  applicability of  i n kraft  bleachery  effluents.  Pap. Puu  12,  Chlorinated  809-814.  V o s s , R . H . , C E . Luthe, B . I . H e m m i n g , R . M . Berry and L . H . A l l e n (1988). insights  into  the  o r i g i n s o f d i o x i n s formed  P u l p Pap. C a n . 89, T 4 0 1 - T 4 1 0 .  modified  22, 285-304.  V o s s , R . H . , J.T. W e a r i n g , R . D . M o r t i m e r , T K o v a c s and A W o n g (1980). organics  the  during  chemical pulp  Some new bleaching.  166  W e b b P . W . and J . R . Brett (1973). pentachlorophenate swimming  on  Effects o f sublethal concentrations  growth  performance  in  rate,  food  underyearling  conversion salmon  of sodium  efficiency  and  (Oncorhynchus  nerkd).  J.  F i s h . R e s . B o a r d Canada, 30, 491-507.  W e s t a l l , J . C . (1985). distribution  Xie,  T . (1983). in  Influence  o f p H and i o n i c strength  o f chlorinated  Determination  sediment.  phenols.  Environ.  Sci. Technol.  o f trace amounts o f chlorophenols  Chemosphere  12,  pentachlorophenol  sorption  on  Chemosphere  1311-1324.  aqueous-nonaqueous 19,  193-198.  and  chloroguaiacols  1183-1191.  X i n g , B . , W . B . M c G i l l and M . J . Dudas (1993).  26,  on the  T h e r m o d y n a m i c parameters for  montmorillonite  in  aqueous  suspensions.  167  PART  7 : APPENDICES  168  Appendix 1  Leech Distinguishing features useful obscura  Identification  in the identification  and Percymoorensis  marmorata.  of the leech  Information  species  from  Nephelopsis  North  American  Freshwater Leeches Exclusive of the Piscicolidae: with a Key to all Species (Sawyer 1972).  1) Nephelopsis  Description:  obscuraNemW  1872  (Family Erpobdellidae)  Length: 5 - 6 cm. Weight: 0.1 - 2.0 g. Colouration: light brown to dark brown with irregularly scattered black splotches dorsally and less commonly ventrally. Distinguishing  anatomical  a) External features:  features: sub-division of most mid-body annuli.  Male and female gonopores separated by two annuli, with pores usually located in furrows (Figure A l ) . b) Internal features:  spirally coiled atrial cornua, pre-atrial loop  of the vas deferans, extending to ganglion XI. Similar species: Erpobdella punctata, Dina dubia  Figure A l :  Relative positions of male and female gonopores for:  1) Nephelopsis  obscura  2) Erpobdella punctata  3) Dina dubia  169  1) Percymoorensis species  is  examination  very  difficult  only.  reliable method  Description:  (Haemopsis)  marmorata.  to  distinguish  Identification  (Family from  by dissection  Hirudinidae).  Haemopsis  and i n t e r n a l  o f i d e n t i f i c a t i o n and i s best conducted  Note  grandis  by  examination  b y a recognized  that  this  external  is  the  only  expert.  Length: 2 - 8 cm. Weight: 1 - 5 g. Colouration:  Highly  variable.  sparse dark  splotches  colouration. Distinguishing  Mottling  anatomical  occurs  Male  to five  and h a l f  the  anterior  third  Internal  positioning  features:  Anterior  o n both  part  and female  The posterior  usually Distinguishing  black  and v e n t r a l  gonopores  o f their may  respective  separated b y  annuli,  however  o f prostate  gland  and slender  located  penis.  between  begins  behaviour:  g a n g l i o n X I I and XIV1/2 ( u s u a l l y  extension  o f the v a g i n a l system  located The intestine  amphibious  habits,  often  found  under  rocks and logs at the waters  Ventures on to shore i n search o f prey at night. flaccid;  drapes  up. species:  Haemopsis  XIII3/4 ).  at g a n g l i o n X I X .  partially submerged  Similar  ganglion  Center o f the ovaries  g a n g l i o n X V and X V I I (usually X V I ) .  extremely  surfaces.  vary.  extraordinarily long  between  between  dorsal  almost  with  a n n u l i , with pores u s u a l l y located on  XIIIi/4 and X V i / 2 (usually X I V ) . located  with  green  features:  five  actual  light olive  to h e a v i l y mottled  a) E x t e r n a l features:  b)  R a n g i n g from  grandis  limply  over  fingers  edge. Body  when  picked  170  A P P E N D I X . 