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Use of the bioconcentration capability of leeches to evaluate chlorophenol pollution Jacob, Cristina 1986

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USE OF THE BIOCONCENTRATION CAPABILITY OF LEECHES TO EVALUATE CHLOROPHENOL POLLUTION by CRISTINA JACOB Dip. Eng., Polytechnical I n s t i t u t e , Bucharest, Romania, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of C i v i l Engineering) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1986 © C r i s t i n a Jacob, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of C i v i l Engineering The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date October 5, 1986 ABSTRACT The objective of t h i s research was to investigate the use of leeches as i n s i t u monitors of the b i o l o g i c a l a v a i l a b i l i t y of chlorophenols and degree of contamination i n the North Arm of the Fraser River Estuary, B r i t i s h Columbia, where chlorophenols are used as wood preservatives by several forest industry operations. The objective was accomplished by : 1) an integrated series of grab and continous samples to determine the s p a t i a l and temporal v a r i a b i l i t y i n chlorophenol contamination, 2) i n s i t u and laboratory experiments to compare leech bioconcentration to water levels of pollutants and to determine environmental factors( temperature, pH, leech size) that regulate and a f f e c t bioassay in t e r p r e t a t i o n . Grab samples were not representative of the average l e v e l of po l l u t i o n i n the r i v e r . High concentrations such as the one reported for M i t c h e l l Island on October 4 (11 ppb TTCP and 2.25 ppb PCP) could give a f a l s e impression of the p o l l u t i o n l e v e l i n the r i v e r . Also, plumes of high concentration of chlorophenols could be missed e a s i l y i f the sampling time does not coincide with the pollutant discharge. High frequency( every 2 hours) automatic water sampling showed a high v a r i a b i l i t y i n chlorophenol contamination( 0.278 ppb to 3.678 ppb TTCP for the March 31- A p r i l 6 f i e l d experiment) which demonstrated . the sporadic nature of chlorophenol discharges. Changes i n the r i v e r flow also affected the level and the pattern of chlorophenol v a r i a t i o n . A method capable of integrating concentration versus time seemed to be the only way to elucidate the i r r e g u l a r pattern of pollutant l e v e l s . Leeches were exposed to the contaminants by submersion in cages at various locations along the North Arm. On the basis of the levels of chlorophenols found i n the leech, estimation of the average chlorophenol concentration i n the water were made. Concentrations as high as 3.2 9 ug/g TTCP and 1.11 ug/g PCP were found i n leeches exposed i n the Mitc h e l l Island area( TTCP and PCP were the only chlorophenols found i n the area of study at any time during our sampling program car r i e d out between August 1984 and September 1985). An average concentration higher than 2 ppb TTCP and 2 ppb PCP was estimated i n the water for the duration of the leech exposure( 7 days) at that location using bioconcentration levels determined i n the laboratory. Laboratory experiments showed that lower pH increased bioconcentration of chlorophenols. Higher bioconcentration i factors were achieved at higher temperatures and regression equations( R= 0.96 to 0.99) were calculated for the f i v e chlorophenols used i n the experiments( 2,4-DCP; 2,4,5-TCP; 2,4,6-TCP; 2,3,4,6-TTCP and PCP). Temperature affected the time needed to achieve steady state which was 4 days at 4 C, 5 days at 12 C and 7+ days at 22 C. Four out of the f i v e chlorophenols tested( 2,4-DCP exhibited d i f f e r e n t bioconcentration c h a r a c t e r i s t i c s ) were bioconcentrated to the same le v e l by leeches, regardless of t h e i r Po/w values. This contradicted the linear, relationships established by various researchers between the bioconcentration factor and Po/w of various compounds — i i i -including chlorinated phenols i n other organisms. Recommendations for setting up a method to use leeches i n a biomonitoring program of the chlorophenol p o l l u t i o n i n the Fraser River Estuary are proposed from the results of t h i s research. The cost of the analyses using a biomonitoring program could be an order of magnitude lower than an adequate water sampling program to assess the chlorophenol p o l l u t i o n l e v e l . - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x ACKNOWLEDGEMENTS x i i i 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 2.1. Introduction 4 2.2. Chlorophenol Presence i n the Canadian Environment 4 2.2.1. Chlorophenol Related Problems i n the Fraser 5 River Estuary 2.2.2. Chlorophenols Levels i n the Fraser River Estuary 7 2.3. Environmental Fate of Chlorophenol 15 2.3.1. Bioaccumulation Process 19 2.3.2. Laboratory Determination of Bioconcentration 20 Factors 2.3.2.1. Bioenergetics and i t s Influence on the 28 Bioaccumulation Process 2.3.2.2. Influence of pH on the Bioconcentration 31 of Pollutants 2.3.2.3. Water Chemistry and i t s Influence on the 41 Bioconcentration Process 2.3.2.4. Pollutant Interaction and i t s Influence 42 on the Bioconcentration Process 2.3.2.5. Ef f e c t of Temperature on the 43 Bioconcentration Factor 2.3.2.6. Ef f e c t of Test Species on the 46 Bioconcentration Factor 2.4. Use of B i o l o g i c a l Indicator Organisms to Quantitate 46 Pollutants i n Aquatic Environments 2.4.1. P o l l u t i o n a l Ecology of Freshwater Leeches 54 3. SAMPLING AND METHODOLOGY 5 8 3.1. Introduction 5 8 3.2. In Si t u Fraser Water Sampling 58 3.2.1. Study Area and Sampling Sites 58 3.2.2. Grab Sampling 60 3.2.3. Automatic Sampling 61 3.2.4. Water Samples Preparation for Chlorophenols 63 Analysis 3.3. Leech Experiments 6 5 3.3•1.In Situ F i e l d Leech Experiments 68 3.3.2. Laboratory Leech Experiments 7 0 3.4. Standards Preparation 72 3.5. A n a l y t i c a l Technique for Chlorophenols Separation, 74 Quantification and I d e n t i f i c a t i o n 3.5.1. I d e n t i f i c a t i o n and Confirmation of the 75 Chlorophenols Presence i n Water and Leech Matrix 3.5.2. Reproducibility and Technique Evaluation 77 3.5.2.1. Recovery from Water 77 3.5.2.2. Recovery from Leeches 78 3.5.2.3. Derivatization Reproducibility 78 - v i -3.5.2.4. Injection Accuracy 79 3.5.2.5. Method Detection Limit 7 9 4. RESULTS AND DISCUSSION 81 4.1. Introduction 81 4.2. A n a l y t i c a l Technique. Reproducibility and Technique 82 Evaluation 4.2.1. Chromatograms of Acetylated Chlorophenols. 85 Standard Mixtures, Water and Leech Samples. 4.2.2. I d e n t i f i c a t i o n and Confirmation of Chlorophenol 94 Acetates by Mass Spectrometry 4.2.2.1. Electron Impact GC-MS Spectra 9 4 4.2.2.2. Positive Ion Chemical Ionization Spectra 95 4.2.2.3. Single Ion Monitoring Spectra 95 4.3. Laboratory Leech Experiments 9 6 4.3.1. Preliminary Leech Exposures 96 4.3.1.1. 24 Hours Bioconcentration Ratios 97 4.3.1.2. Depuration Tests 99 4.3.1.3. pH Experiments 101 4.3.1.4. Summary of Results 104 4.3.2. Bioconcentration Factors Determination 105 4.3.2.1. Bioconcentration of Chlorophenols i n 105 a 7 Day Experiment 4.3.2.2. Bioconcentration of Chlorophenols at 108 Different Temperatures. 4.3.2.3. Species Influence on Bioconcentration 119 Factors 4.3.2.4. Weight of Leech Specimens and i t s 120 Influence on Bioconcentration Factors - v i i -4.3.2.5. Summary of Results 122 4.4. F i e l d Experiments 123 4.4.1. Water Sampling 123 4.4.1.1. Grab Sampling 124 4.4.1.2. Automatic Sampling 127 4.4.1.3. Summary of Results 134 4.4.2. F i e l d Leech Experiments 136 4.5. Sp e c i a l l y Designed Laboratory Leech Experiments 139 4.6. Interpretation of the F i e l d Experiments Results on 142 the Basis of the Specially Designed Laboratory Leech Experiments 5. CONCLUSIONS AND RECOMMENDATIONS 148 5.1. Conclusions 148 5.2. Recommendations 150 BIBLIOGRAPHY 153 APPENDICES I. 162 II. Chromatograms and spectra 164 II I . Symbols 181 - v i i i -LIST OF TABLES Page 2.1 Commercial sapstain formulations i n B.C. 8 2.2 Sediment and water concentrations for chlorophenols, chlorinated benzenes, and pentachloroanisoles 10 2.3 Bioconcentration factors calculated from water s o l u b i l i t y and octariol: water p a r t i t i o n c o e f f i c i e n t 27 2.4 Chlorophenol t o x i c i t y to guppies at d i f f e r e n t pHs 33 2.5 T o x i c i t y of nonionized and ionized forms of 2,4,6-TCP and PCP at various pHs 3 6 4.1 Reproducibility and technique evaluation 83 4.2 Concentration r a t i o s i n leeches a f t e r 24 hours 97 4.3 Chlorophenols i n leeches exposed i n the Fraser RiverlOO 4.4 Concentration r a t i o s as a function of pH for 24 hours exposures 102 4.5 Size of leeches used i n temperature experiments 108 4.6 E f f e c t of temperature on the bioconcentration of chlorophenols 113 4.7 Experimental and calculated BFs 115 4.8 Grab sampling r e s u l t s 125 4.9 F i e l d leech experiments 140 4.10 Special laboratory controlled leech experiments 143 II.1 Single ion monitoring of the chlorinated phenols 165 - i x -LIST OF FIGURES Page 2.1.a Locational map of the Lower Mainland 9 2.1.b Locational map of the Southern Vancouver Island 9 2.l.c Location of the sewage treatment plants 12 2.2 E f f e c t of temperature on the bioconcentration of Aroclor by f i s h 45 2.3 E f f e c t of temperature on the concentration of DDT i n i n rainbow trout 4 7 2.4 Seasonal e f f e c t s of pesticides levels i n f i s h 53 3.1 Sampling s i t e s and industries i n the study area 59 4.1 Chromatogram of acetylated chlorophenols 8 6 4.2 Chromatogram of a 1 ppm standard mixture 88 4.3 Chromatogram of a 0.1 ppm standard mixture 88 4.4 Chromatogram of a Fraser River water sample spiked with a standard mixture of chlorophenols 89 4.5 Chromatogram of a water blank 9 0 4.6 Chromatogram of a Fraser River water sample 90 4.7 Chromatogram of a leech exposed for 6 days i n the Fraser River water 9 2 4.8 Chromatogram of a leech blank 92 4.9 Bioconcentration of chlorophenols by leeches i n a seven-day laboratory experiment 107 4.10 Bioconcentration of chlorophenols by leeches in a laboratory experiment at 4 C 109 4.11 Bioconcentration of chlorophenols by leeches i n a laboratory experiment at 12 C 110 4.12 Bioconcentration of chlorophenols by leeches i n a laboratory experiment at 22 C 111 4.13 Bioconcentration of 2,3,4,6-TTCP by leeches at d i f f e r e n t temperatures 112 - x -4.14 Chlorophenol concentrations at M i t c h e l l Island over a two-day period 128 4.15 Chlorophenol concentrations at M i t c h e l l Island over a six-day period 131 4.16 Chlorophenol concentrations at M i t c h e l l Island over a seven-day period 133 4.17 Chlorophenol concentrations at M i t c h e l l Island over a six-day period 135 I. 1 Separation of chlorophenol acetates 163 II. 1 Total ion chromatogram of a standard mixture 166 11.2 Mass spectra of 2-CP acetate obtained by electron impact 167 11.3 Mass spectra of 2,4-DCP acetate obtained by electron impact 167 11.4 Mass spectra of 2,4,6-TCP acetate obtained by electron impact 168 11.5 Mass spectra of 2,3,4,6 TTCP acetate obtained by electron impact 168 11.6 Mass spectra of PCP acetate obtained by electron impact 169 11.7 TIC chromatogram of a leech extract 170 11.8 Chromatogram of a standard mixture of chlorophenols 171 11.9 Mass spectra of 2-CP acetate obtained by chemical i o n i z a t i o n 172 11.10 Mass spectra of 2,4-DCP acetate obtained by chemical i o n i z a t i o n 172 11.11 Mass spectra of 2,4,6-TCP acetate obtained by chemical i o n i z a t i o n 173 11.12 Mass spectra of 2,3,4,6-TTCP acetate obtained by chemical i o n i z a t i o n 173 11.13 Mass spectra of PCP acetate obtained by chemical i o n i z a t i o n 174 II.14a Total ion chromatogram of a leech extract 175 II.14b Selected ion chromatogram of 2,4-DCP acetate 176 II.14c Selected ion chromatogram of 2,4,6-TCP acetate 176 - x i -II.14d Selected ion chromatogram of 2,3,4,6-TTCP acetate 177 II.14e Selected ion chromatogram of PCP acetate 177 II.15a Total ion chromatogram of a Fraser River water sample 178 II.15b Selected ion chromatogram of 2,4-DCP acetate from a derivatized sample 179 II.15c Selected ion chromatogram of 2,4,6 and 2,4,5-TCP acetates from a derivatized sample 179 II.15d Selected ion chromatogram of 2,3,4,6-TTCP acetate from a derivatized sample 180 II.15e Selected ion chromatogram of PCP acetate from a derivatized sample 180 - x i i -ACKNOWLEDGEMENTS I thank Dr. K. J . H a l l f o r h i s a s s i s t a n c e i n the research and p r e p a r a t i o n of t h i s t h e s i s . I thank Itsuo Yesaki f o r the help provided during the boat sampling t r i p s and f o r d r a f t i n g s e v e r a l f i g u r e s . I am g r a t e f u l to Professor R.W Davies , Head, Department of Biology at the U n i v e r s i t y of Calgary f o r species i d e n t i f i c a t i o n of the leeches. I a l s o thank Paula Parkinson and Susan L i p t a k f o r labo r a t o r y guidance, Timothy Ma f o r the GC/MS work , Guy K i r s c h f o r b u i l d i n g the bioassay chambers and Susan Jasper f o r o b t a i n i n g l a b o r a t o r y s u p p l i e s . This research was supported by NSERC and a U n i v e r s i t y of B.C. graduate f e l l o w s h i p awarded f o r two years, 1984/1985 and 1985/1986. - x i i i -1. INTRODUCTION Chlorophenols, namely PCP and TTCP, are used i n B r i t i s h Columbia for short and long-term wood protection against sapstain and mould. The media has expressed concern related to the health of workers , and investigations on the use and handling of the chlorophenol containing materials have been car r i e d out. The awareness of people d i r e c t l y involved with the product, of ordinary people, and of the goverment authorities has increased through the years. Chlorophenols have been r e c l a s s i f i e d i n Canada in the Category II of P r i o r i t y Chemicals and included i n the l i s t of P r i o r i t y Pollutants i n the United States. There have been numerous studies that have shown CPs presence i n the forest industries effluents and i n the three compartments of the aquatic environment, water, biota, and sediments. An EPS report published i n 1979 as a r e s u l t of an elaborate program of sampling ca r r i e d i n the Fraser River Estuary and on the southeast coast of Vancouver Island(EPS, 1979) revealed the following: " Chlorophenols were found i n the aquatic environment at a l l sampling s i t e s where PCP was used i n i n d u s t r i a l processes. I t i s l i k e l y , therefore, that chlorophenols may be found i n the aquatic environment wherever they have been used i n wood preservation or protection." Concentrations as high as 7.3 ppb PCP and 5.2 ppb TTCP were reported at marine sampling s i t e s . It was further concluded that the data indicated a p o t e n t i a l l y serious environmental problem with respect to the use of CPs( Appendix III contains a l l the symbols and abbreviations - 2 -used i n t h i s t h e s i s ) . In a survey made on f i s h tissue and surface sediments from 10 s i t e s - i n the Fraser Estuary, Hall et al.(1984) reported high levels of chlorinated phenols i n starry flounder tissue ( 0.19-2.52 ug/g TTCP and 0.77-2.77 ug/g PCP on wet weight basis ).-At lower concentrations, chlorophenols were reported i n the tissues of various biota by other researchers(Carey, 1985; Rogers, 1979; Johnston et a l . , 1975; Bawden et a l . , 1973). Chlorophenols are not supposed to enter the aquatic environment, but the available data show t h e i r presence i n the biota, sediments, and the water column. Are the levels found of any immediate danger to the environment, or would they have a long term e f f e c t ? . On the basis of the available data on acute and chronic t o x i c i t y of chlorophenols, safe concentrations of TTCP and PCP( the main ingredients i n the solution formulations) i n water could be established. Fox( 1980) recommended that PCP i n water should not exceed a concentration of 0.04 ppb for the protection of the aquatic l i f e . So f a r , guidelines ex i s t for various municipal and i n d u s t r i a l effluents and for handling of hazardous wastes containing CPs. The study of the chlorophenol presence i n the Fraser River Estuary has two fronts of action: one i s to es t a b l i s h the background leve l s of chlorophenol i n the water and the other i s to monitor t h e i r concentration v a r i a t i o n and compliance with regulatory levels which are s t i l l to be established. The use of biota to assess and monitor the levels of various pollutants makes very much sense when monitoring i s considered. The use of a - 3 -bioindicator organism to monitor i n s i t u levels of contaminants i s not a new technique. However, leeches have received very l i t t l e attention as b i o l o g i c a l monitors. Recent research(Metcalfe et a l . ,1984) found that leeches could accumulate high levels of CPs and i t was suggested that they could be used as "early warning" indicators of contamination of the aquatic environment by organic pollutants. Leeches are invertebrates which belong to phylum Annelida, class Hirudinea. In North America there are 5 families of leeches. They are excellent candidates for bioindicator organisms due to t h e i r resistance to toxic compounds, natural abundance, reasonable s i z e , long survival i n the laboratory, tolerance for brackish water, starvation resistance, bioconcentration c a p a b i l i t y , and simple c o r r e l a t i o n established between t h e i r body pollutant content and concentration of pollutant i n the surrounding water. The objective of t h i s i n v e s t i g a t i o n was to evaluate leeches as a bioindicator organism for chlorinated phenols contamination. Contrary to previous research,the leeches were not c o l l e c t e d from the region of study, but they were col l e c t e d from a p r i s t i n e environment, implanted i n bioassay chambers i n the study region, and exposed for a known period of time. The bioconcentration of CPs i n the leech was used to provide an estimate of the p o l l u t i o n pattern i n a reach of the lower Fraser River. Laboratory studies were conducted to determine the effects of temperature, pH, exposure time, and p o l l u t i o n concentration on the bioconcentration p o t e n t i a l . 2.LITERATURE REVIEW 2.1 Introduction The l i t e r a t u r e review has been divided into three d i s t i n c t i v e sections: the f i r s t section deals with the presence of chlorophenol i n the Canadian environment with emphasis on the Fraser River estuary ; the second section discusses the enviromental fate of chlorophenols with emphasis on bioaccumulation phenomenon, models and parameters which influence laboratory determined bioconcentration factors; the t h i r d section reviews the problems encountered i n interpreting r e s u l t s of f i e l d surveys using biota as p o l l u t i o n indicators and highlights the potential use of leeches as bioindicators as r e f l e c t e d i n some very recent research. 2.2 Chlorophenol Presence i n the Canadian Environment As Jones reported i n a technical review which examined the presence of chlorophenol i n the Canadian environment(EPS,1981), chlorophenols are ubiquitous i n Canada. They have been detected i n surface water, r a i n , snow, l a n d f i l l leachate, sewage ef f l u e n t , sediments, aquatic and t e r r e s t r i a l biota. Major sources of chlorophenols to the environment are wood preservation plant s i t e s , a g r i c u l t u r a l land subjected to the application of phenoxy acid herbicides, and municipal sewage treatment plants. Some other possible sources are improper disposal of contaminated wastes and s p i l l s during manufacturing , storage, and use of , s t o r a g e , and use of h e r b i c i d e s and wood t r e a t m e n t c h e m i c a l s which c o n t a i n c h l o r o p h e n o l s . C h l o r o p h e n o l s have a l o n g h i s t o r y i n t h e environment. TTCPs and PCP have been i d e n t i f i e d i n sediments d e p o s i t e d a n n u a l l y s i n c e 1949 i n the Bay of Q u i n t e a r e a , Lake O n t a r i o ( E P S , 1 9 8 3 ) . A s s i g n e d i n i t i a l l y t o t h e C a t e g o r y I I I of Environment Canada's L i s t o f P r i o r i t y C h e m i c a l s , c h l o r o p h e n o l s a r e now l i s t e d i n C a t e g o r y I I o f t h e l i s t . Compounds i n t h i s c a t e g o r y a r e c h a r a c t e r i z e d as "s u b s t a n c e s which t h e goverment i b e l i e v e s may pose a s i g n i f i c a n t danger t o t h e environment o r human h e a l t h and about which f u r t h e r d e t a i l e d i n f o r m a t i o n i s r e q u i r e d " . On November 28,1980, A g r i c u l t u r e Canada announced i n Memorandum T-l-229(EPS,1981) a r e v i s i o n i n t h e use s t a n d a r d s f o r c h l o r o p h e n o l s . The major uses s t i l l i n f o r c e f o r c h l o r o p h e n o l s a r e f o r l o n g term wood p r e s e r v a t i o n and s h o r t term wood p r o t e c t i o n . 2.2.1 C h l o r o p h e n o l R e l a t e d Problems i n the F r a s e r R i v e r E s t u a r y The F r a s e r R i v e r i s t h e l a r g e s t r i v e r system and one o f t h e most i m p o r t a n t a q u a t i c environments i n B r i t i s h C olumbia. I t i s used i n a m u l t i t u d e of ways as i t c o n s t i t u t e s a major f i s h way, an i m p o r t a n t means of t r a n s p o r t a t i o n , and main r e c e i v e r of i n d u s t r i a l , a g r i c u l t u r a l and m u n i c i p a l d i s c h a r g e s . In t h e p a s t 10 y e a r s i t became c l e a r t h a t an i n t e g r a t e d management p l a n was n e c e s s a r y t o p r e s e r v e t h i s v a l u a b l e r e s o u r c e . As a r e s u l t , p r o v i n c i a l and f e d e r a l goverments undertook a s e r i e s o f s t u d i e s i n t o v a r i o u s a s p e c t s of t h e F r a s e r R i v e r e s t u a r y , such as l a n d - 6 -use, recreation, habitat and water quality(Fraser River Estuary Study,1980). A Water Quality Monitoring Program was i n i t i a t e d i n late 1985 and studies are currently under way, f i n a n c i a l and technical support being provided by both p r o v i n c i a l and federal goverments. In B r i t i s h Columbia, the main source of CPs to the environment i s from t h e i r use in the wood protection and preservation industry. Approximately 900 tonnes of chlorophenols are used annually in B.C.(Garrett,1980). Due to th e i r environmental persistence! e s p e c i a l l y the higher chlorinated congeners) and to the fact that some i n d u s t r i a l formulations contain highly toxic impurities such as dioxins and dibenzofurans, chlorophenols have been subject to a few thorough studies and investigations. In 1981, a Task Force was formed to respond to worker health and environmental impact concerns regarding the use of chlorophenols at saw m i l l s and lumber export terminals. As a r e s u l t of t h e i r investigations a document on recommendations for design and operation of wood treatment f a c i l i t i e s was released! Konasevich et al.,1983). This document outlined recommendations for design features for chlorophenate storage, feed and application f a c i l i t i e s , and recommendations for treated lumber storage areas, recommendations on operating practices, transportation of chlorophenate containing materials, disposal of wastes, and s p i l l contingency plans. The Environmental Protection Service( EPS) i s presently conducting a survey on the stormwater run-off at various forest industry locations using chlorophenols for wood preservation to determine the effectiveness of current recommendations. Several commercial formulations are available i n B.C. Sodium s a l t s of TTCP and PCP at a r a t i o ranging from 2:1 to 4:1 are used primarily i n B.C.,either by spray application or by submerging the lumber i n a dip tank containing the treatment solutions. Table 2.1 presents the most used commercial formulations i n B r i t i s h Columbia. The use, transportation, and disposal of chlorophenols i n B r i t i s h Columbia i s regulated or "could " be regulated under 6 federal and 4 p r o v i n c i a l L e g i s l a t i v e Acts(Konasevich et a l . , 1983). 2.2.2 Chlorophenols Levels i n the Fraser River Estuary One of the most comprehensive studies on chlorophenol evironmental contamination by the wood preservation industry i n B r i t i s h Columbia i s the EPS Regional Report 79-24( EPS, 1979). Areas covered were the lower east coast of Vancouver Island and the lower mainland of B r i t i s h Columbia(see F i g . 2.1.a and F i g . 2.1.b for the sample s i t e s locations). Analysis of contaminants was done on a variety of samples comprising sediments, surface water, ef f l u e n t and b i o t a ( f i s h , molluscs and crustaceans). It was found that PCP and TTCP were present i n th,e aquatic environment at a l l s i t e s where PCP formulations were used. Table 2.2 shows the levels of chlorophenols found i n sediments and water.at various s i t e s . The sediment levels of PCP varied from 5.0 to 187.9 ppb and the water levels from < 0.05 to> 7.3 ppb. The TTCP concentrations ranged from 10.0 to 272.1 ppb i n sediments and from 0.06 to 5.2 ppb i n water. - 8 -TABLE 2.1 COMMERCIAL SAPSTAIN CONTROL FORMULATIONS IN BRITISH COLUMBIA ( from Konasevich et a l . , 1985) COMMERCIAL NAME FORMULATOR C O M P O S I T I O N CHLOROPHENOL ACTIVE INGREDIENTS (Percent by Weight) TETRA* PENTA** OTHER*** ADDITIVES WOODBRITE 24 VAN WATERS 6 ROGERS LIMITED 16.3 7.7 SODIUM TETRABORATE BUFFER DIATOX WOODSHEATH DIACHEM LTD. WALKER BROS. LTD. 19.4 5.4 4.8 1. 5 UNSPECIFIED BUFFERS UNSPECIFIED BUFFERS COLOUR WAX WOODSHEATH (SEABRITE) WALKER BROS. LTD. 11.35 2.85 UNSPECIFIED BUFFERS COLOUR WAX ALCHEM 4135 ALCHEM LTD. 27. 0 UNSPECIFIED BUFFERS TRIBUTYLTIN OXIDE *SODIUM TETRACHLOROPHENA TE •*SODIUM PENTACHLOROPHENA TE ""UNSPECIFIED CHLOROPHENA TES - 9 -Fig.2.1.a. Locational map of the Lower Mainland, B r i t i s h Columbia, showing sampling s i t e s used for chlorophenol survey.(reproduced from EPS Report 79-24). Fig.2.1.b. Locational map of Southern Vancouver Island, B r i t i s h Columbia, showing sampling s i t e s used for chlorophenol survey.(reproduced from EPS Report 79-24). - 10 -TABLE 2.2 AVERAGE(1) SEDIMENT AND WATER CONCENTRATIONS FOR CHLOROPHENOLS, CHLORINATED BENZENES, AND PENTACHLOROANISOLES(2) (reproduced from EPS Report 79-24) S I T E SEDIMENT LEVELS Wat sr Levels (3J 5 CP 4 CP 3CP 6CB 5CB 4CB 5CA 5 CP 4 CP 5CA C I N . D . N . D . N . D . N . D . N . D . N . D . N . D . N . D . N . D . N . D . F r e s h w a t e r I I 3 5 . 0 2 8 . 0 N . D . N .D . N . D . N . D . N . D . 0 . 2 8 0 . 1 0 N . D . I I I 1 0 . 8 2 7 . 4 2 . 3 N . D . N . D . N . D . 2 . 6 0 . 2 5 1.0 N . D . IV 1 8 . 1 2 1 . 9 N . D . 0 . 7 3 N . D . N . D . 3 1 . 1 <0.05 0 . 3 0 N . D . V 5 . 0 1 0 . 0 N . D . N . D . N . D . N . D . N . D . <0.05 0 . 2 0 .006 S a l t w a t e r I 3 4 . 7 3 9 . 8 N . D . N . D . N . D . N . D . 0 . 5 3 0 . 7 5 1 .3 N . D . V I 5 2 . 8 9 8 . 7 5 2 . 1 N . D . N . D . N . D . N . D . 2 . 4 5 . 2 . 020 V I I 1 0 6 . 6 2 7 2 . 1 9 1 . 0 3 .12 1 .9 2 . 76 3 . 5 3 <0.01 0 . 0 6 .005 V I I I 1 6 . 0 1 9 . 5 N . D . N .D . N . D . N . D . 0 . 2 8 <0.05 0 . 0 9 N . D . I X 4 2 . 0 6 5 . 4 N . D . N .D . 0 . 8 3 0 . 71 4 . 3 1 <0.05 0 . 0 6 N . D . X 1 3 . 1 2 2 . 8 N . D . N . D . N . D . N . D . 0 . 5 8 3 .1 3 . 3 . 0 0 5 X I 1 8 7 . 9 1 5 7 . 3 3 7 . 3 9 . 1 9 9 . 7 5 6 4 . 9 1 4 . 8 9 7 . 3 0 . 2 2 N . D . (1) sediment concentrations are expressed on dry weight basis; average of 10 samples i s reported. (2) a l l concentrations are i n ppb, N.D.= not detected. (3) the water concentrations are the r e s u l t of only one sample. 5 CP = P e n t a c h l o r o p h e n o l 4 CP = T e t r a c h l o r o p h e n o l 3 CP = T r i c h l o r o p h e n o l 6 C3 = H e x a c h l o r o b e n z e n e 5 CB = P e n t a c h l o r o b e n z e n e 4 CB = T e t r a c h l o r o b e n z e n e 5 CA = P e n t a c h l o r o a n i s o l e - 11 -Sculpin l i v e r tissue exhibited p r e f e r e n t i a l uptake with bioaccumulation factors( tissue:sediment ratios) of 7-16 for TTCP and 19-33 for PCP with l i v e r tissue burdens averaging 402 and 448 ppb respectively(dry wt). Livers from p r i c k l y sculpins Cottus  asper and staghorn sculpins Leptocottus armatus always contained PCP and TTCP at concentrations 1-3 orders of magnitude greater than s k e l e t a l muscle from the same i n d i v i d u a l s . Cain and Swain(1980) detected 57 trace organic compounds i n the wastewaters coming from the three sewage treatment plants( STP) of the Lower Mainland: Annacis Island, Iona Island and Lulu Island( see F i g . 2.1.c for the plant l o c a t i o n s ) . Chlorophenols were i d e n t i f i e d i n a l l three plant e f f l u e n t s . I n i t i a l sampling showed that PCP levels were s i m i l a r at a l l plants sampled and that TTCP concentrations were much higher at the Iona Island plant than at the other two plants. Additional monitoring for phenolic compounds conducted by the same researchers, indicated that PCP and TTCP leve l s were highest at Annacis Island plant and lowest at Iona Island plant. Mean concentrations of PCP i n sewage samples from Annacis, Iona and Lulu Islands plants were 2.9-9.9, 1.3-1.7 and 2.1-3.7 ppb respectively, while TTCP levels were 2.9-9.9, 0.9-1.3 and 1.2-5.3 ppb, respectively. Rogers I. H. et al . ( 1986) have found chlorophenols i n raw sewage at Iona Island STP. Composite samples were taken for a period of two weeks i n the summer of 1983. TTCP and PCP reached maximum level s of 7.8 and 13.2 ppb, respectively. Swain(1980) has reviewed the i n d u s t r i a l wastewater effluents that discharge d i r e c t l y to the Fraser River. The survey included only companies which had a permit for effluent Fig.2.I.e. Location of the sewage treatment plants i n the Fraser River Estuary.( from Cain and Swain, 1980). 1 - 13 -discharges and the leve l s of contaminants i n the discharges were monitored by the Waste Management Branch whose records were used for the compilation of data. Chlorophenols were detected i n effluents coming from Belkin Packaging Ltd., Burnaby; Scott Paper Ltd., New Westminster; MacMillan Bloedell Ltd., New Westminster; MacMillan Bloedel Ltd., and Canadian White Pine D i v i s i o n , Vancouver, a l l located on the North Arm of the Fraser River. He concluded that for proper monitoring of the effluents from the various industries located i n the Fraser River Estuary, bioassay monitoring was the best a l t e r n a t i v e . Cain et al.(1980) reported the presence of 2,4,5-TCP, 2,4,6-TCP, 2,3,4,6-TTCP and PCP i n wastewaters coming from these industries. The leve l s were i n the low ppb range with the highest values of 7.2 ppb for 2,3,4,6-TTCP and 5.4 ppb for PCP. There are other forest industries along the North Arm of the Fraser River which use chlorophenols for long-term wood preservation and short-term wood protection and they could also contribute to the chlorophenol p o l l u t i o n of the Fraser River. These industries include: B r i t i s h Columbia Forest Products Ltd., Vancouver Sawmill D i v i s i o n , Canadian Forest Products Ltd., Eburne Sawmills Di v i s i o n , Westcoast C e l l u f i b r e Industries Ltd., Fraser River Planning M i l l s Ltd., Terminal Sawmills Ltd., Whonnock Industries L t d . ( a l l these industries are located i n the M i t c h e l l Island area of the North Arm; EPS, personal communication). Sampling by the Waste Management Branch has indicated high levels of TTCP(up to 90 ppm), PCP(up to 525 ppm) , o i l s and greases i n the sediments o f f Koppers International pole t r e a t i n g f a c i l i t y . As a r e s u l t , the Waste Management Branch has ordered - l * r -the termination of discharge and the removal of highly contaminated sediments adjacent to the Koppers fa c i l i t y ( G a r r e t t , 1 9 8 0 ) . Most of the available information on organic contaminants levels i n Fraser River f i s h species was obtained from Westwater Research Centre studies. Garrett (1980) has a complete tabulation of r e s u l t s of chlorophenol analysis compiled from the data published by Westwater Research i n 1978(the biota samples were col l e c t e d i n 1973 and analysed i n 1978); Johnston et al.(1975); Bawden et al.(1973); Rogers (1979). Chlorinated phenols were i d e n t i f i e d with greater frequency and at higher concentrations i n f i s h from the i n d u s t r i a l l y developed lower part of the Fraser River. PCP and TTCP concentrations did not exceed 125.0 ppb and 62.0 ppb wet weight respectively. In more recent studies, Hall et a l . (1984) reported very high levels of chlorophenols i n starry flounder tissue with TTCP from 0.19 to 2.5 ug/g and PCP from 0.77 to 2.77 ug/g wet weight( t h i s gives equivalent concentrations expressed i n ppb as high as 2500 ppb and 2770 ppb for TTCP and PCP r e s p e c t i v e l y ) . A l l samples analysed by these researchers were co l l e c t e d i n November and December 1983. Hall compared the lev e l s of chlorophenols found i n starry flounders during his investigation with the levels of chlorophenols found by Singleton( 1983). His r e s u l t s were one or two orders of magnitude higher than found i n Singleton's in v e s t i g a t i o n . Since a viable explanation for the difference i n chlorophenols concentration i n the two species of f i s h was not found more surveys were suggested i n order to i d e n t i f y the cause of such a v a r i a t i o n . - 15 -Carey( 1985), sampled f i s h and water from the Fraser River estuary area i n 1984, and he found TTCP and PCP(other chlorophenols were present but at very low levels) . The highest concentration of TTCP i n water was 0.133 ppb and of PCP was 0.056 ppb. The highest chlorophenol concentration i n biota was found i n staghorn sculpin at 49 ppb TTCP and 79 ppb PCP(average of 12 samples; concentration reported on wet weight b a s i s ) . 2.3 Environmental Fate of Chlorophenols Callahan et a l . (1979) have reviewed the fate and persistence of several chlorophenols i n aquatic systems and i t was concluded that t h e i r fate could not be predicted from system to system. Laboratory studies provide an i n i t i a l i nsight into environmental factors which can regulate chlorophenol persistence, but the best way to study the fate of chlorophenols i s to study t h e i r d i s t r i b u t i o n and persistence i n a natural environment. Included i n t h i s category are the studies following man created small disasters such as various chlorophenols s p i l l s , leaks and disposal malpractices of materials containing these compounds. Pierce et al.(1977) studied the d i s t r i b u t i o n of pentachlorophenol i n a fresh water system following a s p i l l which resulted i n an extensive f i s h k i l l . The res u l t s of t h e i r investigation showed a short residence time for PCP i n the water column, and higher concentrations of PCP i n the sediments(up to 1200 ppb)for a period of several months with a large reduction within a year. Fish contained concentrations of PCP from 130 to 2500 ppb( dry weight) up to six months following the s p i l l ; - 16 -within ten months the concentration i n f i s h dropped to the background l e v e l s . High levels of PCP persisted i n leaf l i t t e r for at least seventeen months. The persistence of chlorophenols i n sediments and leaf l i t t e r constituted a source of continuous, low l e v e l contamination to the water column and f a c i l i t a t e d b i o l o g i c a l magnification v i a detritus and benthic feeding organisms. In order to b u i l d a predictive model of the d i s t r i b u t i o n of chlorophenols i n the environment t h e i r chemical and physical c h a r a c t e r i s t i c s must be thoroughly evaluated. With a l l the e f f o r t put into t h e o r e t i c a l interpretations and descriptive equations, the end r e s u l t may not provide a useful p r e d i c t i v e function and can often be rendered useless by the complexity of interactions in the natural ecosystem. The main part of the environmental analysis i s the estimation of the probable d i s t r i b u t i o n of a chemical i n the various compartments: a i r , water, sediment/soil, and biota. There are seven major processes to be considered when one evaluates the environmental fate of a chemical: v o l a t i l i z a t i o n , photolysis, oxidation, hydrolysis, sorption, biotransformation/biodegradation, and bioaccumulation(NRCC, 1982). It must be c l e a r l y defined that chlorophenols ex i s t i n two forms: the anionic phenolate and the undissociated phenol at a c i d i c pH. For the p a r t i c u l a r case of PCP the r a t i o between undissociated and dissociated forms changes dramatically between pH 3 and 8 (NRCC, 1982). At pH 5, the r a t i o i s 1:1 and one can e a s i l y see that at regular fresh water pH 6-8 range, most of the - 17 -PCP present i n the water column would be i n the phenolate form. Due to the influence of pH upon the form of chlorophenols, and because of the d i f f e r e n t r e a c t i v i t i e s of the two forms, the rates of photolysis, v o l a t i l i z a t i o n , s o l u b i l i t y and sorptive potential are greatly influenced by changes i n pH. V o l a t i l i z a t i o n a f f e c t s only the undissociated form, so i t can be concluded that for higher chlorinated chlorophenols v o l a t i l i z a t i o n i n the aquatic environment i s not an important process since at the natural occurring pHs they are i n the i o n i c form. Some researchers, Wong(1978) and K i l z e r et al.(1979) mentioned v o l a t i l i z a t i o n of chlorophenols at higher pH but they did not explain the mechanism of the process. The lower chlorinated chlorophenols are more v o l a t i l e than PCP. Photolysis equations developed by various researchers are greatly dependent on l a t i t u d e , time of day, time of year and l i g h t i n t e n s i t y . The absorbtion spectrum varies with pH and the anion absorbs more strongly than does the undissociated form. Wong and Crosby(1978) indicated that the rate of photolysis depended upon pH. The comparative rates at pH 3 and 7 were: -7 -1 -5 -1 5.2*10 s and 2.7*10 s respectively, a f i f t y f o l d difference consistent with the difference i n anion concentrations at the two pHs. The rate constant for primary photolysis i s also controlled by depth of water which i s the major c o n t r o l l i n g factor for photolysis i n any aquatic system. Oxidation as a possible route of transformation of chlorophenols i n the aquatic environment needs more research. It has been suggested that oxidation makes no contribution to the fate of chlorophenols i n the environment(Callahan et a l . , 1979). - 18 -Hydrolysis i s also considered unimportant i n the fate of chlorophenols i n the aquatic environment. Sediment or s o i l sorption processes have received a l o t of attention from s c i e n t i s t s and Ks, the s o i l p a r t i t i o n c o e f f i c i e n t , was related to Kow(octanol:water p a r t i t i o n c o e f f i c i e n t ) through Kom(organic mattertwater p a r t i t i o n c o e f f i c i e n t ) and Koc(organic carbonrwater p a r t i t i o n coefficient)(NRCC, 1982). The equations developed for these relationships cannot be applied to the i o n i c form of chlorophenols. Sorption for the i o n i c form i s estimated to be n e g l i g i b l e , and the only processes that would f a c i l i t a t e the sorption of t h i s form would be chelation and s p e c i f i c s i t e binding. In the case of PCP, Kom i s about 2200 for the a c i d i c molecular form . The s o i l p a r t i t i o n c o e f f i c i e n t Ks can be as low as 22 for a low( 1%) organic s o i l . Microbial degradation has been observed and the general trend i s that the lower chlorinated chlorophenols are more susceptible to microbial attack. At high concentrations the toxic c h a r a c t e r i s t i c s of chlorophenols i n h i b i t any microbial a c t i v i t y . Baker et al.