2  CALCULATION OF REPORTED CHLORINATED PHENOLICS CONCENTRATIONS  Calculation equations  of  chlorinated  adapted  chlorinated  a) Response  Morales  phenolic  analyzed  factors  ( R F ) o f the  (1)  RF  S  (2)  RF  I S  Where:  from  phenolic  T  D  et  during  concentrations al.  1992.  this  were Target  based  on  compound  following  refers  to  target c o m p o u n d  standard  and  the  internal  standard  = Peak A r e a of S T D / [STD]  =  r  e  s  P  o  n  s  e  factor  o f the  target c o m p o u n d  or  surrogate  [ S T D ] = concentration o f target compound or surrogate reference I S  =  i n the G C  standard  response  factor  [IS] = concentration  of  internal  o f the  standard  internal standard  i n the  GC  reference  standard  b) R e l a t i v e response  (3)  Where:  RRF= R F  S  T  D  /  ( R R F ) o f the  RF  target c o m p o u n d  to the  internal  standard  to internal  standard  I S  R R F = relative response  c) C o n c e n t r a t i o n  (4)  factor  o f target c o m p o u n d  factor  o f test c o m p o u n d  [ A ] i n the  original  sample  [A] = ([IS] / R R F ) * ( P e a k A r e a o f A / Peak A r e a of IS) *(Final  Extract Volume  any  project.  = Peak A r e a o f IS / [IS]  ^ S T D  RF  the  / Original  Sample W e i g h t or V o l u m e )  :  171  APPENDIX 3  Raw Data  Laboratory Bioassays  172  Table A1: Bioconcentration of chlorinated phenolics by leeches (N. obscura) as a function of contaminant concentration at T= 12.5 °C and pH = 7.5.  w  a  t  e  Tissue Concentation (ng/kg)  r  Cone. (ug/L)  Leech wt.  1.460  ND  9  0.1  1.430  ND  13  1.240  ND  14  27  (9)  45-DCG  345-TCG  4,5,6-TCG  TeCG  345-TCVR  23  16  2  30  22  1  23  9  0.749  ND  15  42  28  7  0.721  ND  24  55  37  ND  ND  15  35  25  5  5  1 2  7  4  Mean  1.12  Sid Dev.  0.36  1.290  57  30  128  70  18  0.5  1.050  63  31  1 33  79  15  0.590  123  74  217  174  13  1.580  48  24  113  59  17  1.360  59  30  128  72  1 5  Mean  1.17  70  38  144  91  16  Std D e v .  0.38  30  18  37  42  2  0.861  179  95  310  247  21  1.0  0.962  210  99  337  254  49 47  1.770  130  57  293  165  1.650  142  63  315  172  63  0.912  249  98  371  257  ND  Mean  1.23  182  82  325  219  45  Std D e v .  0.43  44  18  27  41  18  1.230  441  194  842  646  223  5.0  1.220  493  249  964  807  118  0.920  603  257  856  724  244  1.750  304  135  615  419  252  1.300  430  199  785  594  194  Mean  1.28  454  207  812  638  206  Sid Dev.  0.30  97  44  1 14  131  48  1.220  872  449  1710  1420  340  10.0  1.020  1150  570  2240  1880  403  0.956  1680  892  3340  2930  319  0.803  1280  596  2210  1810  383  1.150  756  335  1620  1240  302  Mean  1.03  1150  568  2240  1860  349  Std D e v .  0.16  330  187  612  590  38  173  Table A 2 : The depuration of chlorinated phenolics from leeches (N. obscura) over a 28 day period after seven days exposure to a concentration of 1.0  ug/L at a water temperature of 1 2 ° C and water pH 7.5.  Samples taken on day 0, 3 (72 h ) , 7 (168 h.), and 28 (672 h.) after seven day exposure. Results reported on a wet weight basis.  Raw Tissue Concentration  Depuration Time  Leech Wt.  (h)  (g) 0.861  (ng/kg)  4,5-DCG  3,4,5-TCG  4,5,6-TCG  TeCG  3,4,5-TCVR  179  95  310  247  21 49  210  99  337  254  1 .77  1 30  57  293  1 65  47  1.65  142  63  315  172  63  0.912  249  98  371  257  ND  Mean  1 .231  182  82  325  219  45  Std. Dev.  0.441  44  1 8  27  41  1 8  1.350  93  40  244  128  1 1  1 .540  75  31  216  98  1 4  1 .450  91  34  222  107  15  91 147  38 59  238 297  116 179  ND  1 .358  99  40  243  126  1 3  0.228  28  1 1  32  32  2  1.240  11 5  50  228  146  4  1 .350  135  61  300  184  1 9  0.890  1 99  66  299  212  1 3  1.290  108  45  244  149  8  1 .080  136  58  301  198  7  Mean  1 .170  139  56  274  178  1 0  Std. Dev.  0.186  36  8  36  29  6  0.468  227  87  358  287  ND  0.616  147  65  262  202  ND  0.864  1 25  49  236  168  ND  78  283  216  ND  173  ND ND  0  72  0.962  1.480 0.970 Mean Std. Dev.  1 68  672  0.538  174  0.66  161  64  252  Mean  0.629  167  69  278  209  Std. Dev.  0.150  38  48  48  1 5  1 2  174  + ^  cc  > o  +- CJ)  1-  A  "a  C3  o o  H CO  "lis  c o o cu Z3 V) TJ <1) J=  £ o  co o z a.  