(1980) demonstrated that PCP was the most res i s t a n t compound to biodegradation i n the chlorophenol family. Bioaccumulation i s a very important process i n assessing the fate and d i s t r i b u t i o n of chlorophenols i n a natural environment and i t w i l l be discussed separately i n the following section. 19 -2.3.1 Bioaccumulation Process The accumulation of cert a i n chemicals by aquatic biota i s a well known process. Many chemicals are p o t e n t i a l l y dangerous to aquatic l i f e and i n d i r e c t l y to human health, therefore the importance of the r e l i a b i l i t y of the bioaccumulation data avail a b l e to the s c i e n t i f i c world i s very important. The terms of bioaccumulation and bioconcentration have had a multitude of d e f i n i t i o n s i n the past which has created a sense of confusion. More recent terminology and d e f i n i t i o n s have been better defined and today i t i s u n i v e r s a l l y accepted that bioconcentration i s represented by the chemical residue obtained d i r e c t l y from water vi a g i l l or e p i t h e l i a l tissues( Brungs and Mount, 1978). Bioaccumulation i s a broader term which refers to residues obtained from both food and water. F i n a l l y , magnification i s the t o t a l process of bioaccumulation by which tissue residues of toxic material pass through two or more trophic l e v e l s . Kenaga (1972) defined the term of "bioconcentration r a t i o " as the r a t i o between residue values and ambient water concentration. For consistency i n terminology, i t implies that the water i s the only source of toxicant.! Early studies concerning biomagnification processes seem to favour the food chain as the main contributor to the accumulation of chemicals i n biota. More recent studies provide the opposite evidence, namely that the bioconcentration i s one of the main processes. In the aquatic environment, d i r e c t uptake of a chemical from the ambient water w i l l determine the order of - 20 -magnitude of the bioaccumulation factor i n a given organism whereas i n d i r e c t uptake v i a the food chain w i l l increase t h i s factor only moderately( Esser and Moser,1980). Kobayashi et al.(1979) studied the r e l a t i o n between the t o x i c i t y and the accumulation i n goldfish(Carassius auratus), of seven chlorophenols and noticed that the increase of the Cl-atom number i n the chlorophenols caused an abrupt increase i n t o x i c i t y to the f i s h . This observation i s not new to t o x i c o l o g i s t s and the s c i e n t i f i c l i t e r a t u r e ; however, what Kobayashi's re s u l t s revealed was that the increase of t o x i c i t y from the chlorinated phenols i s mostly due to t h e i r r e l a t i v e accumulation in f i s h and not to t h e i r i n t r i n s i c t o x i c i t y . Chlorophenol concentrations i n f i s h achieved a cer t a i n l e t h a l l e v e l , roughly 100-200 ug/g body weight and only the time to achieve t h i s concentration i n the f i s h tissue d i f f e r e n t i a t e d the t o x i c i t y of various chlorophenols. 2.3.2 Laboratory Determination of Bioconcentration Factors Three procedures are used to estimate the bioaccumulation or the bioconcentration factor i f only exposure to the contaminant in water i s considered. These include: deduction from the Po/w, n-octanol:water p a r t i t i o n c o e f f i c i e n t . -determination of steady-state p a r t i t i o n i n g . -determination of the r a t i o of uptake and depuration rates. As the bioconcentration i s based on p a r t i t i o n / s o r p t i o n phenomena, l i p o p h i l i c i t y of the chemicals i s expected to play a major r o l e . Consequently, the Po/w, as a measure of l i p o p h i l i c i t y , should be - 21 -correlated to the bioconcentration factor. There are several methods of experimentally determining Po/w and a l i s t of d i r e c t and i n d i r e c t methods of determination of log Po/w and the problems and l i m i t a t i o n s existent i n each case i s presented by Esser and Moser(1980). Estimation of Po/w from water s o l u b i l i t y has a l o t of drawbacks. Correlation established between the two parameters seem to be r e l i a b l e only for a series of s t r u c t u r a l l y related chemicals. Also ,as Todd et al.(1980) reported i n t h e i r study, s o l u b i l i t y data for organic compounds of low s o l u b i l i t y i n water are very unreliable. They concluded that a given s o l u b i l i t y value i s strongly dependent on the experimental method used to obtain i t . The same observation i s v a l i d for the determination of Po/w by other methods. The l i t e r a t u r e reports a variety of Po/w values for each compound and care must be used when they are considered i n bioconcentration factor relationships( Esser and Moser, 1980). Ionizable compounds such as chlorophenols pose another problem for the determination of Po/w. It i s known that the introduction of charges into a molecule decreases l i p o p h i l i c i t y . The degree of i o n i z a t i o n of the weak acids and bases depend on t h e i r pKa values. A model compound with pKa 4.2, i s 99.9% ionized at pH 7.2, which results i n a concomitant decrease of the o r i g i n a l Po/w by three orders of magnitude. For reasons of better standardization, Esser and Moser(1980) recommend that the Po/w of weakly ionized chemicals be measured by using a buffer of physiological pH and sodium chloride concentration as the aqueous phase. The influence of pH upon Po/w may explain the large - 2 2 -v a r i a b i l i t y of t h i s parameter value reported for chlorophenols. Most values are reported without mentioning the experimental pH, which makes the reported values hard to compare. Some of the regression equations published for the re l a t i o n s h i p between the bioconcentration factor(BF) and Po/w of various chemical compounds are: -Log BF=0.542*Log Po/w + 0.124 ; equation was developed by Neely et al.(1974) using the r a t i o between the rate constants of uptake and depuration i n trout. -Log BF=0.85*Log Po/w - 0.70 ; equation was developed by Veith et al.(1979) using the steady-state approach. Organism of study was fathead minnow. -Log BF=0.935*LogPo/w - 1.495 ; equation was developed by Kenaga and Goring (1978) using the steady-state approach on various biota. -Log BF=0.83*LogPo/w -1.71 ; equation was developed by Ellgehausen et al.(1980) using the steady-state and depuration k i n e t i c s . Organism of study was c a t f i s h . If one considers a chemical with a Po/w of 1000, the corresponding bioconcentration factors calculated from these equations d i f f e r by roughly one order of magnitude. The BF values determined from the above equations are 56, 78, 24, and 6, respectively. These short calculations also show that values of - 23 -Po/w above 1000 are necessary before the corresponding BF exceeds 100 . If experimental determination of the BF by the steady-state approach i s used, a very s t r i c t standardization of the experimental conditions must be imposed. Factors such as v a r i a t i o n i n species, biomass/volume r a t i o , temperature, water c h a r a c t e r i s t i c s , a n a l y t i c a l methodology, samples analyzed, and test chemical properties such as weak i o n i z a t i o n , high s o r p t i v i t y , molecular s i z e , v o l a t i l i t y , and degradability may contribute to the v a r i a t i o n of the measured BF's by factors of up to 5 and more( Esser and Moser,1980). If the BF i s determined from the r a t i o of uptake to depuration rate constants, i t means that f i r s t - o r d e r k i n e t i c s are assumed for both processes. Some studies have shown that depuration i s best described by second-order rate equations(Esser and Moser,1980).Their studies on c a t f i s h showed that the h a l f - l i f e of depuration depended upon the i n i t i a l concentration. The most commonly used k i n e t i c description of the process of bioaccumulation i s based on the "one . compartment" model, described by the equation! Hamelink,1977): CF=K1*CW*(1-EXP(-K2*T))/K2 where: CF,CW = concentration of the chemical i n f i s h and water, respectively Kl,K2 =uptake and excretion rate constants[1/time], respectively. T = temperature The bioconcentration factor i s given by: BF=Kl/K2. The rate constants Kl and K2 have to be determined. A computer program - 2b -BIOFAC may be used to f i t the data to the equation. This computer program or others that are commercially available require i n i t i a l estimates of Kl and K2. Often experimental scatter i s such that the use of sophisticated f i t t i n g routines i s not warranted and simpler procedures produce adequate r e s u l t s . If Kl i s expressed as a function of BF and K2 the following equation i s obtained: (CF/CW)/BF=1-EXP(-K2 *T) It means that i f K2 i s known the BF can be calculated. The same equation can also be used i f the accumulation curve shows signs of reaching equilibrium. Curtis et al.(1977) used the "two compartment" model and obtained an empirical function to describe the accumulation of methyl mercury i n blue g i l l sunfish(Lepomis macrochirus). The disadvantage of the empirical function i s that the parameters used have no physical meaning. The one compartment model i s an ov e r s i m p l i f i c a t i o n but the application of two or more compartments(Moriarty 1975;Robinson 1975) i s d i f f i c u l t to use since the experimental data are seldom detailed and accurate enough to warrant t h e i r use. In a l l models developed so far i t has been assumed that the uptake and excretion rate constants Kl and K2, and consequently BF, are independent on concentration of the chemicals. Experimental data indicate that t h i s i s frequently not the case and that the bioconcentration factor usually decreases(but sometimes increases) with increasing concentration of the chemical(Zitko,1980). Majori and Petronio(1973) demonstrated that the decrease in - 25 -BF with increasing CW follows the law of mass action. They also assumed that the concentration i n fish(CF) cannot exceed a certain maximum concentration CT("concentration of binding centers") and that the rate of uptake i s proportional not only to the concentration of the chemical i n water(CW) but to the difference CT-CF("concentration of free binding centers") as well. Using t h i s approach means that the excretion rate constant i s higher during the accumulation phase than that during the excretion phase. According to S t r e i t and Schwoerbel (1976) the equilibrium concentration of atrazine i n the leech(Glossiphonia complanata) i s given by the following power function for a CW range from 0.001 to 1 mg/L : CFE=8.690*CW**0.74 0 The above equation was approximated by the function : CFE=K1*CW*CT/(K1*CW+K2) for CT=6 and K2/Kl=0.201. K2 was estimated from excretion data; consequently Kl=0.91. CFE i s the concentration i n f i s h at equilibrium, therefore, CFE=BF. Comparison between the experimental and calculated data showed a very good agreement. From the data of Hansen et al.(1974) the CFE's of Aroclor 1016 i n American oysters(Crassostrea v i r g i n i c a ) and grass shrimp(Palaemonetes pugio) but not those i n the pinfish(Lagodon  rhomboides) can be expressed as a function of CW. Attention should be paid to the p o s s i b i l i t y that the BF values reported i n the l i t e r a t u r e may not be for an equilibrium BF.This can be e a s i l y checked, i f the excretion rate constant i s known, by applying the equation:(CF/CW)/BF=1-EXP(-K2*T) (Zitko,1980). As - 26 -also i l l u s t r a t e d , the BFs often depend on the concentration of the chemical i n water and i t i s expected that they increase with decreasing concentration. Generally speaking, compounds that have BFs of more than about 200-300 i n laboratory t e s t s , and excretion rate constants of less than 0.02 day-1, are potential suspects for bioaccumulation problems. Even.in the absence of demonstrated toxic e f f e c t s such compounds must be scrutinized carefully,because there i s always a potential for subtle chronic ef f e c t s when compounds reside i n an organism for considerable periods of time. Zitko,(1980) presents a very comprehensive l i s t of values for Kl , K2 and log Po/w compiled from the existent l i t e r a t u r e . The values quoted for pentachlorophenol are the ones reported by Glickman et al.(1977): Kl=74 ; K2=2.4 and logPo/w=5.01. One has to bear i n mind that the general equations established for BF give only rough estimates. They have been derived from data obtained on a variety of compounds, under d i f f e r e n t conditions and by various techniques. For a quick orientation, some predicted values of BF are given i n Table 2.3. It appears that accumulation problems are l i k e l y i n case of compounds with s o l u b i l i t i e s in water below 1 mg/L and octanol/water p a r t i t i o n c o e f f i c i e n t higher than 3*10^ (log Po/w higher than 4.5). If one needs an accurate value for BF, the experimental method i n which the chemical under study i s tested on the species of i n t e r e s t appears to be the most appropiate technique. - 2? -TABLE 2.3 BIOCONCENTRATION FACTORS CALCULATED FROM WATER SOLUBILITY AND OCTANOL:WATER PARTITION COEFFICIENTS Water s o l u b i l i t y BCF [mg/L] KOW LOG(KOW) from WS from K0W(18) 0.001 300,000 5.5 30,000 4,400 0.01 110,000 5 8,300 1,500 ! 0.1 40,000 4.6 2,300 640 i 1 14,000 4.1 600 200 10 5,000 3.7 170 90 100 2,000 3.3 50 40 BCF = bioconcentration factor KOW = octanol:water p a r t i t i o n c o e f f i c i e n t WS = water s o l u b i l i t y BCF values were calculated using relationships established by Kenaga and Goring(1978): LOG(BCF) = -1.495 + 0.935 LOG(KOW) LOG(BCF) = 2.791 - 0.564 LOG(WS) - 2!;8-2.3.2.1 Bioenergetics and Its Influence on the Bioaccumulation  Process The influence of age, body weight, and l i p i d content on chemical residue levels i n biota have been evaluated by many authors. No consistent pattern has emerged and i t i s not established yet i f residue concentrations should be reported on a l i p i d basis or a whole body weight basis. In numerous cases reporting the residues as a function of the l i p i d content helped in reducing the v a r i a b i l i t y among samples. As Hamelink and Spacie(1977) remarked, the r e s i d u e - l i p i d content correlations do not necessarily prove a cause and e f f e c t r e l a t i o n s h i p . That i s , the presence of more l i p i d s may not cause greater quantities of residues to be accumulated ;rather the factors that r e s u l t s in l i p i d deposition may also promote residue storage. Fish must obviously expend energy i n order to acquire and store energy. Since l i p i d s have a greater c a l o r i c content than muscle, a fat f i s h must do more eating, swimming and r e s p i r i n g than a lean f i s h of the same age. As a r e s u l t a fat f i s h has more opportunity to take up residues than a lean f i s h . The fact that a fat f i s h could re t a i n greater concentrations of residue was probably just related to the l i p o p h i l i c nature of the chemicals. The fat just happened to be a convenient place to store the chemicals, but the presence of the l i p i d s did not cause the compounds to be taken up and stored. The factors of age and weight cannot be separated because they normally co-vary i n biota. Correlations established between residue concentration and weight of fishes suggest that the - 29 -mechanism of residue uptake i s ultimately linked to the metabolic a c t i v i t i e s . Norstrom et al.(1976) reasoned that the uptake of pollutants should f a l l within the l i m i t s set by those factors that control metabolism and growth, as modified by environmental factors such as temperature and food a v a i l a b i l i t y . To evaluate t h i s concept they developed a pollutant accumulation model based on f i s h bioenergetics combined with some data on pollutant b i o k i n e t i c s . In essence they devised an equation that stated that pollutant body burden changes with respect to time were equal to the uptake from the water plus the uptake from the food minus the depuration rate, wherein each rate was modified by complex body weight-dependent functions. The obvious disadvantage of t h i s approach i s the d i f f i c u l t y one encounters i n measuring a l l of the various metabolic rates constants needed for the model.The authors demonstrated that for PCBs and mercury i n yellow perch (Perca flavesens) the model can be approximated by: dP/dt = AW0'70- kPW- ° ' 5 8 where A i s a constant which combines a l l the c o e f f i c i e n t s i n the uptake parts of the model, W the body weight, and P the pollutant body burden. Since t h i s i s e s s e n t i a l l y the equation for a simple compartment model, one might expect an equilibrium state to be reached. However the exponents operating on the changes i n body weight prevent an equilibrium from a c t u a l l y being achieved. That i s , i f the r e l a t i v e value of the exponent term on body weight i s correct, then the uptake factors for methyl mercury and PCBs have increasing power over the depuration factors as the weight of the aquatic animal increases with time. Thus, i f a compound i s - 30 -persistent, has a high bioconcentration p o t e n t i a l , and the animal gains appreciable weight i n time, there i s more opportunity to c a p i t a l i z e on these seemingly small differences and steady state cannot be reached. The absolute value of these exponents must vary between species and between various l i f e - s t a g e s for each species( Hamelink and Spacie,1977). Kinetic rates and the r e l a t i v e importance of the body weight change as the f i s h grows, matures, spawns, and f i n a l l y reaches senescence . Seasonal factors(such as temperature) a l t e r food consumption, habitat se l e c t i o n , etc. Each of these factors contributes to the bioenergetics of the f i s h and i t s subsequent bioaccumulation of various contaminants. Thus, to be precise would require a combined biomass-bioenergetics model which incorporates various subroutines for i n d i v i d u a l f i s h stocks that might be further subdivided on the basis of sex and year class.As Hamelink and Spacie (1977) concluded t h i s level of refinement would be a monumental task which, with the exception of being informative, would achieve very l i t t l e i n answering more important questions pertinent to the environmental behaviour of various pollutants. By applying simpler s t r u c t u r e - a c t i v i t y concepts developed by Hansch or Veith (Veith et al.