CM CM  T-  T-  CO L O CM O) CM  CNJ CM  LO i - CO if) CM  (!)  W  CO CM O) CO CM CM CM CM CM  LO iD CM  CM O C> CO l O (D N CO CM CV] CO  CO CO CM . LO LO  r*-  CM  CM CM  o  CD CM  in"  G O H  CO  00  CM  CO CD io  2  00 N i -  O  O)  CO  CO C O  1  CD IO  co  LO  N  co  in •tf m"  00  CD O  CM  o  CD  Tf  LO  Tf LO  LO  CO CO  CO CO CM  CM CO CO  T-  O  o  CM CM CM CM  LO CD CO CO CO CD Tf Tf Tf Tf  LO  2 "a»  CD  CO COi LO T  LO O O  T  f—  CM CM CM CM I  CM  r-  co  •H  LO  CM  r»- LO Tf CO  w o CM CM  CO Tf  E 8  O  c o  "5 c  ^3) <  ^ 1=  CO  < - CO CJ CD N CO (D Tf Tf ""fr Tf  O  i?  S-DCC  3~  »-  LO CD  CO  CO  ra £ TI « o co  O  •et  CO  Tt~  ii CO  O  CM ^  "a 3  CO CM l O  Tf  CO CM  cn  LO  CM  n -r-  T—  CM CO LO  CM CO CD -tf , i - CM CO CM I CO CM CM CO  r—  T— T—  If) S S ^ CO CD Tf LO CM CM CM  CO  CM i CD CO CO CO CM t- -r-  Q  Q  Q  r*-  CD  CM  Q  O  <t  Q  IS  LO Tf Tf CO CM T CO CM CM CO  N CM  CM  CO o CO CM Tf i CM CO  CD  LO N T - CD CD CM CM CO  O) CD hCD CD CM CM  CO CO O  N  t  O  r-.  co  r-  CM  s  co  s s  co O) V  o  CD Q_  - ° b oo ^ cd C 0)  tj (D "D  CD  * " *  cb N  CO CM 00 ai CM T} ID t  8 CO 2? o Q o •= a> § -s -5 « t  T-  00 W  N  LO  ^ N -sr  o  CO  CO t CO O) CO co  CM  lO  1  U) CD U) Tr Tf co I -. LO CD S  Cll S  CO -r-  FT-  CO O)  CD ' TJ  t f IO N (O N r*- LO TCM CM  C  TCD CO  o  (0 O  9> c  s  o CM . 2 II "&>  CM CD O)  o o o o o If) T f Ifl CO I io " t ' " f o s  o  o  o  o  o  CO CD tf d  (D Tf CO i — CD CO CD CO L O o d o  . o •H C3 ^ o  o f ft  o> O  : Q O I Ul 111 I LU CC CC •: CC CL 0- Q •  U)  .2 £ § "5 .2> -g 3  co  ) CD CD  "> TJ «.fi c x: _ CO CJ)  til  Ife 8 t S rs » « IB •a "& "S 11  (fj  5  CO N Tco co CM CM  •  18  r  o co  ^1 s  •<t CO -t H CO  175  Table A 4 : Bioconcentration of chlorinated phenolics by leeches (N. obscura) as a function of leech weight at water temperature 12 °C, pH 7.5 and a contaminant concentration of 1 ug/L.  Tissue Concentation (ng/kg) Leech wt. Group  (9)  45-DCG  0.162  705  1  0.123  805  0.168  573  345-TCG  4,5,6-TCG  TeCG  345-TCVR  325  992  810  50  395  1 120  920  29  246  669  592  27  0.139  673  273  779  741  21  Mean  0.148  689  310  890  766  32  Std. Dev.  0.021  96  66  204  137  1 3  488  218  644  542  76  391  186  504  424  30  0.380  301  130  435  377  52  0.322  471  197  628  559  113 90  0.207 2  0.282  ;  0.322  51 1  231  691  616  0.303  432  192  580  504  72  0.064  86  39  107  99  32  0.861  179  95  310  247  21  0.962  210  99  337  254  49  0.912  249  98  371  257  ND  1.770  130  57  293  165  47  1.650  142  63  315  172  63  Mean  1.231  182  82  325  219  45  Std. Dev.  0.441  49  21  30  46  18  1.740  85  33  219  120  34  1.550  92  32  200  116  42  2.010  76  32  207  110  88  2.010  62  23  1 73  90  36  1.300  102  56  223  162  33  1.722  83  35  204  120  47  0.306  1 5  1 2  20  26  26  Mean Std. Dev.  3  4  Mean Std. Dev.  176  gacup)  Table AS- Bioconcentration of chlorinated phenolics by leeches (N.  as a function of  water p H after seven days exposure at water temperature 12 - C and contaminant concentration  0.1 u.g/L. Tissue Concentation (u.g/kg)  Water L e e c h wt.  45-DCG  345-TCG  4,5,6-TCG  TeCG  345-TCVR  11.0  13.0  12.0  9.7  11.0  0.575  13.0  16.0  13.0  11.0  9.3  0.491  11.0  13.0  9.8  11.0  7.8  0.592  19.0  15.0  13.0  14.0  10.0  0.428  8.6  21.0  10.0  7.1  0.535  13.0  7.6  11.0  7.0  4.9  0.718  4.1  8.9  8.9  6.0  9.2  0.690  4.3  13.0  24.0  16.0  5.2  