(1975), one can achieve a quicker insight into the problem. The environmental behaviour of the pollutants appears to depend more on t h e i r chemical-physical properties than on the b i o l o g i c a l - e c o l o g i c a l features of the receiving body. As Hamelink and Spacie(1977) remarked , d i r e c t a p p l i c a t i o n of a p a r t i t i o n c o e f f i c i e n t regression appears to underestimate bioaccumulation of compounds having P values - 31 -greater than log 6. Compounds in t h i s class often display bioconcentration factors greater than log 5 i n whole f i s h from natural environments. In t h i s case the time required to reach a steady state i s extremely long and growth and fat deposition have to be considered i n the analysis. Consequently the models developed for these super-pollutants should incorporate the bioenergetics of the f i s h . 2.3.2.2 Influence of pH on the Bioconcentration of Pollutants It i s well-known that pH does a f f e c t the t o x i c i t y of ionizable substances such as phenol derivatives. It i s surprising how l i t t l e attention has been paid i n so many studies to the influence of pH. QSARs(Quantitative Structure-Activity Relationships) are becoming increasingly helpful i n aquatic t o x i c i t y research. Several authors established the t o x i c i t y of phenol derivatives and calculated QSARs. Meister(1977) used the mathematical Free-Wilson method and others( Hansch and Fujita(1964), Zitko(1976), Durkin(1978), Kopperman et al.(1974) have used the Hansch approach to calculate QSARs. However, no attention has been paid to the influence of the pH upon the QSARs. Explanations to account for t h i s influence have been published by Lloyd and Herbert (1960) and Tabata(1962). Variations of the two approaches have been presented i n the l i t e r a t u r e i n past years. Most researchers share the opinion that the Tabata method - 32 -i s the most useful(Konemann and Musch, 1981; Saarikovski and V i l u k s e l a , 1981). Tabata studied the influence of pH on the t o x i c i t y of ammonia to aquatic organisms. He developed the concept that both ionized and unionized forms of a compound contribute to i t s t o x i c i t y , but the unionized form was usually more toxic than the ionized one (due to the differences in uptake).Konemann and Musch (1981) have used Tabata's model to develop a formula describing the influence of pH on the t o x i c i t y of chlorophenols to f i s h . They used 11 chlorophenols in t h e i r study on guppies(Poecilia r e t i c u l a t a ) and determined t o x i c i t i e s at 3 pH values. Using t h e i r formula they determined t o x i c i t y at another pH. QSARs were established for the LC50's at a l l the pH values considered, with log Poet(octanol:water p a r t i t i o n c o e f f i c i e n t ) and pKa as variables(Table 2.4 summarizes t h e i r r e s u l t s ) . As can be seen the t o x i c i t y of the chlorophenols increases with decreasing pH. Maximum t o x i c i t y i s reached when LC50=l/Tm (Tm i s 1/LC50 of the pure molecular form). The pH of 3 represents t h i s s i t u a t i o n for a l l the chlorophenols. Experimental determination of the LC50 at t h i s pH are not possible so Tm can be calculated from the graphical representation as the intercept when p l o t t i n g 1/LC50 against Ka/([H+] + Ka). QSARs have been obtained at various pHs using Po/w as a sole parameter or including pKa values as well. The QSARs with pKa were better than the corresponding equations without pKa. According to the authors there are at least three possible causes of the influence of the pKa on the t o x i c i t y . F i r s t , i s - 33 -TABLE 2.4 CHLOROPHENOL TOXICITY TO GUPPIES AT DIFFERENT pHs (from Konemann and Musch, 1981) LC„'s AND OTHER DATA FOR QSAR CALCULATIONS Substance P>0 log LC « at pH log l /*" m 7.8 7.3 6.1 Phenol 1.55 9.92 2.52 2.50 2.59 2.55 2-Chlorophenol 2.27 8.52 2.02 1.94 1.74 1.77 3-Chlorophenol 2.27 8.97 1.79 1.70 1.70 1.69 2,4-Dichlorophenol 2.93 7.90 1.56 1.41 1.30 1.30 3,5-Dichlorphenol 2.93 8.25 1.46 1.22 1.20 1.18 2,3,5-Trichlorophenol 3.69 6.43 1.38 0.90 0.65 0.52 2,3,6-Trichlorophenol 3.69 5.80 1.83 1.41 0.68 0.23 3,4,5-Trichlorophenol 3.69 7.55 1.08 0.76 0.76 0.73 2,3,4,5-Tetrachlorophenol 4.42 5.64 1.00 0.52 0.28 -0.19 2,3,5,6-Tetrachlorophenol 4.42 5.03 1.23 0.77 0.23 -0.81 Pentachlorophenol 5.19 4.74 0.46 0.15 -0.32 -1.60 'Calculated after Rekker (ref. 13), for the undissociated form. From Drahohovsky (14). lInMmol/l. Poet i s the octanol:water p a r t i t i o n c o e f f i c i e n t Tm i s the 1/LC50 of the pure molecular form - j>k -the e f f e c t of pH on the accumulation: the molecular form of an acid can pass through membranes (the g i l l s or e p i t h e l i a l tissue) in both directions faster than the i o n i c form. Thus , the uptake and elimination rate constants,with respect to the t o t a l concentration of a p a r t l y dissociated acid, depend on both pKa and pH and therefore bioaccumulation also depends on these factors. At a given t o t a l concentration of the acid, the t o t a l uptake rate increases with decreasing external pH, because the r e l a t i v e concentration of the molecular form outside the f i s h increases, as the r e l a t i v e concentration inside the f i s h can be assumed to remain constant. When the int e r n a l pH i s higher than the external pH, i o n i z a t i o n can play a role inside the f i s h , and t h i s w i l l reduce the elimination and therefore increase the accumulation. Second, the toxic action of the phenolic compounds can be caused by either the molecular or the i o n i c form or both of them; At a given t o t a l concentration i n f i s h the concentration of the more active form depends on pKa. Third, the i n t e r r a c t i o n with the receptor of the active form of the chemical can depend on i t s electron configuration, which also governs the pKa. In t h i s way pKa and t h i s i n t e r r a c t i o n can be correlated. In contrast to the f i r s t one, the l a s t 2 pKa influences w i l l not depend d i r e c t l y on the external pH. Saarikovski and V i l u k s e l a (1981) have studied the influence of pH on the t o x i c i t y of substituted phenols to the guppy(Poecilia r e t i c u l a t a ) . The 96-hr LC50 values were determined by a semistatic method i n the pH range of 5 to 8. The pH did not a f f e c t appreciably the t o x i c i t y of 4-chlorophenol, but the t o x i c i t y of more a c i d i c phenols decreased as the pH - 35 -increased. The changes i n t o x i c i t y were su b s t a n t i a l l y smaller than they would be , i f only the nonionized form were toxi c . The r e s u l t s could be explained by assuming that the phenate ion also contributes to the t o x i c i t y , but i t s molar t o x i c i t y decreases with r i s i n g pH.The authors main assumption was that the absorption of phenols from water i s the main factor influencing the t o x i c i t y , which i s p r i n c i p a l l y , i f not s o l e l y , affected by pH changes. The change i n the pH of the surrounding water i s not l i k e l y to a f f e c t the d i s t r i b u t i o n , i n t r i n s i c a c t i v i t y , o r metabolism of phenols i n the f i s h since the pH of the guppy's blood i s kept within very narrow l i m i t s ; because phenols are excreted from fishes mostly as neutral conjugates( Kobayashi,1978), t h e i r elimination should also be rather independent of the water pH. Saarikovski and V i l u k s e l a f i t t e d the following equation to t h e i r r e s u l t s : L C 5 0 ( p H l ) / L C 5 0 ( p H 2 ) - (1 + 4>"V ( 1 • 4 P " 2 - * a , The above equation predicted the changes i n t o x i c i t y better than the so-called pH-partition hypothesis, which states that a c i d i c and basic drugs penetrate b i o l o g i c a l membranes only as uncharged molecules( Shore et a l . 1957; Hogben et al.1959; Jollow and Brodie 1972). The explanation given by Saarikovski and V i l u k s e l a was that both the molecular and the i o n i c form contribute to the t o x i c i t y , conforming to the equation! see Table 2.5 for abbreviation d e s c r i p t i o n ) : 1/LC50 = T H A *[HA]/C + T A_ *[A-]/C With t h i s kind of model, Levy and Gucinski(1964) arrived at the r e s u l t that the absorption rate of ionized secobarbital! pKa - 36 -TABLE 2.5 TOXICITY OF THE NONIONIZED AND IONIZED FORMS OF 2,4,6-TRICHLOROPHENOL AND PENTACHLOROPHENOL AT VARIOUS pHs (reproduced a f t e r Saarikovski and V i l u k s e l a , 1981) 2,4,6-Trichlorophcnol |HA|'C Total pH x 100 x 100 toxicity ' ! « . , x ||IA|/C A k 100 (1 344 S . 94 6 323 323 6 61 39 222 210 NS 7 14 86 86 4K 38 44 8 1.6 98.4 25 5.5 19.5 20 Pentachlorophenol |HA|/C |A |/C total pH x 100 x KM) toxicity T«.v x |HA|/C A Tv k 100 0 15800 5 33 67 6250 5214 6 4.R 95.2 2275 758 1517 1593 • i n 7 0.5 99.5 602 79 523 526 .* ,U 1 u 8 0.05 99.95 292 8 284 284 This table was calculated on the basis of the equation: 1/LC50 = T R A * [HA]/C + T A- * [A~]/C T R A = t o x i c i t y of the molecular form T - = t o x i c i t y of the phenate ion [HA] = concentration of the molecular form [A ] = concentration of the i o n i c form C = t o t a l chlorophenol concentration D = difference between t o t a l t o x i c i t y and t o x i c i t y of molecular form k = decrease i n t o x i c i t y of phenate ion as pH increases _ 37 _ 7.92) i n gold f i s h i s 9.6 times lower than that of the molecular form. S i m i l a r l y , Broderius et al.(1977) obtained a r a t i o of 2.3 for the t o x i c i t i e s of HCN and CN- and a r a t i o of 15 for those of H2S and HS-. Table 2.5 reproduced aft e r Saarikovski and Viluksela(1981) shows calculated values for the t o x i c i t i e s of phenate ions for PCP and 2,4,6-TCP. The t o x i c i t y of the phenate ion was calculated by substracting the t o x i c i t y due to the phenol form from the measured o v e r a l l t o x i c i t y and d i v i d i n g with the proportional concentration of the phenate ion. The t o x i c i t y of the phenate ion was not constant, but decreased with r i s i n g pH. This means that the changes in the t o x i c i t y i s caused, i n part, by changes i n the degree of i o n i z a t i o n and, i n part, by changes i n the absorption rate of the phenate ions. With very a c i d i c phenols, the t o t a l t o x i c i t y should be caused s o l e l y by the phenate ions and follow the changes i n ion t o x i c i t y . On t h e o r e t i c a l grounds, the change i n the t o x i c i t y of the phenate ion should be anticipated. An increase i n pH r e s u l t s i n a negative surface potential for the outer membranes of the e p i t h e l i a l c e l l s , which should decrease t h e i r permeability to organic as well as inorganic anions(Wright and Diamond 1968; Deuticke 1977). The increase i n pH may also s h i f t the potential gradient across the g i l l membrane to a negative direction(McWi1liams and Potts 1978), which impedes the d i f f u s i o n of phenate ions from water to f i s h without a f f e c t i n g the absorbtion of the uncharged molecules. In a study published i n 1982, Saarikovski and Viluksela presented more results on t h e i r studies upon the r e l a t i o n - 38 -between the physic-chemical properties of phenols and t h e i r t o x i c i t y and accumulation i n f i s h . This time they determined 96-hr LC50 values of 21 substituted phenols for the guppy (Po e c i l i a r e t i c u l a t a ) i n the pH range 6-8.They determined regression equations of t o x i c i t y on log Po/w and dpKa(difference between the pKa of a substituted phenol and pKa of phenol) at various pH levels i n a manner sim i l a r to Konemann and Musch( 1981). They concluded that l i p o p h i l i c i t y i s the primary parameter determining t o x i c i t y , and that the c o r r e l a t i o n between t o x i c i t y and dpKa i s at least p a r t l y a consequence of i n t e r c o r r e l a t i o n of the parameters. They went even further i n establishing an empirical equation to approximate the t o x i c i t y of a phenol to guppy at some given pH i n the range 6-8: l o g ( l / L C 5 0 p H ) = l o g ( l / L C 5 0 H A ) - l o g ( 4 p H ~ p K a +1), where LC50^ A i s the LC50 value of the nonionized phenol form, pH the pH of the test solution,and pKa the logaritmic i o n i z a t i o n constant of the phenol. When t h i s formula i s used to correct the t o x i c i t y values for ionization,and new values employed i n the . regression analysis, the r e s u l t i n g equations are, i n p r i n c i p l e , independent of the pH of water. They can, for example, be derived from t o x i c i t y values measured at d i f f e r e n t pH l e v e l s . B a s i c a l l y they r e l a t e the t o x i c i t y which phenols would have, i f they were nonionized i n water, to t h e i r physiochemical properties. With proper substitutions they can also predict t o x i c i t y at any pH within the range where the empirical equation i s v a l i d . The same authors correlated the bioconcentration factor to the Po/w. The log BF value showed a good c o r r e l a t i o n to the Po/w value only - 39 -when corrected for io n i z a t i o n using the following equation: logBF(corrected) = 1.021ogPo/w - 1.82 The excellent correlations between t o x i c i t y and log Po/w are very puzzling since the Po/w values used i n deriving these equations are for the nonionized form of the phenols. The less a c i d i c phenols are i n t h i s form , even at pH 8, but the most a c i d i c phenols appear at and above pH 6 for the most part as phenate ions. These ions do dissolve i n octanol as ion pairs, but the apparent p a r t i t i o n c o e f f i c i e n t of the ion i s at least three orders of magnitude smaller than that of the nonionized form (Hansch and Leo, 1979). For thi s reason a d i s t r i b u t i o n c o e f f i c i e n t should be used to determine BF. Scherrer and Howard( 1977) assumed that weak acids appear in the organic phase only i n the neutral acid form (ignoring ion pairs) and calculated the d i s t r i b u t i o n c o e f f i c i e n t s at each pH from the p a r t i t i o n c o e f f i c i e n t s by making use of the Henderson-Hasselbach equation. When values of the Po/w i n the regression equations r e l a t i n g t o x i c i t y to Po/w were replaced with the new values of the d i s t r i b u t i o n c o e f f i c i e n t s no improvments to the regressions were observed(Saarikovski and Viluksela,1982). Fujita(1966) has used another approach, i n which log Po/w values are used as such, but the measured LC50s are corrected for i o n i z a t i o n i n the same way as Sherrer and Howard corrected the log Po/w values. If t h i s approach i s used on the regression equations established by Saarikovki and Viluksela(1982) they show weaker correlations than the uncorrected equations or the corrected regression equations using the empirical formula established by the same authors. - ko -The reason for these unexpected r e s u l t s may be found i n the multitude of in t e r r a c t i o n s between the parameters considered and the very complex toxic action of the ionizable chemicals. The corrections based on the Henderson- Hasselbach equation f a i l to improve the c o r r e l a t i o n because they presume that t o x i c i t y i s proportional to either neutral or ionized form, when i t has been established that both forms contribute to the tox i c i t y , ( S a a r i k o v s k i and Vi l u k s e l a , 1981).It i s also quite possible that the neutral and ionized form have e n t i r e l y d i f f e r e n t modes of action. Konemann and Musch(1981) estimated the t o x i c i t y of the nonionized phenol form based on the assumption that the t o x i c i t i e s of neutral and i o n i c form of phenols are additive and independent of water pH. Saarikovski and V i l u k s e l a agreed on the a d d i t i v i t y aspect but proved that t o x i c i t y of the i o n i c form changes according to the pH. The importance of l i p o p h i l i c i t y and i o n i z a t i o n i n determining the t o x i c i t y of phenols may be rather d i f f e r e n t i n various animal groups. McLeese et al.(1979) measured the t o x i c i t y of various phenols to a shrimp(Crangon septemspinosa)in sea water. The re s u l t s showed a poor c o r r e l a t i o n with log Po/w and dpKa. When corrected for io n i z a t i o n according to the Henderson-Hasselbach equation, the c o r r e l a t i o n was quite good. Their r e s u l t s showed that the e f f e c t of i o n i z a t i o n may be greater i n the shrimp than i n the guppy. - 1*1 -2.3.2.3 Water Chemistry and i t s Influence on the Bioconcentration Process Water c h a r a c t e r i s t i c s other than pH, such as s a l i n i t y , hardness, suspended s o l i d s load and composition act upon the accumulation of contaminants by the biota. Various species could be affected d i f f e r e n t l y by changes i n water qua l i t y and a generalization of the observed trends i s d i f f i c u l t to do. Muir et al.(1980) showed the importance of the water chemistry on the uptake of organic pollutants by mosquito fish(Gambusia a f f i n i s ) i n r i v e r water. Concentrations of a l l compounds tested were lower i n f i s h i n r i v e r water than i n lab water. Their r e s u l t s suggest that the suspended s o l i d s , p a r t i c u l a r l y the organic carbon content of the suspended material, have a great influence on the a v a i l a b i l i t y of hydrophobic organic chemicals to f i s h i n natural waters. They also recommended the introduction of a corrective term for the uptake rate constants to account for the ef f e c t s of suspended s o l i d s or suspended organic carbon. The e f f e c t s of s a l i n i t y on the uptake of pollutants plays an important r o l e i n estuarine environment. Results of various research work indicate that s a l i n i t y decreases the uptake of pollutants(Murphy,1970). Whatever the reasons for the difference i n the residue r a t i o s , the e f f e c t of s a l i n i t y i s extremely important i n terms of the i n t e r p r e t a t i o n of results from indicator surveys which use samples from areas d i f f e r i n g in s a l i n i t y . The e f f e c t s of s a l i n i t y on the uptake of residues were suggested to be caused by differences i n osmoregulation by f i s h at d i f f e r e n t s a l i n i t i e s or by an e f f e c t of s a l i n i t y on the - 42 -lipid-water p a r t i t i o n of chemicals. Eisler(1972) reported that small variations of temperature, pH, or s a l i n i t y of test waters could change the LC50 values for organochlorines by at least one order of magnitude. Water hardness has shown an e f f e c t on t o x i c i t y tests as well. Holden(1972) and Henderson et al.(1959) showed that DDT and d i e l d r i n are both more toxic i n soft water than i n hard water at the same exposure concentration. 2.3.2.4 Pollutant Interaction and i t s Influence on the R i o n o n c e n t r a l - i o n Process Interactions between organochlorines i s a proven phenomenon and i t s e f f e c t upon the organochlorines levels i n biota have been studied. Organisms which are susceptible to bioconcentration v a r i a t i o n due to pollutant interactions must be eliminated from monitoring programs as no l o g i c a l interpretations of the a n a l y t i c a l results may be made to account for the i n t e r a c t i o n . Macek(1975) tested the acute t o x i c i t y of twenty nine two-chemical mixtures to bluegilIs(Lepomis macrochirus). Eleven of these mixtures exhibited synergism, seventeen exhibited additive t o x i c i t i e s , and one exhibited antagonism. However, Veith et al.(1979) found that the uptake of one chemical was independent of the other chemicals dosed i n the exposure solution, providing that the metabolism of the organism subjected to the experiment was not s i g n i f i c a n t l y altered by the presence of one of the chemicals. The p o s s i b i l i t y of te s t i n g more than one chemical in a - 43 -single exposure(if the chemicals can be separated for accurate analysis i n the presence of each other and they do not i n t e r a c t ) i s a very a t t r a c t i v e proposition. Moreover i f an int e r n a l standard i s adopted, the c a l c u l a t i o n of a r e l a t i v e BF to the BF of that i n t e r n a l standard would reduce the d i f f i c u l t i e s i n comparing the res u l t s of laboratories where d i f f e r e n t experimental conditions and species are used. 2.3.2.5 Ef f e c t of Temperature on the Bioconcentration Factor. Temperature i s another important variable which has a large influence on the bioconcentration factor. It can a f f e c t bioconcentration i n two ways. F i r s t , the water s o l u b i l i t y of the pollutants changes as a function of temperature. In general, the s o l u b i l i t i e s of organochlorines i n water increase with increased temperature. Organochlorines ex i s t i n water mostly adsorbed to pa r t i c u l a t e material. An increase i n water temperature leads to an a l t e r a t i o n of the soluble/particulate r a t i o by the di s s o l u t i o n of some of the pa r t i c u l a t e associated compounds. Such a process i s probably not r e s t r i c t e d to situations where the s o l u b i l i t y maxima for a pollutant i s exceeded, as temperature changes may be expected to a l t e r the soluble/particulate r a t i o independent of the concentration i n the two phases. I t follows that organisms that accumulate at least part of t h e i r t o t a l body load of pollutant d i r e c t l y from water w i l l contain higher concentrations of organochlorines i n waters of higher temperature even i f the t o t a l organochlorine concentrations at a l l locations are the same. _ 44 -Second, there i s the case where the concentrations of pollutants are far below the s o l u b i l i t y maxima which i s ac t u a l l y the most frequent case. The universal explanation accepted i s that temperature af f e c t s the net uptake of pollutants by a l t e r i n g the metabolic rate of the organisms. Fish are poikiothermous,so rates of metabolism and chemical uptake are linked to the temperature of t h e i r environment. Thus, we find that the uptake of chemicals d i r e c t l y from water by f i s h increases with temperature i n proportion to t h e i r oxygen consumption( Hamelink and Spacie,1977). Fig.2.2 reproduced aft e r Veith et al.(1979) shows the v a r i a t i o n i n BF of Aroclor 1254 with exposure temperatures for fathead minnows, green sunfish, and rainbow trout. It i s clear from t h e i r r e s u l t s that the temperature has a p o s i t i v e e f f e c t on the bioconcentration. The three species used i n his experiment show d i f f e r e n t uptake rates for Aroclor 1254 but a l l exhibit higher BF with temperature increase. Reinert et al.(1974) ran tests on rainbow trout (Salmo gairdneri) at various temperature level s and proved the increased bioconcentration with the increased temperature(see Fig.2.3 ). The temperature e f f e c t must be considered when data obtained at d i f f e r e n t times of the year are used to evaluate the levels of contaminants i n the environment. In combination with other seasonal ef f e c t s such as contaminant use and f i s h growth and development , temperature contributes to the variable pattern of contaminant leve l s i n biota. - 45 -5.0 4 8 -4.6 -Lu 4.4 _ O 03 4.2 -4.0 • o 3.8 • 3.6 -• Fathead minnow • Green sunfish o Rainbow trout 5 10 15 20 25 Test Temperature (°C) F i g . 2.2. E f f e c t of temperature on the bioconcentration of Aroclor by f i s h ( reproduced from Veith et a l . , 1979). - 46 -2.3.2.6 E f f e c t of Test Species on the Bioconcentration Factor The use of d i f f e r e n t test species i n determining bioconcentration factors makes the results from d i f f e r e n t laboratories very d i f f i c u l t to compare. It has been found that d i f f e r e n t species bioconcentrate the same chemical at d i f f e r e n t rates, and even within the same species the c h a r a c t e r i s t i c s of each individual(such as age, s i z e , physiological state) influence the bioconcentration c a p a b i l i t y . Fig.2.2 (Veith et a l . , 1979) shows the d i f f e r e n t values of the bioconcentration factor obtained under i d e n t i c a l experimental conditions for the three species used i n the tests 2.4 Use of B i o l o g i c a l Indicator Organisms to Quantitate  Pollutants i n Aquatic Environments Surveys based on the use of b i o l o g i c a l indicators must be designed very c a r e f u l l y and t h e i r results must be interpreted with a p r a c t i c a l knowledge of the effects of the i n t e r f e r i n g parameters. The term " b i o l o g i c a l indicator organism" has been used in several d i f f e r e n t ways i n the l i t e r a t u r e . Some authors have even used t h i s term i n ecological studies which make no reference to p o l l u t i o n of any kind. There are mainly two d i f f e r e n t methods by which biota have been used to assess p o l l u t i o n . This thesis employs a modification of one of these methods. An organism may be used as an indicator of p o l l u t i o n by i t s presence or absence in a given environment, or as a member of the biota indigenous to - 47 -60 ) i i i i i i '— 0 2 4 6 8 10 12 TIME, weeks. w i g . 2.3. E f f e c t of temperature on the concentration of DDT i n rainbow trout. Exposure levels were 176 mg/L at 5 °C, 137 mg/L at 10°C and 133 mg/L at 15 °C( from Reinert et a l . , 1974). - 48 -the study r e g i o n , by the accumulation of p o l l u t a n t s from water and/or ingested food. In the second case, the organism acts as an i n d i c a t o r by means of the p o l l u t a n t content of i t s t i s s u e s , which may be assumed to be an index of the average p o l l u t a n t a v a i l a b i l i t y at the b i o t a c o l l e c t i o n s i t e . The t h i r d mode of p o l l u t i o n assessment with an i n d i c a t o r organism i s a v a r i a t i o n of the second one i n which the accumulation of p o l l u t a n t s i n the t i s s u e s of the chosen organism i s used to q u a n t i t a t e p o l l u t i o n . However, the organism does not belong t o the b i o t a of the study region and i s purposely implanted at the monitored s i t e . The necessary a t t r i b u t e s f o r an organism to act as a b i o l o g i c a l monitor of aquatic p o l l u t a n t s were f i r s t suggested by B u t l e r et al.(1971) and amended by Haug et a l . (1974) and P h i l l i p s (1978). A summary of those a t t r i b u t e s i s l i s t e d below: - the organism should accumulate the p o l l u t a n t without being k i l l e d by the l e v e l s encountered i n the environment. the organism should be sedentary i n order to be r e p r e s e n t a t i v e of the study area. - the organism should be abundant throughout the study area. - the organism should be s u f f i c i e n t l y l o n g - l i v e d to al l o w the sampling of more than one y e a r - c l a s s , i f d e s i r e d . - the organism should be of reasonable s i z e , g i v i n g adequate t i s s u e f o r a n a l y s i s . - the organism should be easy t o sample and hardy enough to s u r v i v e i n the l a b o r a t o r y . - the organism should t o l e r a t e b r a c k i s h water. - a simple c o r r e l a t i o n should e x i s t between the p o l l u t a n t content of the organism and the average p o l l u t a n t c o n c e n t r a t i o n - 49 -i n the surrounding water. - a l l organisms of a given species used i n a survey should exhibit the same c o r r e l a t i o n between t h e i r pollutant content and the average pollutant concentration i n the surrounding water, at a l l locations studied, under a l l conditions. When using biota as a p o l l u t i o n indicator a series of factors a f f e c t i n g t h e i r pollutant content must be taken into consideration. The way those factors influence the pollutant bioaccumulation must be c a r e f u l l y studied and understood before any int e r p r e t a t i o n i s given to the r e s u l t s of various monitoring surveys using biota. The major factor that seems to determine the concentrations of organochlorines i n biota i s the amount of l i p i d present i n the organisms studied. Other factors include species variables such as age, sexual condition , behaviour, and water qua l i t y variables such as temperature, pH, a l k a l i n i t y , oxygen content. Miscellaneous factors such as seasonality , d i e t and interactions between the pollutants have t h e i r influence on the bioconcentration v a r i a b i l i t y . Differences i n the wet weight based concentrations of organochlorines between d i f f e r e n t teleost species have been ascribed by many authors to differences i n the l i p i d content of these species(e.g. Duffy and O'Connell, 1968; Hannon et a l . , 1970; Reinert, 1970,-Sprague and Duffy, 1971; Carr et al.,1972; Jensen et a l . 1972 ; Frank et al.,1974; Linko et a l . , 1975). S i m i l a r l y , v a r i a t i o n of organochlorine concentrations i n the d i f f e r e n t tissues of a single f i s h species has been correlated to the l i p i d content of the tissues(Holden,1962;Reinert, 1969; - 50 -Stout et al.,1972; Yoshida et a l . , 1973; Ernst et al.,1976). Although some data suggest that the va r i a t i o n of organochlorine concentrations i n any one species of teleost i s markedly less on l i p i d weight basis than on a wet weight basis t h i s i s not always the case (Anderson and Fenderson, 1970). The p a r t i a l f a i l u r e of many authors to reach u n i f i e d conclusions on t h i s matter i s due to the i n t e r f e r i n g e f f e c t s of other differences(e.g. size or age, metabolic rates,migration, etc.) between individuals i n any given teleost population. Data concerning the eff e c t s of l i p i d on species or tissue differences i n organochlorine concentrations amongst organisms other than te l e o s t are sparse. Ernst et al.(1976) reported a co r r e l a t i o n between the tissue d i s t r i b u t i o n s of DDT,DDE,DDD, and PCBs and the l i p i d contents of the tissues i n the scallop (Chlamys opercularis) and similar data are available for shrimp(Nimmo et al.,1971), sea-lions(Delong et al.,1973) and seals and porpoises(Holden and Marsden, 1967). Problems such as differences between l i p i d types i n sequestration of organochlorines(Addison and Smith,1974), and the i n a b i l i t y of solvent extraction methods to extract certain types of 1ipid(Vreeland, 1974) are additional concerns i f the l i p i d weight i s used as a basis for organochlorine concentrations i n indicator organisms. However i t i s strongly recommended (Phillips,1978) that authors report data concerning the concentrations of organochlorines i n biota by both wet weight and l i p i d weight. The e f f e c t of l i p i d s on the concentration of organochlorines found i n aquatic biota are not only important i n th e i r own ri g h t , - 51 -but are also the main cause of the variations seen i n such concentrations related to other factors such as age or season. The data available on the ef f e c t s of age or related factors on the concentration of organochlorines i n f i s h are very contradictory. Zitko(1971) reported higher concentrations of organochlorines i n whole herring (Clupea harengus)of average weight 222 g than i n those of average weight 59 g. In studies were data were reported by both wet weight and l i p i d weight the concentrations of organochlorines by wet weight increased i n older f i s h and showed no variations with age i f the l i p i d weight was used to calculate concentrations.(Hannon et al.,1970;Kelso and Frank, 1974 ) . Murphy and Murphy(1971) correlated the uptake of DDT by f i s h of d i f f e r e n t sizes to the v a r i a t i o n i n oxygen uptake rates of these f i s h . The regression of DDT uptake with weight and of oxygen uptake with weight had almost i d e n t i c a l slopes. The conclusion was that the uptake of DDT was proportional to the metabolic rates of f i s h and i t translates into higher organochlorines concentrations i n younger f i s h . With the available data, no universal available conclusion can be reached on the r e l a t i o n s h i p between age and organochlorine content of the biota. It i s evident that the age of the organisms sampled i s a major i n t e r f e r i n g parameter i n indicator surveys using biota. Sampling for such surveys should aim at taking organisms of sim i l a r ages or sizes from a l l locations studied or at selecting a range of size of f i s h of each species and subject the results to a detailed s t a t i s t i c a l analysis.(Anderson and Fenderson(1970). Data on other organisms than teleosts do not c l a r i f y the problem - 52 -any further. Seasonality of the concentrations of organochlorines in biota i s due to two major factors. The f i r s t i s the seasonal difference i n a v a i l a b i l i t y of organochlorines to aquatic organisms which may be correlated to pesticides application times in a g r i c u l t u r e , to i n d u s t r i a l discharges of organochlorines, to r a i n f a l l or run-off or to other factors. The second major factor causing organochlorine seasonality i s the seasonal fl u c t u a t i o n of l i p i d contents i n biota. These two factors need not be synchronized with each other and seasonal p r o f i l e s of organochlorines may exhibit multiple annual peaks i n biota due to lack of synchronization. Robinson et al.(1967) suggested that the seasonal fluctuations of DDE and d i e l d r i n i n sand eels and cod taken from the Fame Islands (Fig. 2.4) may have been due to migration of f i s h i n and out of the population sampled. However, thi s hypothesis appears un l i k e l y because of the s i m i l a r i t i e s between the seasonal p r o f i l e s of the two species. It would seem more l i k e l y that the differences reported r e f l e c t r e a l differences i n the a v a i l a b i l i t y of organochlorines throughout the year( Phillips,1978). Bivalve molluscs have been widely used as indicator organisms and the data r e f l e c t the importance of the pollutant source on the seasonal a v a i l a b i l i t y of those pollutants. Seasonality of organochlorines has been studied i n the sand crab(Burnett,1981) in the amphipod(Gammarus pulex ,Sodergen et al.,1972)in phytoplankton (Jensen et a l . , 1972), and i n zooplankton(Williams and Holden,1973). Their data showed the wide v a r i a t i o n of pollutant content i n biota as a function of season. - 53 -Figure 2.4. Seasonal ef f e c t s of pesticides levels i n f i s h . Th f i s h , cod( Gadus morrhua) and sand eel( Ammodytes lanceolatus were caught at d i f f e r e n t times of the year around the Farne Islands. - 54 -The e f f e c t of temperature on the uptake of organochlorines by organisms probably also contributes to the seasonality of these compounds i n biota(Kellog and Bulk ley, 1976).Temperature e f f e c t s are d i f f i c u l t to separate from those of l i p i d changes and variations i n the available ambient concentrations of organochlorines, both of which also contribute to the seasonality. Only controlled laboratory studies can c l e a r l y defined the r e l a t i o n s h i p between temperature and bioconcentration. 2.4.1.Pollutional Ecology of the Freshwater Leeches Sawyer(1974) commented that there i s the quantitative, rather than the q u a l i t a t i v e , composition of the leech fauna which characterizes the d i f f e r e n t types of normal or disturbed habitat. Few leech species, i f any, are so e c o l o g i c a l l y r e s t r i c t e d that a single environmental factor can determine t h e i r d i s t r i b u t i o n s . Among the environmental factors that influence the leech d i s t r i b u t i o n , i n approximate order of s i g n i f i c a n c e , are: a v a i l a b i l i t y of food organisms, the nature of substrate, the depth of water, water currents, size and nature of the body of water, hardness, pH, temperature of the water, minimum concentration of dissolved oxygen, s i l t a t i o n and t u r b i d i t y , and the s a l i n i t y of the water(Sawyer, 1974). Species of leeches which feed upon aquatic oligochaetes and dipteran larvae respond i n d i r e c t l y to organic enrichment. The abundance of these leeches i n zones subjected to raw sewage discharges(Richardson,1928) r e f l e c t s t h e i r response to the - 55 -increased food supply. Except at extremely low le v e l s , hardness, t o t a l a l k a l i n i t y , and pH have l i t t l e or no influence on the d i s t r i b u t i o n or r e l a t i v e abundance of leeches. Water temperatures play an important r o l e i n the reproductive biology of leeches, primarily by determining the onset of the breeding season. In the laboratory, leeches die at temperatures of 33-35C( Sawyer,1974). High summer temperatures(about 30C or higher) l i m i t the d i s t r i b u t i o n of leeches i n both naturally and thermally polluted environments. Most leeches can withstand anaerobic conditions for long periods and are not r e s t r i c t e d by temporary oxygen depletion. S i l t a t i o n and t u r b i d i t y have a profound e f f e c t on the ecology of leeches. Water t u r b i d i t y reduces the natural predation of other organisms on leeches and leads to high densities and even epidemics. S i l t a t i o n changes the nature of the substrate to such an extent that leeches have d i f f i c u l t y moving and depositing cocoons. Leeches are r e l a t i v e tolerant to bunker o i l and exhibit a very high tolerance for pesticides and other pollutants. LC50 values for various species of leeches exposed to pesticides are readil y available i n the l i t e r a t u r e . The LC50's of the major pesticides, such as dinex, chlordane, diazinon, lindane, mirex , and inalathion, vary i n the laboratory from 0.5 to 10.0 ppm(Sawyer, 1974). Besides t h e i r high resistance to the toxic action of various chemicals , leeches have also been found to have a considerable bioaccumulation potential for synthetic organic chemicals. According to Navqui and de la-Cruz(1973), levels of mirex i n the leech Erpobdella punctata were among the highest found in - 56 -invertebrates and f i s h c o l l e c t e d from areas treated with the chemical(up to 1.76 ppm).Webster(1967) reported residues of DDT in leeches three months afte r a e r i a l spraying of the pesticide; no residues were found i n the tissues of amphipods and copepods from the same area. D'Eliscu(1975) found 8.1 times higher DDT levels i n the tissues of leeches than i n clams co l l e c t e d from the Lake Tahoe basin. Other results from the work of the same researcher indicate that the bioaccumulation potential of leeches varies from species to species. Carey et al.