0.766  9.9  7.4  22.0  21.0  8.0  0.655  18.0  11.1  16.0  12.3  8.1  Mean  0.606  10.2  0.111  5.2  4.5  2.1  Std Dev.  3.7  4.3  6.6  9.7  9.5  4.3  25.0  0.743  6.9  15.0  12.0  6.9  6.7  0.859  12.0  22.0  17.0  7.7  5.7  0.514  5.0  16.0  12.0  7.5  3.7  0.707  5.0  14.0  9.1  6.4  6.4  0.772  5.5  8.1  3.3  0.995  6.9  13.0  12.0  15.0  16.0  9.6  6.8  0.451  8.1  7.9  7.1  4.2  4.7  0.670  13.0  11.3  10.5  0.714  6.9  6.0  Mean  0.175  4.9  3.3  7.2  Std Dev.  2.7  1.6  8.0  8.7  5.8  4.7  32.0  0.696  5.1  7.5  2.7  6.4  0.682  2.4  0.677  4.2  7.5  2.5  2.4  13.0  5.6  6.0  2.9  19.0  0.585  3.5  3.0  12.0  2.1  1.8  6.7  0.815 0.630  4.3  9.4  2.5  2.1  10.0  4.0  11.0  4.0  2.3  11.0  0.801  6.0  5.7  3.4  33.0  0.622  2.0  8.2  9.8  5.9  15.0  0.567  3.0  10.0  3.2  9.6  0.726  4.5  3.7  0.680  5.3  3.6  2.7  Mean  8.8  15.6  1.7  2.1  9.7  0.084  0.9  Std Dev.  1.3  PH  (g)  5.1 ± 0.2  7.1 ± 0.1  9.0 ± 0.2  177  Table A 6 • Bioconcentration of chlorinated phenolics by leeches obscura) as a function of water temperature after seven days exposure at p H 7.0 and contaminant concentration.0.1 ug/L.  Tissue Concentation (u.g/kg)  Water Tpmri 1  Cl 1  l\J-  (° C )  20.0  L e e c h wt. (g)  45-DCG  345-TCG  4,5,6-TCG  TeCG  345-TCVR  0.664  6.5  8.6  6.8  4.4  4.7  0.826  6.0  16.0  4.5  3.8  5.0  2.7  15.0  0.678  6.5  8.1  4.0  0.559  8.3  9.3  4.7  3.3  12.0  4.1  3.4  2.4  12.0  7.4  3.1  13.0  9.1  7.3  18.0 29.0 16.0  0.554 0.494 0.986  6.8 10.0 7.3  6.1 9.1  0.602  5.4  3.7  4.1  2.7  0.479  8.4  4.9  7.1  4.9  0.507  6.3  5.5  8.3  4.7  9.5  5.9  3.9  13.4 7.0  Mean  0.635  7.2  7.5  Std Dev.  0.162  1.4  3.6  2.0  1.5  0.447  ND  4.3  4.1  2.9  9.0  0.635  5.0  5.9  5.3  4.8  22.3  0.755  5.8  8.3  6.0  5.7  13.9  0.426  8.8  5.7  5.8  4.4  11.6  0.664  7.3  4.4  4.1  9.0 18.6  11.8  Mean RtiH  nou  4.4  4.9  0.672  10.3  7.7  9.5  7.2  0.759  3.6  6.2  5.5  4.2  7.8  0.502  9.5  7.1  6.5  5.4  10.5  0.683  8.3  7.1  6.5  5.7  11.4  0.436  2.9  4.1  5.4  4.6  12.7  0.598  6.1  6.1  5.9  4.9  12.7  0.132  3.3  1.4  1.5  1.2  4.6  0.633  6.2  2.9  3.8  3.8  24.0  0.535  7.3  3.3  3.5  3.5  48.0  5.8  3.9  2.3  2.5  31.0  6.0  2.4  2.3  13.0  4.7  26.0  0.627 0.563  7.8  0.410  8.0  4.2  5.0  0.421  6.3  2.4  2.9  2.2  35.0  4.4  4.8  34.0  4.3  4.3  21.0 24.0  0.493  7.6  0.600  5.3  4.4 4.3  0.494  6.4  2.8  2.9  2.8  0.623  6.4  5.1  6.4  5.4  29.0  Mean  0.540  6.7  3.9  3.8  3.6  28.5  Std Dev.  0.083  0.9  1.1  1.3  1.2  9.4  178  Table A 7 : Bioconcentration of chlorinated phenolics by leeches (N. obscura) as a function of water temperature after s e v e n days exposure at pH 7.0 and contaminant concentration 0.01 ^ g / L .  Tissue Concentation (ug/kg)  Water Temp.  L e e c h wt.  (° C )  (g)  45-DCG  345-TCG  4,5,6-TCG  TeCG  345-TCVR  0.6  ND  4.1  ND  0.5  3.3  0.6  ND  3.3  1.8  1.3  1.4  0.7  ND  2.5  0.9  1.0  1.6  0.5  ND  3.7  2.6  2.0  3.3  0.7  ND  2.4  1.1  0.9  1 .5  0.6  ND  1.7  1.8  0.7  2.2  0.8  ND  2.0  1.1  0.8  1.1  0.6  ND  1.7  1.1  0.9  2.0  0.6  ND  1.5  1.4  0.8  1.4  ND  3.5  3.5  1.7  1.6  0.0  2.6  1.5  1.1  1.9  0.5  0.8  20.0  0.7 Mean Std Dev.  12.6  Mean Std Dev.  0.640 0.100  0.0  0.9  1.0  0.770  ND  1.6  1.0  1.3  ND  0.546  ND  5.1  1.5  1.7  ND  0.417  ND  3.2  2.9  2.3  ND  0.799  ND  2.8  0.8  2.0  ND  0.666  ND  3.7  0.9  0.9  ND  0.692  ND  4.6  0.8  1.4  ND  0.893  ND  6.4  1.1  ND  ND  0.460  ND  6.8  1.0  1.2  ND  0.631  ND  4.6  3.0  1.9  ND  ND  4.3  1.4  1.4  ND  1.7  0.9  0.7  ND  ND  ND  3.3  ND  0.4  4.6 3.5  0.653 0.158 0.533  4.4  1  0.647  ND ND  0.9  0.421  ND  1.9  ND  0.6  0.365  ND  0.9  ND  0.8  5.8  0.689  ND  0.3  ND  0.2  2.6  0.533  ND  ND  0.8  0.9  4.3  0.58  ND  0.3  0.5  0.4  2.9  ND  ND  ND  0.3  2.4  1.4  0.7  0.4  2.6 3.2  0.525 0.62  ND  0.469  ND  1.0  0.9  1.1  Mean  0.538  ND  0.7  0.3  0.5  3.5  Std Dev.  0.101  0.7  0.4  0.3  1.1  179  Table A8a : Bioconcentration of chlorinated phenolics by leeches (J±. obscura) as a function of bioassay water stirring, suspended sediment concentration and composition, at water temperature 12 °C, pH 7.1 and contaminant concentration 0.1 iig/L. Suspended Sediment Cone. (g/L)  Tissue Concentation (|xg/kg) Leech wt. (g)  45-DCG  345-TCG  4,5,6-TCG  TeCG  345-TCVR  0.486  ND  3.08  2.50  2.61  35.0  0  0.482  ND  3.71  2.54  3.67  26.7  (Unstirred)  0.394  ND  4.56  4.67  3.91  17.3  0.629  ND  3.47  3.38  2.83  9.5  0.346  ND  5.16  2.45  4.51  9.0  Mean  0.467  ND  4.00  3.11  3.51  19.5  Std Dev.  0.108  0.85  0.95  0.78  11.3  0.425  7.91  17.4  17.6  15.1  15.5  0  0.475  10.4  17.3  • 17.7  11.8  15.8  (Stirred)  0.285  12.5  27.7  26.0  22.6  14.5  0.364  8.55  21.7  21.2  18.7  14.5  0.448  9.70  18.0  18.4  15.4  14,9  0.440  10.0  23.0  25.1  18.5  11.5  0.293  16.9  22.6  25.9  17.4  15.3  0.476  15.9  26.2  28.9  20.1  13.4  0.235  ND  27.4  30.9  21.9  12.9  0.384  19.2  30.4  31.3  22.1  13.2  Mean  0.383  12.3  23.2  24.3  18.4  14.1  Std Dev.  0.086  4.9  4.7  5.3  3.5  1.3  0.025  g/L*  (Stirred)  0.278  10.2  20.3  25.0  21.3  10.0  0.443  15.4  22.1  28.2  16.7  53.1  0.381  20.9  38.5  38.3  28.8  15.1  0.477  20.1  34.4  39.2  31.1  16.1  0.379  22.3  41.7  50.0  38.2  7.98  0.414  21.9  45.7  51.4  34.3  11.7  0.342  22.6  42.8  48.9  34.1  10.9  0.403  25.2  45.7  48.8  38.5  8.32  0.438  18.9  35.1  39.6  24.0  7.45  Mean  0.395  19.7  36.2  41.0  29.7  15.6  Std Dev.  0.059  4.5  9.5  9.7  7.6  14.4  0.406  .6.57  23.7  27,6  16.1  6.0  0.543  11.5  21.8  24.2  16.6  22.1  0.08  g/L*  (Stirred)  0.296  ND  15.7  24.6  18.3  8.07  0.380  13.20  26.1  32.7  26.7  8.18  0.384  6.81  22.8  26.8  18.5  14.8  0.492  8.16  24,0  29.4  19.4  25.6 7.44  0.614  9.23  27,2  29.9  20.6  0.345  10.1  22.9  27.7  23.5  10.1  0.448  9.38  22.3  26.8  19.6  8.40  0.415  6.21  22.2  26.1  19.5  8.27  Mean  0.432  9.0  22.9  27.6  19.9  11.9  Std Dev.  0.095  2.4  2.9  2.4  3.0  6.4  180  Table A8b : Bioconcentration of chlorinated phenolics by leeches (N_. obscura) as a function of bioassay water stirring, suspended sediment concentration and composition, at water temperature 12 °C, pH 7.1 and contaminant concentration 0.1 iig/L. Suspended Sediment Cone. (g/L)  Tissue Concentation (|xg/kg) Leech wt. (g)  45-DCG  345-TCG  4,5,6-TCG  TeCG  345-TCVR  0.401  21.2  36.6  40.7  21.8  8.43  0.08 g/Lt  0.351  15.1  28.2  35.1  19.5  7.77  Inorganic  0.418  12.8  30.4  38.4  19.3  23.2  (Stirred)  0.302  14.9  32.5  37.6  20.2  9.81  0.414  14.6  36.2  43.1  26.1  6.56  0.468  18.6  35.9  45.4  24.6  12.3  0.438  12.8  33.8  38.7  26.5  7.57  0.342  14.3  37.1  48.6  27.1  9.66  0.370  11.3  23.0  34.0  17.8  8.33  Mean  0.389  15.1  32.6  40.2  22.5  10.4  Std Dev.  0.052  3.1  4.7  4.8  3.6  5.1  0.451  26.0  43.1  47.4  36.1  14.79  0.15 g/L*  0.302  11.0  20.2  24.8  17.8  7.21  (Stirred)  0.325  13.2  29.8  37.0  21.6  9.9  0.444  15.5  35.0  43.8  33.3  8.54  0.305  19.5  34.2  43.7  28.7  11.01  0.367  11.8  28.0  31.7  19.6  7.1  0.367  12.5  33.5  35.0  25.4  39.38  0.376  6.6  25.0  28.8  20.3  10.69  0.341  9.0  29.5  32.7  24.4  8.35  Mean  0.364  13.9  30.9  36.1  25.2  13.0  Std Dev.  0.054  5.9  6.6  7.6  6.3  10.1  * Suspended material: size < 63 pm, 5% organic : 95% inorganic material, t Suspended material: size < 63 pm, 100% inorganic material.  .  