(1983) reported extremely high concentrations of chlorophenols i n leeches c o l l e c t e d from Canagagigue Creek located in Ontario. Concentrations were 20 times higher than was found i n f i s h sampled at the same locations. As a r e s u l t of t h i s observation, another study by Metcalfe et al.(1984) emphasized the use of leeches as a potential bioindicator of organic chemical pollution.Their data showed that leeches accumulated very high levels of pollutants (in t h i s case chlorophenols) and the levels found i n t h e i r tissues were one or two orders of magnitude greater than the levels found i n thirteen other benthic invertebrates as well as f i s h and tadpoles. At one of t h e i r sampling s i t e s , leeches accumulated chlorophenols to levels ranging from 40,000 to 140,000 times the average water concentration. Metcalfe et al.(1984) were f i r s t to suggest that leeches be used as a "early warning " indicator of organic p o l l u t i o n . .Their work on leech bioconcentration c a p a b i l i t y constituted the s t a r t i n g point of the present thesis. Chlorophenols are widely used i n B r i t i s h Columbia by the wood industry and a bioindicator of chlorophenol p o l l u t i o n i n the - 57 -water system of the province would be a major contribution to present monitoring programs which a l l r e l y on discrete, grab water samples. - 58 -3.SAMPLING AND METHODOLOGY 3.1 Introduction The experimental work can be divided i n three major sections: i n s i t u Fraser water sampling, i n s i t u f i e l d leech experiments, and laboratory controlled leech experiments. This chapter w i l l describe each section i n d e t a i l with respect to sampling technique, a n a l y t i c a l procedures and experimental setup and design. 3.2 In Situ Fraser Water Sampling Two methods of water sampling were used, namely: grab sampling and automatic sampling. The grab sampling was i n i t i a l l y performed i n order to obtain basic information upon the CP concentration lev e l s i n the study area and to determine the general pattern of CP contamination i n the surveyed portion of the Fraser River. Automatic sampling was performed only at one location which was selected on the basis of previous grab sample r e s u l t s , s i t e a v a i l a b i l i t y and equipment safety. 3.2.1 Study Area and Sampling Sites The study area consisted of a stretch of Fraser River, the North Arm, between New Westminster and Iona Island. F i g . 3.1 - 59 -F i g . 3.1. Sampling s i t e s and industries i n the study area sampling stations are i d e n t i f i e d i n Table 4.7 and industries i the text, section 3.2.1). - 60 -i l l u s t r a t e s the sampling s i t e s and forest industry m i l l locations. This reach of the Fraser River has a high density of m i l l s using CPs and was considered suitable for a study of chlorophenol p o l l u t i o n : (A) :B.C. Forest Products Ltd., Vancouver; (B) :Canadian Forest Products Ltd., Eburne Sawmills Di v i s i o n ; (C) : West Coast C e l l u f i b r e Industries Ltd.; (D) : Fraser River Planning D i v i s i o n ; (E) : MacMillan Bloedel Ltd., Canadian White Pine Di v i s i o n ; (F) :Terminal Sawmills Ltd., Richmond; (G) : Whonnock Industries Ltd., S i l v e r t r e e D i v i s i o n ; (H) : Scott Paper Ltd., New Westminster; (I) : Belkin Packaging Ltd., Burnaby; Note: the l a s t two m i l l s do not use CPs i n t h e i r process but t h e i r e f f l u e n t s contained CPs( Cain et a l . 1980). Sampling s i t e s locations are indicated i n F i g . 3.1 by numbers from 1 to 7 and w i l l be referred to when res u l t s are discussed. 3.2.2 Grab Sampling To assess the general level of contamination i n the study area, a series of grab samples were c o l l e c t e d between June and October 1984. In June and August samples were co l l e c t e d at access points along the r i v e r bank and i n September and October samples were c o l l e c t e d i n midstream using a boat. The water samples were c o l l e c t e d i n 4L amber bottles with t e f l o n l i n e d screw caps, and preserved with NaOH( 5 p e l l e t s / L ) . - 61 -The bottles had been previously cleaned using a three step procedure: step one included washing the bottles with tap water and regular laboratory detergent followed by thorough r i n s i n g with tap water; step two consisted of cleaning the bottles with hexane followed by r i n s i n g with tap water; step three consisted of applying the f i n a l rinse to the bottles using d i s t i l l e d water. The samples were transported to the UBC Environmental Engineering laboratory and stored i n a cold room at 4 °C. Extraction and further preparation of samples was completed within 7 days as s p e c i f i e d i n Test Method 604 for phenols (U.S.EPA, 1982). Replicate analyses were performed on many samples to evaluate the r e p r o d u c i b i l i t y of the extraction and a n a l y t i c a l technique. Blank samples were prepared and analysed for each sampling t r i p . A blank consisted of d i s t i l l e d water placed i n a regular sample bo t t l e with preservative. This blank was transported to the f i e l d and stored i n a s i m i l a r manner to the Fraser River samples. Transportation of blanks to the f i e l d was performed i n order to subject the blanks to the same outdoor temperature v a r i a t i o n and transportation conditions as the f i e l d samples. 3.2.3 Automatic Sampling For automatic sampling, an ISCO automatic sampler which contained 28(500ml) polyethylene bottles was used. A l l the automatic sampling was done from the loading dock of West Coast C e l l u f i b r e Industries Ltd. The sampler was sheltered i n a small shack located on the f l o a t i n g dock and was operated by a 12 V battery.The suction tubing was immersed i n the water and the - 62 -exact point of sampling was 4 feet below the water surface. Every 2 hours a 500 mL sample was taken. After each sample was taken, a purging cycle cleared the suction l i n e to avoid contamination between succesive samples.The sampling process including purging lasted approximate 3 minutes. The concern of adsorption or leaching problems with p l a s t i c bottles was checked by spiking both p l a s t i c and amber glass bottles with known amounts of chlorophenols and analysing samples after one week. Very comparable r e s u l t s were obtained and the p l a s t i c bottles were considered acceptable for use. NaOH p e l l e t s were s t i l l used for sample preservation. Standard deviations of the combined samples(contained i n p l a s t i c bottles and amber bottles) were 3.78% and 12% for TTCP and PCP respectively, values well within the recovery and r e p r o d u c i b i l i t y v a r i a b i l i t y . Similar values were obtained for the lower chlorinated phenols. The i n d i v i d u a l samples were analysed as such, or a composite sample was made manually. For the composite samples, three consecutive i n d i v i d u a l samples were combined i n equal volumes (400 mL each),in one 1200 mL sample which thus contained combined chlorophenol concentrations over a period of 6 hours. This technique was used i n order to reduce the number of samples to be analysed during longer sampling periods( 6 or 7 days). In th i s mode the high frequency of sampling was s t i l l maintained( i . e . every 2 hours) without t r i p l i n g the number of samples to be analysed. With the exception of the f i r s t 2-day automatic water sampling experiment, a l l the other automatic sampling experiments were performed by using 6 hour composite samples. - 63 -3.2.4 Water Sample Preparation for Chlorophenols Analysis The extraction of chlorophenols and further sample preparation for GC analysis followed the methodology outlined by Metcalfe et al.(1984). This technique was o r i g i n a l l y developed by Chau and Coburn(1974). The technique was further improved by Fox(1978) with two modifications: 1) Alkaline storage at pH 12 to increase s o l u b i l i t y , which in turn decreases adsorption to surfaces, followed by a c i d i f i c a t i o n to pH 1-2 immediately p r i o r to analysis. 2) Replacement of benzene with toluene as the extraction solvent, because of the suspected r o l e of benzene as a carcinogen. Extraction e f f i c i e n c i e s of the two solvents are approximately equal. The water samples were a c i d i f i e d with 50% H^SO^ to pH 1-2 just p r i o r to extraction. The i n i t i a l volume of the water sample analyzed was d i f f e r e n t at various experimental times( 400 mL, 1000 mL and 1200 mL). The following procedure describes the various reagents quantities used i f i n i t i a l water sample i s 1000 mL. S l i g h t l y modified quantities were used for the other sample volumes. Appropiate correction factors were used when c a l c u l a t i n g the concentration factor from the i n i t i a l sample volume to the f i n a l extract volume which remained constant( 10 mL) regardless of the s t a r t i n g sample volume. A c i d i f i e d water samples were extracted 3 times with chromatographic grade toluene( 40, 30, 3 0 mL). The combined toluene extracts were back extracted 3 times with 0.1 M I^CO^ ( 40, 30, 30mL). - 6k -D e r i v a t i z a t i o n using acetic anhydride was used to prepare the extracts for GC analysis. Underivatized phenols do not possess favourable properties for gas chromatographic analysis due to t h e i r high p o l a r i t i e s and low vapour pressures( Krijgsman and van de Kamp,1977). Deriv a t i z a t i o n i s recommended to form less polar compounds and help overcome the t a i l i n g e f f e c t and the assymmetric peaks obtained by running pure phenols through the columns. Many d e r i v a t i z a t i o n methods have been published. They include formation of ethers, esters, phosphorus esters and s i l y l derivatives( Krijgsman and van de Kamp,1977). A summary of derivatives with appropiate references i s available( NRCC,1982). Deriva t i z a t i o n also helps to accomplish sample clean up by eliminating other i n t e r f e r i n g compounds and gives better separation on the chromatographic column. To the aqueous K CO extract, 10 mL of hexane and 1 mL of r e d i s t i l l e d a c e tic anhydride were added. Acetylation was performed by shaking the above reagents for one hour i n a 2 50 mL separatory funnel on an automatic shaker(Model:Wrist Action Shaker; Manufacturer:Callahan). After shaking, the hexane layer which contained the acetylated chlorophenols was removed with a Pasteur pipet and analyzed by high resolution c a p i l l a r y gas chromatography. The d e r i v a t i z a t i o n agent , acetic anhydride, was always fre s h l y r e d i s t i l l e d ( b.p.=139°C). It i s a stable and strong acetylation agent. Stoichiometric calculations showed that 1 mL acetic anhydride used for acetylation was well i n excess to the amount of trace chlorophenols found i n the sample extracts, so i t was assumed that a l l chlorophenols were t o t a l l y reacted with the - 65 -d e r i v a t i z a t i o n agent and were present as acetylated derivatives in the f i n a l volume. 3.3 Leech Experiments As previously mentioned, the leeches used as bioindicator organisms were c o l l e c t e d from an unpolluted environment and implanted i n bioassay chambers i n the study region or used for laboratory controlled experiments. Leeches were c o l l e c t e d from the l i t t o r a l zone of a p r i s t i n e lake, Kentucky Lake, located some 300 km east of Vancouver, between Princeton and Me r r i t t , B.C. C o l l e c t i o n t r i p s were made during the i c e free period, usually A p r i l to October. The l a s t t r i p i n the f a l l of 1984 was at the end of September and the f i r s t t r i p i n spring of 1985 was mid A p r i l which proved to be early for that year which was characterized by a very late spring, with lower than normal temperatures. The lakes i n the Mer r i t t area were s t i l l frozen at the end of A p r i l . Only early i n May, were enough leeches co l l e c t e d to permit the continuation of the leech experiments. For the purpose of i d e n t i f i c a t i o n , leeches were preserved i n 85% solution of alcohol. In order to get the leech specimens stra i g h t , moderately extended, and undistorted, alcohol was added gradually and used as an i n i t i a l anesthetic( Moore ,1959). Five indiv i d u a l s of each species were sent to Calgary for i d e n t i f i c a t i o n . Leeches are never i d e n t i f i e d on the basis of host or habitat. Only the morphology w i l l lead to secure i d e n t i f i c a t i o n s ( Moore,1959). With the help of Prof. Davies, Head of the Department of Biology at the University of Calgary the - 66 -species of leeches found i n Kentucky Lake were i d e n t i f i e d as follows: Percymorensis marmorata ,Nephelopsis obscura and  Glossiphonia complanata. Only the f i r s t two species were used i n the experimental work. Our intention was to use only one species i n a l l the experiments in order to keep variables to a minimum. Unfortunately, during May leech c o l l e c t i o n t r i p s , the slim brown leeches(N.obscura) were i n great abundance whereas the large, black leeches(P.marmorata) which had been used the previous f a l l were in very li m i t e d supply and very small i n s i z e . For t h i s reason the temperature/bioconcentration factor experiments run i n May, 1985, were done with the brown leeches. A l l other experiments, f i e l d or laboratory controlled were done with the black leeches which were f i r s t selected because of t h e i r apparent abundance( during summer and f a l l ) and large size( 0.1g-4.0g). The leeches were transported to the U.B.C. laboratory i n p l a s t i c p a i l s , and were stored i n a 50 L aerated aquarium containing native lake water. Sand, pebbles, and some wood debris taken from the lake were used to create a more natural environment. A very small percentage of leeches died during the f i r s t days of acclimation(5%). Previous storage of leeches i n reconstituted fresh water, soft type, as formulated by US EPA (1978) gave us a higher mortality rate( 20%), so i t was decided to store leeches i n water c o l l e c t e d from t h e i r native environment. Species i d e n t i f i e d i n Kentucky Lake prefer a harder water than the soft type used i n the early storage and that explains the d i f f i c u l t y i n ad a p t a b i l i t y experienced by the leeches at f i r s t . The majority of leech species are most abundant - 67 -i n water with t o t a l a l k a l i n i t y above 60 ppm CaCO^- Few species occur i n an a l k a l i n i t y below 18 ppm. S i m i l a r l y , a l l leech species are most abundant i n water with a pH of 7.0 or higher, and few species occur at pH 6.0 or below( Sawyer, 1974). Leeches survive well i n media of low oxygen content and no bubbling of the water i s usually recommended for the maintenance of the adults( Fernandez, 1982); however, the aquarium was aerated to eliminate any foul odours from decomposition processes. No food was supplied to the leech stock. The species present in the supply lake are reported to feed on oligochaetes,insect larvae and snails( Sawyer, 1974). Since the leeches survive without food for a long period of time (4-6 months), i t was elected that no food be provided to them i n order to eliminate possible contamination v i a food source, or possible concentration of the chlorophenols i n the ingested food. As a r e s u l t of feeding, leeches store large amounts of blood or tiss u e f l u i d i n t h e i r gut. Degradation of t h i s food takes days, weeks, or months depending on the species of leech (Fernandez,1982). Bioaccumulation of pollutants v i a the food chain i s considered to play a minor r o l e as compared to the d i r e c t uptake from the water matrix and i t was not included i n the experimental setup. The aquarium was kept only two th i r d s f u l l with water to allow the black leeches (P. marmorata), which are amphibious, to get out of the water and crawl on the walls. The other two species are completely aquatic and do not usually leave the water. This c h a r a c t e r i s t i c of the black leech made the laboratory controlled experiments a l i t t l e d i f f i c u l t since the leeches - 68 -tended to leave the water and were not exposed to the contaminant i n the water for the entir e period of exposure. This phenomenon was e s p e c i a l l y encountered at room temperature( approx. 23°C). At 12 and 4°C the black leeches seemed to remain i n the water. 