181  APPENDIX 4  Raw Data Field Monitoring Trials  182  183  c CJ)  c 'co  CO T - O) O) ID CO S  9 9 9 9 9* 9  9 9 9 S9 99  CO  9 999  9 9 99  9999999  3  O cri  _co co ^ £  i—  CD C CM CD O CD  co  o  tr  O  co  1—  CO <N CO . LL °2 CD sz n CD — CD <D C LL ^ TJ " ° *-  C3)  . CQ  §  £o a  CD CJ  CD as IS « TJ CO  « >- o  T -  C L CJ) CD CD  CO .CJ -tf  CO  o  c  co-B  c o O *  8  I  D  Q  o  T _  Q  Q  o  o  o  CM CM O O  9 i 9 i 8  Q  O tf tf CM CM  o  CO CO CO tf CM CM <M CM O O O O  o  o  o  o  o m o M C O in CM r- O) C i- -r- i- O To- O T-  o  — i CM O CO CO 1Ti O O o  o  o  o  fg  5  2  o o o  o  T- C M CO tf o o o o o o o o o o o o o o o  in  O)  o C O CO T- O O o o o  CO i- O  c o C L CM o o £ E o  o o o o d d  8  o o o  CJ  CO  CM  o  o o co vn  in  O TJ-  in in  D  O  Q  Q  O  D  z  z  tf  m  co  r-  i -  <N  co co o  o o o  CO T-  o  r- co »tf tf o o  CO CD 00  CM  -° 5 CD LL "5 C  o  co  CO • "TJ o •5 <" o> o> © XJ •to c c E p CD CD CD  co O  CL  O  O)  o  o  CP  CO CO CD= CO CD E  E  C ' CD »  Q  z z z z z z z  c  O co o  S|  "  o  s  8S  .Q  Q  c o  E E l  o  o  00  CO co i_ CM c CD > CD CM  CD  CO CO CO tf  O) cn co  co  c  oo ?  i_ CD  co  c  00 "CJ  L.-5 0 5 =  O <D O T- C D o O CO  0  0 Q.  E  CO o E CD)D o Q  H >= CO CO o  Q  T-  CM co  z z  Q  z  99  8 a »  Q) OS <» " O  *1  D  2  >> &  CD . £ TJ  S- ^ &  CO CC CC CO CO CO ^ O O O O O O O  to  Q  co  CO  •<*  co  to  Q  Q  0>  -r-  L/>  CD  CO CO Q O O  h-  T-  co m  O  O  co a  o o  CO  o-.Q - i - Cf) 3 «  ICJSTO  CO CM  5  CD  N  °>  c  CO  a>  >  CD  a  •6  55  184  D_ O CD H CD •tf" co"  D  tficOWh-COO-i-CM  D  D  Q  D  Q  Q  Q  D  Q  cocMc\icMco'tfcotf  CM  CD > tr  0_  i_  OinOCMCMfNOJTf  o  CD CO CO  CO<DO)<OLOr^U)CO  •  CO •tf"  CM"  T3  CD CO  o  999999999  9999999999  ississsss  9999999999  o o o o o o o o o  Q  CJ)  CL  X CD 3 O CO  n o  c CD o c O O  CO CD sz o CD CD CO  XJ CD  o 2 0  Q  Q  D  D  Q  Q  D  Q  Q  z z z z z z z z z z  0  3 CO CO o o o o  E  T-  O  999 9 9 9 9 9 9 9  CNJ  ^  XJ CO 7  •2 o O cfj  o o o o o o o o o  C  0  -  *  «  W  W  O  J  T  N  -  r  C  O  N  O  )  C  O  O  O  J  )  C  -  9999999999  J  O  CD CO 03  «  c —  o Q Q Q Q Q Q Q Q Q  CJ  999999999  CO CJ)  o "55  •5  2 oo  C XJ CD C O CO  o c o CJ  o o o o o co co in in co CVJ T- O O CM  St St  a> - § CD 1  CM 1/5 f- CO O i-  T-  r- -i-  i - O o - i - T - r - r - O T -  *—' CD •C O 3 +-* CO CO CO  c o 0 CD  <o 0 C  00  co"co  0  5  CD O) tf CO O i-  O  CD  CD c  E o E co  o o  c? L_  CD  O  9. » CO CO CD w '—' CD  "5"  >, »- CO  * —  Q "CD  CO  st w  O  _  " 5 i: co g CD  O  To O co ~— LU  c5 CD  > Q  5 -oi  55  185  a o CD  H co  °  CM  °  9  °  o in  9 9  in  C M W o J C O ^ o J O l O  o  CO  CO CM" l —  CD  >  'tr  in  O O O Q O O Q Q Q Q z z z z z z z z z  i _  CD CO C C I  co d  CD  £Z CZ  §s  g g g i g g 9 i z §  TJ CD CO  O OX CD  =L  C C i _  Z3 o CO  .Q cn  o  ±  CD  co CM CD  1  sz o  o  o  o  o  o  o  o  o  O) O)  9 S  cs  s§  ^  c o cc c  d  CD O  c o O  ( 0 I i r i N C O i i i o i ^ n O ) ©  CD  CD CD  .£  .O.  x. 2 CD O O O  O)  £ E ® o "° £ co o r % O c CQ  o  T- O) ^  o  T  CD LO Lf) N  o  o  o  o  o  <o in  s  I/)  o  o  ^  T) CO CD iii  i  i  ss  O i h t C D T - W O ^ N r r N r - N N W r -  Q>  T-^  1 -c  c O o o  §s  CO CD  c = o a>  co C TJ  ® o c5 c  c o -p » o  CD  ® S|  CD W CM CO CD C f-O CO CO  §1  d  CO  CO CMO) CJJ O CO CO o  O  CD >  a> o "55 CD  -  2  Cl) O  CM < c a) B 5 co CC ^  E o co o  CO CO  H - co  CO O  cu >  j- w  CO —i  o  CD  5  >  ><  JO  V.  W CO  CD  CO  186  CD  s co »  H  I  1^  CO  CM  •tf" co"  CM  O CM  CM  CM"  CD >  0_ 0 I-  cc — i  co  T-  CJ  T-  CO  co  O  Q  1  CD CO CO  CO  T-  T-  r  CO  CO  CO  ^_  <D CM  CO  U) CO  U) CM  •tf"  CM  CD  SI  Q  D  D  Q  Q  Q  99999  D  TJ  CD CO O CL X  CJ)  CD  O)  «? k-  co o  CO CO  CM  1-  Q  D  Q  Q  z z z z  Q  <v-  c  CD CJ  co  c o  CD CJ 3 CD E CD Si — CD  o  LL  •o E ® o o CD  .  co o  CO  §6  l.i  <£> Oi  co co CO co r*C M CM CM 1- Lf) )Z  s s s ss  CD CO CO  I -  CO  O)  CM O) CO CO s - - < - c o c o - * * c o - * - o h -  CO  CM  o 9 § §S  C M C M C M C M C M T - C O C M C M C M  „  CL CO cu 03  co un un h - <o CO O) CO r-- CM h- 1CO CM CO CM CO  a  Zlc  C  •r-  c o  O  N  C  O  N  O  J  N  W  O  u  O  ©  Lnmh-mio-fl-h-irjcoLjo  y C  c o •c o o ~—' x:  999999999  "5 «  o 9 99  9 9 9 99  J2 00  .2 "a 13 CO c c CD o O ' c o co o  sz sz CD  CD  00  co  CJ) CM  CM  tf  tf  r»  CO  in  CO  co tf  CO  co  co  CO CO  CM  cn I—  1- O) O h CO O CO h- r- co a o o •<- 0  T-  OO  O) tf CO  r-  CO  § To CO ^ CO  ..  CO  c o  500 CO — I CO  CD  CD >  CO C  GL*  c  g  g- 13  E o CO o co —'  CD  CD  13 co  co c7j a) to  C0  LU  t  -  II >. — co  >  _0> O _ "CD CD O  Q  co to §  CO  sz ~o  to O  co '—' LU  CD  "d  187  s  CD H CO  l f ) C > J C O O ) C O C \ I C \ I N W C 0  CM  T-  T t L O T f T j - L O L O L O C D T j -  co"  CO  CL O  o o o o o o o o in  > O Q CD LO"  > 3. o  H LO -*" CO  c o  O  O  O  O  O  O  O  oo  CM  O  CO  o o0> o o oO)o i o- o  CO  CO  o o o o oo  LO  OJ  CM  OJ  CM  CM  CO»  CD  OJ  CO1  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  O  o o o o o o o o o  Q  L0l  CO  0  O O O O  Q Q  CD  c o IO  o o o o o  o o o o o o o o o o  o o o o oo  O i ^ i f l o s c O T f c o c o n l  C V J C M C O C M C M C O C O C O C O C O  f A l l M t M C M O I C O W C l l  CD 13 CO CO CD O H CD to  CO  o o o o oo T-  O  CD O H  O  CO  O  O  O  O  O  T-  CM  O  O CM  O CO CM  O LO CM  O  O  ICM S  O  CM CM  O  a>r-  CO  CD  Tf  LO  LO  O  O  LO  O 00  O CO  O  O  O  CO  CO  OJ  T -  o o co o coo o o  O O O  o  O  O  CT)  CM  CD LO  O i— \  o o o o oo  TJ- C D CO CO TJ- CO  1-  Tf  LO IO  O  Tj-  co"  CD O Q LD  o o o o o oo •M- co cotf)tf)co <t  x: . c  S »  o o o o o o o o o o o o o o o o o o o z z z z z z z z z z z z z z z z z z z l  o o o o o o o o o z z z z z z z z  C  1~  O  D  C  M  I  *~ *~  -  O  O  C  r-  M  C  r-  O  T  o o o o o o o o o o o  o o CM  -  i-  O  L O C O r - S N N C O L O t M O l t M  CM OJ  o o oCO TT CD OJ  TJ-  CM  CM  I  T - i - i - O J O J C D C ^ C O C O C O C M  O  O  O  O  CD  >  CJ) C .5 CO  CO  o o -I  CD O  w co  3  D  CD c o  o  CD TJ W CO "co CC  > CD Q  188  °O CD H CD •tf" crf CM" 0_ O  O co  CO •tf"  o  CM"  CO CD SZ o CD  cu  T3  ^CD —' o CD CD  T3  CO CJ  "5  c  C CD CJ C  CD  o O  CD  CO CM CD -i— CO 00 CJ) tf c\i n CM CM LO LO  8  CM CD  CD ZJ CO  4—' CO  c  co  o °2 1  CZ , •glCO i v— i_ C CD CD _ Q CJ O  o  LO •>-  o  o  o  o  O O O O O ONt-LOOjT-(0(DlMO)  o  •*-*  C  o o § w co  " O •= o •£=  .E O <o  Q O Q Q Q Q z z z z z z  m  CD >O CD C C CD O CD  1  o o o  CMCDr^LO'M-l -COi-CD CO < r ) T - C M C M C M C 0 ^ f - * C O s  CT) CD O tf' tf (D U) IO  ° coco CM  0 0 0 0  z  z  z  z  z  z  z  z  z  CO  15 C  £ JZ CD >  §<*  31  '«a-ooo'^-h-cooLO LOCOCOCO^-h-CMCOCD o o o o o o  CDLOh-Oi-r^-OOCR CM CO CD TCO To o o o T- T- O 1-  _£= »O  CD  - «  S2  CO CO iO LL  6 SZ  CD  cp C  j= o E o co o co —i  -9 °CO  X  r— CD  CO  CD  > ir  (5 o  O -g co co  to  S  <D  O) Q  > ir CD  >  O  CD D  CO CO  CO  I*  189  O O O O ^  00 ^  oj  CM T-  D  Q  Q  O  O  Q  Q  O  Q  D  y-  ^ CD  m CM  O) 3s  Q  O  c o  s  a) m 0)  Q  •  Q  Q  tf)tf)CO  Q  Q  Q  tf)  co  CO CM  tf) C\i  CD  CM CO  190  Lfl N (O oi oi oi co  ©  o  9 9 9 9 9 9 9 9 9 9  CM 1-  o q CM CT)  o o o o o to- TC O - OC M 1C M ^ - i -1C CO - r in -CM T-  XJ 031 T 3 o in CO  oo  CM" CM *~  9 9 9 9 9 9 9 9 9  9 9 9 9 9 9 9 9 9 9  9 9 9 9 9 9 9 9 9  9  o o o o o o o o COfljlfiCOrcitOO) (MT--r-'-OJCMi-CM  co  ™ <0  •>, CO — Q 3 O -3  *  E 2  Q CO * -  %o  CO ^ CD CO CJ 2 CO  co 5 o  o  CQ  ec  o  CO  CO CD CD C X o a>  3.  9 9 9 9 9 9 9 9 9  c  o — t• ' CC  c CD O c  o O  o tf cn in o to •<- o i n C M i n c o c o ^ t c o in cd  CD 3 CO CO CO  CC  >. — 1  CMO)h-T-CM-T-tfO)CD CTCMCMCMCOCOCOCvicO  xi CD CO > CJ CC  o k _ CD  c CD x QXJ CD 03 C  i_ o  X  CO CC LL CD X  C XJ CD CO  03 k_  cc  rm  £ 5 o  c  CC  o  z z  CD  ce  c  tf  Q Q Q D Q  9 9 9 9 9 9 9 9  xp  o  1-  "•^  o o o c  q cd  2 S tf » £ « S t ? Sf- r CM • co _; o)  ( O t t i M S T - N O C D OIOJOJ^COCOOJCOCM  E  CO CQ CO  < XI >CC 0)  •S 3 CD CD  o o tf CO CD CM l - T 0  CM in O O 0 0 0 r- co CO IO CO tf o 1- o CM CO - - r - < - T - 0 ^  o O CM O co CO CM CO CO  O in co in O CO 0 Oi CMc Co O in CM CO CM CO CM CM  CO  CD O CD CL CO  o CO O  > co Q CD 2 TJ CO  To o £  > co O CD 5 -d  55  191  Table A 1 8 : Bioconcentration of chlorinated phenolics by the leech species Percvmoorensis marmorata after seven days laboratory exposure to various contaminant concentrations at T = 12.5 °C and pH = 7.5.  