3.3.1 In-situ F i e l d Leech Experiments Exposure experiments were ca r r i e d out for periods of s ix or seven days and 5 leeches were placed i n each bioassay chamber. The bioassay chambers were suspended i n the water, at 3-4 feet depth, at various locations. The majority of leech f i e l d experiments were done at the location where automated water sampling was performed as well. Some leech f i e l d experiments were run concurrently with automatic water sampling while others were conducted independently. The c y l i n d r i c a l bioassay cages were made of f i n e aluminum mesh reinforced on the sides with steel straps. The top of the cylinder was secured with a large hose clamp to f a c i l i t a t e transfer of leeches. The mesh allowed the water to flow f r e e l y through the cage. Approximate dimensions of the c y l i n d r i c a l cage were 100 mm i n diameter and 300 mm i n length. A f i s h i n g l i n e was used to t i e i t securely to a stationary platform or pole. Leeches survived very well the conditions of exposure and none succombed during the f i e l d experiments. Upon completion of the exposure period, leeches were taken to the laboratory where they were immediately weighed and digested for chlorophenol analysis. The leeches were dried with a paper towel and wet weights were determined on an a n a l y t i c a l balance. Mann( 1953) showed that the amount of water carried over - 69 -was s u f f i c i e n t l y constant to enable the wet weight to be accurately determined to the nearest 10 mg. Digestion was performed by using concentrated HCl i n a r a t i o of 1 mL acid to 0.1 g of leech( Metcalfe et al.,1984). The digestion was completed i n several hours and usually during the next day the chlorophenols were extracted from the digested leech matrix and the extract prepared for GC analysis.The acid digested leech was extracted with hexane, back extracted i n 0.1 M K 2 C 0 3 and acetylation performed as for the water samples. The f i n a l sample volume was 10 mL of hexane which contained the acetylated derivatives of the chlorophenols found i n the leech. Phase separation was often d i f f i c u l t when chlorophenols were being extracted from the digested leech matrix. Centrifugation had to be performed on a few occasions to accomplish separation. Centrifugation had to be perfomed as well for a set of water samples that contained high l e v e l s of suspended s o l i d s . These samples were c o l l e c t e d towards the end of A p r i l when the Fraser River was i n flood due to the melting snow. Recovery of chlorophenols i n the presence of high sediment load was also lower than under normal circumstances and corrections were applied to the r e s u l t s . - 70 -3.3.2 Laboratory Leech Experiments For the controlled exposure of leeches to various concentrations of chlorophenols, one l i t e r of reconstituted fresh water was added to 1400 mL beaker, followed by additions of known concentrations of chlorophenols( non-derivatized standards contained i n toluene were used for spiking). The medium hard water formulation recommended by EPA was sim i l a r to the Fraser water c h a r a c t e r i s t i c s and i t was used as d i l u t i o n water i n the control experiments( pH 7, a l k a l i n i t y 50 mg/L CaCO^, hardness 60 mg/L CaCO-} ) . Various concentrations of chlorophenols were used for 24 hours exposure studies and a mixture of 5 chlorophenols at 10 ppb each was used for one week exposure t r i a l s . The same mixture of chlorophenols was used for other controlled exposures at lower levels of chlorophenols. Since experiments were conducted under s t a t i c conditions, leeches were transferred to a fresh solution of 10 ppb chlorophenols each day throughout the bioassay period; the concentration of chlorophenols i n water in which leeches were immersed was checked and i t did not change much due to volatilization,photodegradation or bioconcentration of chlorophenols by leeches. At pH 7.0 most chlorophenols are ionized and the process of v o l a t i l i z a t i o n applies only to the small f r a c t i o n of undissociated molecules. Photodegradation was not a problem since most laboratory controlled experiments were run i n dark rooms or enclosures. Mass balance was calculated as well, and the amount of chlorophenols accumulated i n 24 h by - 71 -leeches was i n s i g n i f i c a n t as compared to the t o t a l amount contained i n the spiked water. Analysis of a spiked one l i t e r d i l u t i o n water at an i n i t i a l concentration of 10 ppb was performed (container covered with a glass l i d and l e f t on the counter top for one day ). After 24 hours, chlorophenols concentrations were 8-9 ppb as compared to an i n i t i a l concentration of 10 ppb. When a l l errors due to the extraction, recovery and instrument performance were considered the values indicated that a r e l a t i v e constant concentration of chlorophenols was maintained by d a i l y replacement of the solution. In the 6 or 7 day experiments, every 24 h, three leeches were digested and extracted for analysis. Parameters considered during leech experiments were contact time, chlorophenol concentrations, temperature, and d i l u t i o n water pH. Leech size was monitored and i t was kept i n a very narrow range. A s p e c i f i c experiment having leech size as variable parameter was not carried out. Constant temperature was obtained by either running the experiments i n an enviromental temperature controlled chamber or by using a temperature regulated water bath. pH adjustments were made with additions of 50% s u l f u r i c acid or concentrated caustic( 10 N NaOH). For the duration of the pH experiments( 24 h), the pH remained within 0.5 units of the i n i t i a l value. Since only the trend of v a r i a t i o n of the bioconcentration factor with pH .was monitored t h i s was considered s a t i s f a c t o r y . - 72 -3.4.Standards Preparation Stock solutions of a l l the 19 congeners of the chlorophenols family were made at a concentration of lmg/mL. Isopropanol was chosen as a solvent because i t s higher p o l a r i t y favored the solution of the polar chlorophenols. Ten mg of pure material was weighed and dissolved i n pestic i d e q u a l i t y 2-propanol( isopropanol) and di l u t e d to 10 mL volume. The chlorophenols were supplied l o c a l l y by BDH which acted as a d i s t r i b u t o r for A l d r i c h Chromatographic Chemicals. Since purity of the compounds was c e r t i f i e d at 96% or greater, no correction was made to calculate the concentration of the stock solutions. The stock solutions were kept i n amber teflon-sealed screw-cap b o t t l e s , and stored at 4°C i n the r e f r i g e r a t o r . Stock standard solution were not used for more than 6 months as recommended(U.S.EPA,1982).Al1 the organic solvents used were of chromatographic pur i t y . The stock solutions of each chlorophenol( 1 mg/L),.were used to prepare various mixtures of chlorophenols at desired concentrations. In order to determine the retention times of a l l 19 chlorophenols, 5 standard mixtures of chlorophenols were prepared and analyzed separately for proper compound i d e n t i f i c a t i o n When i n doubt, single compounds were run to confirm a retention time. Non-derivatized standards were made from stock solutions using toluene as solvent and they were generally used for spiking various samples, to make additions to the controlled bioassay chambers i n which leech exposures were performed, and - 73 -to make derivatized standards. Derivatized standards were made for every large series of samples and they were used for the c a l i b r a t i o n f i l e s , q u a l i t y control and monitoring of the detector s e n s i t i v i t y . A concentrated derivatized standard was usually made and d i l u t e d to cover the possible range of concentrations encountered i n the samples. The most frequently used standard was a derivatized mixture of 5 chlorophenols( 2,4-DCP; 2,4,6-TCP; 2,4,5-TCP; 2,3,4,6-TTCP; PCP) at 1 ppm concentration. For c a l i b r a t i o n , the external standard technique was used. The most frequently used c a l i b r a t i o n f i l e was made at three concentration l e v e l s , namely: 0.01 ppm; 0.1 ppm; 1 ppm. The r a t i o of instrument response to amount injected( c a l i b r a t i o n factor) for the 5 chlorophenols was quite constant over the entire working range which indicated a straight c a l i b r a t i o n l i n e for the concentrations i n question.(if the r e l a t i v e standard deviation of the c a l i b r a t i o n factor i s less than 10%, l i n e a r i t y can be assumed! U.S.EPA, 1982). Standards were stored i n amb er v i a l s ( 10 or 40 mL) with t e f l o n caps and kept i n the r e f r i g e r a t o r , at 4 °C, when not i n use. A l l the sample v i a l s used i n the autosampler were amber with special EC detector caps. The working c a l i b r a t i o n curve was v e r i f i e d each day that analysis were made and i f the response for any compound varied more than 10%, the tes t was repeated using a fresh c a l i b r a t i o n standard, or a new c a l i b r a t i o n factor was determined. In order to achieve consistency of the r e s u l t s , a few older samples which had been analysed and quantified on another occasion were run together with the new set of samples and requantified according to the new c a l i b r a t i o n curve. In case that - 74 -quantitation of those samples varied too much between the two runs(20% v a r i a b i l i t y ) correction measures were taken and sometimes enti r e old runs were rerun together with the new run. 3.5 A n a l y t i c a l Technique for Chlorophenols Separation, Quantification and I d e n t i f i c a t i o n The acetylated extracts were analysed for chlorophenols by c a p i l l a r y gas chromatography using the electron capture detector.The temperature program developed allowed a complete separation of the 19 congeners of the chlorophenols family(with the exception of 2,4 and 2,5-DCP which eluted together and could only be separated at very low concentrations). The method of determination of chlorophenols by c a p i l l a r y gas chromatography developed by Krijgsman and Van de Kamp(1977) served as a s t a r t i n g point i n setting up the methodology. Appendix I( F i g . I . l ) shows the chromatogram and the r e l a t i v e retention times of the chlorophenol acetates presented by Krijgsman and Van de Kamp. As stressed by these researchers several aspects make t h i s method very a t t r a c t i v e for the determination of chlorophenols i n environmental samples: s e l e c t i v i t y of the extraction; high separation power of the c a p i l l a r y column; and s p e c i f i c i t y and s e n s i t i v i t y of the electron-capture detector. Analysis of the derivatized chlorophenols were performed on a Hewlett Packard 5880 A ,gas chromatograph, equipped with an electron capture detector and an autosampler. A DB5 c a p i l l a r y column ( 30 m x 0.332 mm I.D.) provided good isomer - 75 -separation.The DB5 l i q u i d phase of the fused s i l i c a c a p i l l a r y column i s crosslinked and bonded. I t i s of non-polar composition: 95% Dimethyl-(5%)diphenylpolysiloxane. Helium was used as c a r r i e r gas at 2mL/min and nitrogen as make-up gas at 20mL/min. S p l i t l e s s i n j e c t i o n was necessary because of the trace lev e l s involved. The i n l e t was purged afte r 0.5 min i n order to diminish the solvent peaks. Detector temperature was set at 310 °C and i n j e c t i o n port temperature was set at 2 50°C. Oven temperature was programmed i n three lev e l s as a r e s u l t of repeated t r i a l s to separate the 19 constituents of the chlorophenols family. The temperature program developed was: - l e v e l one, from i n i t i a l oven temperature of 75°C to 148 °C (15°C/min); - l e v e l two, from 148 °C to 180 °C (3 °C/min); - l e v e l three, from 180°C to 2 25°C (10 C/min). A post temperature of 2 50°C was used for 5 minutes. A t o t a l time of 3 0 minutes was required to separate a l l the chlorophenols. Standard mixtures with CP concentrations as low as 0.01 ppm gave sharp, symmetrical and completely separated peaks of a l l the chlorophenols. 3.5.1.Identification and Confirmation of the Chlorophenols  Presence i n the Water and Leech Matrix Confirmation and i d e n t i f i c a t i o n of chlorophenols present i n various samples was obtained by GC/MS (Hewlett-Packard 5985 B), with a mass scanning range of 1-1000 amu. The MS was interfaced to a 584 0 gas chromatograph equipped with a DB5 c a p i l l a r y column. A simpler temperature programme was used since only 5 congeners - 7 6 -used i n laboratory experiments were run through the GC/MS( 2,4-DCP; 2,4,5-TCP or 2-CP; 2,4,6-TCP, 2,3,4,6-TTCP, and PCP). Standard mixtures at a higher concentration level (100 ppm) were used for chlorophenols i d e n t i f i c a t i o n . This was due to the fact that the f u l l scanning mode of the GC/MS has a s e n s i t i v i t y 100 times lower than the electron-capture detector used for the chromatographic analyses.Since the spectral l i b r a r y (31,500 spectra to date) did not contain the acetylated chlorophenols,the spectra of the chlorophenols standards were f i l e d . A set of electron impact spectra are presented i n Appendix I I . The use of po s i t i v e ion chemical i o n i z a t i o n was explored as an a l t e r n a t i v e method for chlorophenols i d e n t i f i c a t i o n . This method gives a much higher molecular i n t e n s i t y . Appendix II contains the t o t a l ion chromatogram and a complete set of posi t i v e ion chemical i o n i z a t i o n spectra of the chlorophenols monitored during the study . Leech extracts were also analysed on the GC/MS system and the chlorophenols presence confirmed. A tentative i d e n t i f i c a t i o n of other major peaks present i n the leech matrix was performed( see Appendix II for a l l the pertinent spectra). Negative chemical i o n i z a t i o n i s a method of combined chromatographic and mass spectra analysis i n the range of the ECD s e n s i t i v i t y . This technique was not explored but single ion monitoring was conducted, which i s known to enhance s e n s i t i v i t y up to 1000 times. Appendix II contains spectra obtained through t h i s method. I d e n t i f i c a t i o n and quantitation of the leech control( blank) samples were done and they confirmed the levels found with the regular GC . - 77 -3.5.2 Reproducibility and Technique Evaluation. To determine r e p r o d u c i b i l i t y of the sample preparation for the GC analysis, the extraction step and the d e r i v a t i z a t i o n step were assessed separately by c a l c u l a t i n g recovery of chlorophenols from water and leech matrix and by c a l c u l a t i n g the d e r i v a t i z a t i o n r e p r o d u c i b i l i t y . Reproducibility of the r e s u l t s as a function of i n j e c t i o n technique, instrument parameters and sample v i a l s condition was evaluated by c a l c u l a t i n g the i n j e c t i o n accuracy. F i n a l l y , the method detection l i m i t (MDL) was calculated through a method defined by Glaser et al.(1981). 3.5.2.1 Recovery from Water At le a s t four 1 L water samples were spiked with 1 mL of 1 ppm mixture of 5 chlorophenols.(2,4-DCP; 2,4,6-TCP; 2,4,5-TCP; 2,3,4,6-TTCP; PCP). The respective spiked water samples were extracted for chlorophenols using toluene and back extracted using the 0.IM solution of R^CO^. The extracts were derivatized and analysed for chlorophenols. Results were compared with the deriv a t i z e d standard of 0.1 ppm concentration.(1 mL of 1 ppm chlorophenols added to 1 L of water and contained at the end of d e r i v a t i z a t i o n i n 10 mL of hexane leads to a t h e o r e t i c a l l y concentration of 0.1 ppm chlorophenols i n the derivatized e x t r a c t ) . - 78 -3.5.2.2 Recovery from Leeches At least four leeches were spiked with 1 mL of 1 ppm mixture of 5 chlorophenols. Spiking was done i n the v i a l s which contained the acid digested leech. Normal extraction steps were followed and the derivatized chlorophenols contained i n 10 ml of hexane were compared with a 0.1 ppm standard of derivatized chlorophenols. 3.5.2.3 Derivatization Reproducibility Deri v a t i z a t i o n r e p r o d u c i b i l i t y was determined at 2 levels of concentration, namely 0.1 ppm and 1 ppm of the acetylated derivatives. For the 0.1 ppm derivatized mixture of the 5 chlorophenols, one mL of 1 ppm non-derivatized standard mixture of the 5 chlorophenols was added to 100 mL of 0.1 M I^CO^ and acetylation performed as usual. The standard deviation was calculated on the basis of 5 derivatized samples. Same procedure was followed for the 1 ppm l e v e l . Various concentrations of derivatized standards were obtained i n a s i m i l a r manner. It should be mentioned that an absolute derivatized standard, what i s c a l l e d a check standard which can be usually supplied by the chromatographic material supplier was not a v a i l a b l e . Hence i t was necessary to evaluate the d e r i v a t i z a t i o n r e p r o d u c i b i l i t y as part of the process of standardization. - 79 -3.5.2.4 Injection Accuracy Twelve i n j e c t i o n s of a 0.1 ppm standard of the mixture of 5 derivatized chlorophenols were made and the mean and the standard deviations of the r e s u l t s were used to calculate the accuracy of a single i n j e c t i o n reported within the 95% confidence i n t e r v a l . The i n j e c t i o n v a r i a b i l i t y evaluation contained some of the v a r i a b i l i t y a t t r i b u t a b l e to instrumental parameters and d i f f e r e n t sample v i a l s . Proper preparation of the GC p r i o r to running a set of samples was found to be very important. The instrument required at least 4 hours of e q u i l i b r a t i o n before any of the readings could be used. Those 4 hours did not include the column preparation or detector s t a b i l i z a t i o n . Various standards of chlorophenols were run during the 4 hour period and an assessment of the c a l i b r a t i o n curve was made on the basis of previous runs. New c a l i b r a t i o n f i l e s were used for each day of operation. As a q u a l i t y control measure af t e r every 10 samples, a standard of known concentration was run i n order to confirm the accuracy of the c a l i b r a t i o n f i l e . 3.5.2.5 Method Detection Limit Detection l i m i t i s one of the most important performance c h a r a c t e r i s t i c s of an a n a l y t i c a l procedure. The detection l i m i t of the method used i n t h i s study was assessed according to the method described by Glaser et a l . , 1981. They defined MDL as an error d i s t r i b u t i o n implying that 99% of the t r i a l s measuring the - 80 -analyte concentration at the MDL must be s i g n i f i c a n t l y d i f f e r e n t from zero analyte concentration. The procedure of determining the MDL contains the following steps: -MDL i s f i r s t estimated. -samples containing the analyte at the estimated MDL are analysed. - on the basis of the standard deviation of the seven re p l i c a t e s measurements the MDL i s computed as: M D L = ^ - 1 , 1 ^ . 9 9 ) * 3 where S i s the standard deviation of r e p l i c a t e determinations at a fixed concentration and t,„ , , _ n n . i s the student's t (N-1,1-°^ = .99) value for a one-tailed t e s t at the 99% confidence l e v e l with N-1 degrees of freedom. If i n i t i a l estimates of the MDL are not proximate to the true MDL, the calculated MDL w i l l be much i n error. This f a c t can be e a s i l y tested knowing that the estimated MDL i s equal to the calculated MDL, i f the 95% confidence i n t e r v a l of the calculated MDL contains the MDL value. Detection l i m i t i s usually provided by the instrument.manufacturer. However t h i s method assesses the MDL to an a n a l y t i c a l method and to a sample matrix. - 81 -4. RESULTS AND DISCUSSION 4.1 Introduction This chapter presents r e s u l t s under three main sections: a n a l y t i c a l technique evaluation, laboratory experiments, and f i e l d experiments. A series of chromatograms and spectra pertinent to the i d e n t i f i c a t i o n and confirmation of chlorophenols i s presented i n Appendix I I . Laboratory leech experiments are discussed before the leech f i e l d experiments but controlled laboratory experimental conditions were established from preliminary f i e l d r e s u l t s . Special laboratory experiments are presented separately at the end of the chapter. These experiments were designed to take place concurrently with f i e l d experiments and t h e i r parameters were set i n such a way that the f i e l d experimental r e s u l t s would be easier to i n t e r p r e t . These special experiments are part of the recommended methodology i f leech bioconcentration c a p a b i l i t y i s to be used for monitoring purposes. A general discussion of the experimental data i s presented at the end of each section and i t constitutes the basis of the f i n a l conclusions presented i n the following chapter. - 8 2 -4.2 A n a l y t i c a l Technique. Reproducibility and Technique  Evaluation. A l l the data gathered to evaluate the a n a l y t i c a l technique used to quantify the chlorophenols are presented i n Table 4.1. As expected, lower detection l i m i t s were obtained for the higher chlorinated compounds of the chlorophenol family, with values as low as 2 ppt for 2,4,5-TCP, 2,3,4,6-TTCP and PCP. Krijgsman and Van de Kamp(1977) report the l i m i t of detection for the pentachlorophenol acetate at 1 pg( equivalent to 1 ppt). In our study we assumed that the d e r i v a t i z a t i o n reaction was 100% complete and r e p r o d u c i b i l i t y of the d e r i v a t i z a t i o n referred s o l e l y to the technique used. Derivatization r e p r o d u c i b i l i t y calculated at two lev e l s of concentration, namely 0.1 ppm and 1 ppm, was better at the higher concentration l e v e l , with the exception of PCP were a higher deviation was found for the 1 ppm standard. The y i e l d s of PCP acetate from the acetylation of PCP on d i f f e r e n t stationary phases were determined by Chau and Coburn (1974). They reported values higher than 93% for three d i f f e r e n t types of columns. The 6'x 1/4" OD glass column packed with 3.6% OV-101-5.5% OV-210 on 80-100 mesh Chromosorb had c h a r a c t e r i s t i c s s i m i l a r to the c a p i l l a r y column used. Average recoveries from water were a l l i n the 90 % range with standard deviations within ± 10%. The i n i t i a l evaluation of the recovery from water was done using d i l u t i o n water. Subsequent recovery values have been determined for the r i v e r water samples at the time of each f i e l d experiment. Recovery values obtained TABLE 4.1. REPRODUCIBILITY AND TECHNIQUE EVALUATION(1) Recovery Reproducibility Compound Detection Limit Water^2) Leeches^3^ Derivatization^4) Injection^5) (ppt) 0.1 ppm 1 ppm (accuracy) 2,4-DCP 10 100 + 9.6 83 + 12.4 + 10.6 + 5.7 100 + 8.4 2,4,6-TCP 10 98 + 8.3 99.7 + 6 + 9.9 + 5.0 100 + 4.6 2,4,5-TCP 2 95.6 + 6.0 92 + 8.5 + 14.5 + 6.0 100 + 11.5 2,3,4,6-TTCP 2 96.5 + 7.3 87.5 + 0.7 + 10.3 + 9.0 100 + 4.8 PC? 2 95 + 6.8 84.7 + 7.5 + 12.3 +18.8 100 + 7.7 (1) All values expressed as percent unless Indicated. (2) Water samples were spiked with 1 ppm mixture of the 5 CPs; mean and st. dev. calculated for n=4. (3) Leeches were spiked with 1 ppm mixture ol the 5 CPs; mean and st. dev. calculated for n=4. (4) Derivatization reproducibility was determined at 2 concentrations. A mixture of the 5 CPs at 0.1 ppm and at 1 ppm was derivatized . The standard deviation was calculated on the basis of 5 derivatized samples (n=5). (5) This is the accuracy for one injection reported with 95% confidence. The accuracy is calculated on the basis of 12 injections (n=12). - 84 -for the r i v e r water were usually i n the same range as those obtained for the d i l u t i o n water. Chau and Coburn( 1974) reported s i m i l a r recoveries of PCP from d i s t i l l e d and tap waters( 88% average). Lake Ontario water spiked by them resulted i n recoveries from 84 to 93 %. Recovery of chlorophenols from the extraction-acetylation step was 80-100% i n the work of Krijgsman and Van de Kamp and 90% or better i n the work of Metcalfe et al.