Tissue Concentation (p.g/kg)  Water Cone.  Leech wt.  (ng/L)  (g)  45-DCG  345-TCG  2.200  110  190  2.230  75  122  2.410  128  2.290 2.283  0.5  Mean  TeCG  345-TCVR  1 55  128  32  11 1  97  20  151  138  131  58  1 11  139  142  122  46  106  151  137  120  39  1 8  1 5  17  4,5,6-TCG  0.093  22  29  2.230  288  287  319  240  39  3.070  466  443  529  350  43  2.240  242  235  266  228  40  3.090  249  247  302  204  56  Mean  2.658  311  303  354  256  45  Std Dev.  0.488  105  96  119  65  8  1.660  1300  1070  1440  673  143  5.0  3.780  1290  1290  1270  813  163  0.779  1520  1070  2130  1630  257  2.050  168  1380  1870  1 180  191  2.067  1070  1203  1678  1074  189  1.260  610  157  393  428  48  1.370  3990  2360  4930  3370  289  3.270  2250  1820  2740  1230  452 203  Std Dev.  1.0  Mean Std Dev.  10.0  1.560  3060  2100  3410  2680  2.980  2510  1960  3070  1330  94  Mean  2.295  2953  2060  3538  2153  260  Std Dev.  0.969  770  230  968  1047  151  192  Appendix 5 Predictions of field water contaminant concentrations using measured leech bioconcentration and laboratory regression  relationships  Sample Calculation  Predicted water concentration, 3,4,5-TCG; July  ug/L  1991.  Predicted water concentrations were calculated from the laboratory relationship between water contaminant concentration and bioconcentration, with rearrangement of the equation to yield water concentration from observed bioconcentration. Correction factors for differences in water temperature, pH, leech weight and water current between measured field and laboratory experiments were multiplied by the initial predicted concentration. No formal regression relationship water was developed for the effect of water current on bioconentration. The correction factor for the effect of water current divided by the water concentration predictions was calculated as the inverse of the percent increase observed between bioconcentration in static and stirred bioassays. Measured Field Values Average Measured leech bioconcentration = 28 fig/kg (Table 4.14) Average measured water concentration = 0.052 ug/L (Table 4.11) Water temperature = 12.0 °C (Table 4.8) pH = 7.8 (Table 4.8) Average leech weight = 1.07 g (Table 4.14). Predicted Water Concentration, |xg/L. 1) Water concentration as a function of leech bioconcentration at water temperature 12 °C, pH 7.5, average leech weight 1.17 g (n = 25). a) 3,4,5-TCG (ng/kg) = 74(water cone. ng/L)expt.0.77 = 74(0.052)expt.0.77 (Table 4.1) Rearranged to: Water cone. (iig/L) = 0.77 Bioconc (ng/kg)/74 Water cone (ug/L) = 0.77 28/74 = 0.283  2) Correction factors used for adjustment of calculated value for differences in laboratory and field conditions.  Correction factor = Predicted lab value/Predicted field value  b) Water temperature: no significant effect 11.8 - 20 °C c) Water pH: 3,4,5-TCG (ng/kg) = 33.1 (0.86)expt. pH (Table 4.3) Field water pH = 7.8: 33.1(0 86)expt.7.8 = 10.21 Laboratory water pH = 7.5: 33.1(0.86)expt.7.5 = 10.67 Correction factor = 10.67/10.21 =  1.045  d) Leech weight: 3,4,5-TCG (jig/kg) = 68(weight)expt.-0.83 (Table 4.4) Field leech weight = 1.07 g: 68(1 07)expt -0.83 = 64.28 Laboratory leech weight = 1.17 g: 68(1 17)expt.-0.83 = 59.69 Correction factor = 59.69/64.28 = 0.929  3) Predicted water concentration (static bioassay water) 3,4,5-TCG (ug/L) = 0.283 ug/L * 1.045 * 0.929 = 0.275 ug/L. 4) Water current: no laboratory regression relationship: Correction factor for presence of water current in laboratory = 5.80 * bioconcentration without current (Table 4.7) Inverse of correction factor = 1/5.80 =  0.172  5) Predicted water concentration (continuous current) 3,4,5-TCG (ug/L) = 0.275 ug/L * (1/5.80) = 0.047 jig/L.  (Table 4.20).  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0086733/manifest

Comment

Related Items