(1984), which makes our r e s u l t s very comparable. Recovery values lower than the above mentioned range were obtained for the r i v e r water co l l e c t e d during the experiment between A p r i l 18 and A p r i l 25,1985. The r i v e r water contained a higher sediment load at that time of the year and the recovery values were 52% for TTCP and 44% for PCP( suspended s o l i d s load in the Fraser River water varies from 18 to 118 ppm, with the highest values during freshet ( Drinnan and Clark, 1980). For that p a r t i c u l a r run, corrections were applied to the concentrations of chlorophenols i n the water. Average recoveries from leeches were also very good with over 80% recovery and a maximum of 12 % deviation from the mean for the DCP. The f a c t that the derivatized extracts did not need to be further concentrated enhanced the recovery both from the water and the leech matrix. Reproducibility of r e s u l t s as a function of i n j e c t i o n technique, instrument parameters and sample v i a l condition was very good. The degree of accuracy obtained by using one i n j e c t i o n per sample was i n the same range as other s t a t i s t i c a l c h a r a c t e r i s t i c s of the a n a l y t i c a l technique therefore quantitative determination of chlorophenols was done on the basis - 85 -o f o n e i n j e c t i o n p e r s a m p l e . 4.2 . 1 C h r o m a t o g r a m s o f A c e t y l a t e d C h l o r o p h e n o l s : S t a n d a r d  M i x t u r e s , W a t e r a n d L e e c h S a m p l e s . T h e w o r k i n g r a n g e o f c o n c e n t r a t i o n f o r t h e s t a n d a r d s o l u t i o n s w a s f o u n d t o b e 0.01 t o 1 ppm. T h i s r a n g e s e e m e d t o c o n t a i n t h e f i e l d c h l o r o p h e n o l s c o n c e n t r a t i o n s a s w e l l a s t h e c h l o r o p h e n o l s l e v e l s b i o c o n c e n t r a t e d i n t h e l e e c h d u r i n g t h e e x p o s u r e e x p e r i m e n t s . S h a r p , s y m m e t r i c a l l y a n d c o m p l e t e l y s e p a r a t e d p e a k s o f a l l t h e c h l o r o p h e n o l s w e r e o b t a i n e d o v e r a l l t h i s r a n g e . F i g . 4.1 p r e s e n t s a c h r o m a t o g r a m o f a s t a n d a r d m i x t u r e o f a l l t h e c o n g e n e r s i n t h e c h l o r o p h e n o l f a m i l y , a t 0.01 ppm c o n c e n t r a t i o n . C o r r e s p o n d i n g t o t h e i r e l u t i o n t i m e s t h e p e a k s a r e i d e n t i f i e d i n t h e f i g u r e f o o t n o t e s . A s t h e s e n s i t i v i t y o f t h e d e t e c t o r r e s p o n d s t o t h e n u m b e r o f c h l o r i n e a t o m s i n t h e m o l e c u l e , o n e c a n s e e t h a t t h e h i g h e r p e a k s c o r r e s p o n d t o t h e t r i , t e t r a a n d p e n t a c h l o r o p h e n o l s a n d t h e v e r y s h o r t p e a k s c o r r e s p o n d t o t h e m o n o - c h l o r i n a t e d c h l o r o p h e n o l s . C h r o m a t o g r a m s a t a l o w e r c o n c e n t r a t i o n l e v e l , s u c h a s 0 . 0 0 1 ppm, c o n t a i n e d o n l y p e a k s o f t h e h i g h e r c h l o r i n a t e d c h l o r o p h e n o l s . I n t h e c a s e o f 2,4 a n d 2 , 5 - D C P w h i c h e l u t e t o g e t h e r a n d w e r e n o t s e p a r a t e d i n t h e c h r o m a t o g r a m a t 0.01 ppm, a c o m p l e t e s e p a r a t i o n w a s o b t a i n e d a t l o w e r c o n c e n t r a t i o n o f t h e s t a n d a r d m i x t u r e . S i n c e a t 0 . 0 0 1 ppm a n d l o w e r , t h e m o n o c h l o r i n a t e d c h l o r o p h e n o l s d o n o t a p p e a r o n t h e c h r o m a t o g r a m , t h e c h r o m a t o g r a m o b t a i n e d a t 0.01 ppm w a s c h o s e n f o r i l l u s t r a t i v e p u r p o s e s . - 86 -F i g . 4.1. Chromatogram of acetylated chlorophenols. A l l the 19 chlorophenol congeners were separated i n t h i s run with the exception of 2,4 and 2,5-DCP which eluted together. Concentration of the standard mixture was 0.01 ppm of each congener. Peaks i d e n t i f i c a t i o n according to retention time: 9. 81 min : 2-CP 10. 35 min : 3 -CP 10 .50 min: 4-CP 13. 01 min : 2 ,3-DCP 13 .52 min: 2,4-DCP 13. 52 min : 2 ,5-DCP 13 .86 min: 2,3,5-TCP 14. 51 min : 2 ,6-DCP 15 .25 min: 2,3,4-TCP 16. 52 min : 2 ,4,6-TCP 17 .92 min: 3,5-DCP 18. 10 min : 2 ,3,6-TCP 18 .27 min: 2,4,5-TCP 19. 47 min : 3 ,4-DCP 19 .77 min: 3,4,5-TCP 21. 29 min : 2 ,3,5,6-TTCP 21 .40 min: 2, 3, 4,6-TTCP 22. 76 min : 2 ,3,5,6-TTCP 26.05 min: PCP - 87 -If one compares the i l l u s t r a t i v e chromatogram(Fig. 4.1) with the chromatogram from Appendix I ( F i g . I . l ) obtained by Krijgsman and Van de Kamp(1977) one can notice the high l e v e l of resolution achieved i n both. One difference i s that t h e i r chromatogram contains only 13 congeners and they are at various concentrations i n order to keep the peak height uniform. They do report the r e l a t i v e retention times of a l l the 19 congeners, however no chromatogram accompanied t h e i r r e s u l t s to v e r i f y complete separation of a l l the compounds. For the laboratory controlled experiments a mixture of only 5 chlorophenols was used. Their chromatogram i s represented i n Fig.4.2. The l e v e l of concentration used i n t h i s run was 1 ppm and the peaks are i n order of e l u t i o n : 2,4-DCP(14.40 min) 2,4,6-TCP(17.46 min) 2,4,5-TCP(19.10 min) 2,3,4,6-TTCP(22.21 min) and PCP(27.25 min). One can see that the size of the peaks corresponding to t h i s concentration i s much larger than for the 0.01 ppm standards( see F i g . 4.1). Most sample concentrations were found to be i n the neighbourhood of 0.1 ppm, the intermediate concentration( see F i g . 4.3). A t y p i c a l chromatogram of a Fraser River water sample spiked with a mixture of chlorophenols was very s i m i l a r to a standard solution chromatogram( see F i g . 4.4 ). The extraction and acetylation steps manage to clean up the water samples of most i n t e r f e r i n g and unwanted compounds. One can also see that, the retention times of the chlorophenols are very reproducible even when run on d i f f e r e n t days. A t y p i c a l water blank chromatogram i s represented i n F i g . 4.5. Four peaks of i n t e r e s t were i d e n t i f i e d : 2,4,6-TCP; - 8 8 -Fig.4.2. Chromatogram of a 1 ppm standard mixture chlorophenols. Z.M-QCJP TCP, •7> i n 7* Fig.4.3. Chromatogram of a chlorophenols. 0.1 ppm standard mixture Fig.4.4. Chromatogram of a Fraser River water sample spiked with a mixture of 5 chlorophenols! same chlorophenols as i n the standards from Fig.4.2 and 4.3. Spiking l e v e l : 10 ppb. - 90 -Fig.4.5. Chromatogram of a water blank. Fig.4.6. Chromatogram of a Fraser River water sample. Date of sampling: September 13, 1985. - 91 -2,4,5-TCP; 2,3,4,6-TTCP and PCP. Only the TTCP and PCP were quantified at 8 ppt and 6 ppt respectively. The other i d e n t i f i e d peaks were present i n concentrations below the detection l i m i t . Generally, the chlorophenol values for water blanks were i n the neighbourhood of 10 ppt. These values were considered as background concentration and substracted from the values of chlorophenols found i n the r i v e r water samples. A chromatogram of acetylated CPs extracted from a Fraser River water sample i s represented i n F i g . 4.6. Peaks of in t e r e s t are TTCP(at retention time: 22.22 min) and PCP (at retention time: 27.27 min). Quantification of those peaks gave 2.268 ppb TTCP and 0.345 ppb PCP. This water sample was concentrated 120 times by the extraction technique p r i o r to the GC analysis. A chromatogram of acetylated CPs extracted from a leech i s presented i n F i g . 4.7. This p a r t i c u l a r leech had been exposed to Fraser River water for 6 days i n the i n t e r v a l September 12-18/1985. The leech extract was d i l u t e d 10 times p r i o r to the GC analysis. The leech chromatogram i s much more complex than the chromatogram of a water sample taken during the same time i n t e r v a l ( F i g . 4.6). A t y p i c a l chromatogram of the acetylated CPs extracted from a control(blank) leech i s presented i n F i g . 4.8. If compared with a water blank chromatogram (see Fig.4.5), the control leech chromatogram contains very many peaks. In the chromatogram from Fig.4.8, four peaks of i n t e r e s t were i d e n t i f i e d and t h e i r q u a n t i f i c a t i o n gave the following values: 47 ppb for 2,4-DCP; 24 ppb for 2,4,5-TCP; 36 ppb for 2,3,4,6-TTCP and 76 ppb for PCP. The le v e l s of chlorophenols found i n the control leeches - 9 2 -Fig.4.8 Chromatogram of a leech blank. - 93 -were always high, and a viable explanation for these high background leve l s i s s t i l l to be found. The peaks were confirmed by GC/MS analysis to be chlorophenols and not some other compounds with si m i l a r retention times. Control leeches were co l l e c t e d together with a l l the other leeches from Kentucky Lake and analysed at a r r i v a l i n the Vancouver laboratory. The lake i s located i n a remote area and should be contaminant free, but as other researchers have reported, low background concentration of CPs were present i n p r i s t i n e environments(Pierce et a l . , 1977). They reported a 0.5 ppb of PCP i n the water column of t h e i r control pond. If a sim i l a r or even lower concentration was present i n Kentucky Lake, given the high bioconcentration c a p a b i l i t y of leeches, the high level i n the control leech could be explained. Unfortunately, the Kentucky Lake water was never tested for chlorophenols. The consistency of CPs i n the control leeches made i t necessary to accept t h i s contamination as an experimental condition( average and standard deviation of the CPs found i n six control leeches were 16 ± 13 ppb for 2,4,5-TCP, 2 5 + 8 ppb for TTCP and 91 ± 3 6 ppb for PCP). The only step taken to improve the res u l t s r e l i a b i l i t y was to s e l e c t the siz e of the control leeches. Size s e l e c t i o n was done by keeping the control leech s i z e i n the same size range with leeches used i n the experiment for which the control leech was to be considered. The size range and influence of size on the bioconcentration c a p a b i l i t y w i l l be discussed further i n a separate section. - 94 -4.2.2 I d e n t i f i c a t i o n and Confirmation of Chlorophenol Acetates  Through Mass Spectrometry A l l the i l l u s t r a t i v e spectra pertinent to t h i s section are contained i n Appendix I I , to which references are made throughout the text. As outlined i n the previous chapter, three methods of mass scanning were used for i d e n t i f i c a t i o n and confirmation of chlorophenols i n various sample matrices: electron impact, p o s i t i v e ion chemical i o n i z a t i o n and single ion monitoring. 4.2.2.1 Electron Impact GC-MS Spectra Standard mixtures used for t h i s method were scanned and spectra of the acetylated derivatives of the chlorophenols present i n the standards used were f i l e d i n the spectral l i b r a r y of the Applied Science GC-MS laboratory. F i g . II.1 presents a chromatogram of a 100 ppm standard mixture containing: 2-CP(mol wt=170); 2,4-DCP(mol wt=204); 2,4,6-TCP(mol wt=238); 2,3,4,6-TTCP(mol wt=272); PCP(mol wt=306). Fig. II.2 to II.6 present the EI spectra of each acetylated chlorophenol i n t h i s standard. A leech extract was scanned by t h i s method and a tentative i d e n t i f i c a t i o n of unknown peaks was made. Fig.II.7 presents the chromatogram of a leech extract which was exposed to a standard mixtures of chlorophenols for 7 days( 2-CP; 2,4-DCP; 2,4,6,-TCP; 2,3,4,6-TTCP and PCP). Three other peaks were i d e n t i f i e d as dibromophenolacetate, tribromophenolacetate and underivatized - 95 -pentachlorophenol. No explanation was found for the presence of the brominated chlorophenols i n the leech matrix. Underivatized pentachlorophenol indicated that t h i s p a r t i c u l a r phenol was not exhaustively derivatized during the preparation step, however, the peak was very small i n comparison with the derivatized chlorophenol peak. 4.2.2.2 Positive Ion Chemical Ionization Spectra The same standard mixture was used for t h i s scanning method. Fig. II.8 presents a t y p i c a l t o t a l ion chromatogram of the standard mixture and F i g . II.9 to 11.13 present the spectra of each i n d i v i d u a l acetylated chlorophenol obtained through t h i s method. The ion source pressure used was 0.2 x 10-4, using methane as i o n i z i n g gas, and temperature programming had a constant rate of 8 C/min from 75 C to 280 C. 4.2.2.3 Single Ion Monitoring Spectra Parameters used for t h i s method are contained i n Table II.1 from Appendix I I . The method was applied to various samples such as leech extracts, Fraser River water samples and standard mixtures. A semiquantitative analysis of the samples was also t r i e d . F o r t y - f i v e picograms appeared to be the detection l i m i t of the method. Fi g . II.14a to II.14e present the chromatogram and the spectra from a leech extract, and F i g . II.15a to II.15e present - 96 -the chromatogram and the spectra pertinent to a Fraser River water sample. A control(blank) leech extract was analysed through t h i s method of increased s e n s i t i v i t y and the presence of chlorophenols at high le v e l s was confirmed. This confirmation was necessary for the assessment of the background concentration i n a l l the leech experiments. 4.3 Laboratory Leech Experiments A series of laboratory experiments was set up at the beginning of the experimental work i n order to determine the range of concentrations to be used to make the exposure solutions. These laboratory data, together with the levels of chlorophenol found i n the Fraser River during the preliminary grab sampling t r i p s , constituted the basis to es t a b l i s h the parameters for the laboratory controlled experiments. 4.3.1 Preliminary Leech Exposures The preliminary exposures covered three areas of importance to help set the experimental parameters, namely, 24 hour exposure, depuration and pH influence on bioconcentration a c t i v i t y . - 97 -4.3.1.1. 24 Hour Bioconcentration Ratios The concentration of chlorophenol found i n leeches a f t e r 24 h exposure was divided by the solution concentration i n order to obtain the bioconcentration r a t i o s . The term of bioconcentration factor was avoided since the equilibrium state was not reached i n such short time and i t would have been improper to use the term of bioconcentration factor i f i t s d e f i n i t i o n was respected( see section 2.3.1). The r e s u l t s of the 24 h exposure tests at various chlorophenol concentrations are summarized i n Table 4.2. TABLE 4.2 CONCENTRATION RATIOS IN LEECHES AFTER 2 4 HOUR EXPOSURE Compound Exposure solution concentration 10 ppb 2 0 ppb 3 0 ppb 2,4-DCP 40 40 54 2,4,6-TCP 58 50 79 2,4,5-TCP 60 72 88 2,3,4,6-TTCP 60 72 100 PCP 60 88 135 Note:solutions contained 10 ppb, 20 ppb and 30 ppb of each of the CPs l i s t e d i n the composition ; average leech weight = 1.35 g, standard deviation = 0.37 g ; experimental temperature = 2 2 C. pH = 6.5. - 98 -Only one leech was exposed at each concentration of the solutions used for the exposure tests and the r e s u l t s have no s t a t i s t i c a l value. However, one can see that the general trend i s to exhibit higher bioconcentration r a t i o s as the concentration of the exposure solution i s increased and the trend i s more evident for the higher chlorinated chlorophenols(i.e. TTCP and PCP) . For the same concentration of the exposure solution the lowest r a t i o corresponds to the least chlorinated chlorophenol. This i s expected since i t i s known that the bioconcentration factors r e l a t e p o s i t i v e l y to the Po/w values, which increases with the number of chlorine atoms i n the molecule( Konemann and Musch, 1981). No attempt was made to determine i f these r e s u l t s f i t equations developed by various researchers( Saarikovski and V i l u k s e l a , 1982) since the values determined i n t h i s preliminary experiment were not true equilibrium bioconcentration factors. Exposure of leeches to a solution of 1 ppm TTCP (1000 ppb) for 24 h gave a bioconcentration r a t i o of 100. This would indicate that at higher exposure concentration, the bioconcentration a c t i v i t y levels off and does not continue to increase with increased concentration. This behaviour follows the theory developed by Majori and Petroni(1973) who stated that the concentration i n biota cannot exceed a c e r t a i n maximum concentration( concentration of binding centers). They also stated that the rate of uptake i s proportional not only to the concentration of the chemical i n water but to the concentration of free binding centers. - 9 9 -The same experiment was repeated for 1 ppm PCP , but the PCP concentration proved to be deadly to the leeches and no conclusions were drawn from that experiment, except that the dead leeches seem to loose the bioconcentrated chlorophenol once they die( very low levels of PCP were found i n the dead leeches). The same observation was made on other occasions when dead leeches were analysed a f t e r various exposure experiments when some deaths occurred . I t was therefore decided that chlorophenol concentrations i n dead leeches were never to be used . The phenomenon of chemical release upon death by leeches was also observed by J.H. Carey from Canada Centre for Inland Waters,Burlington, Ontario (personal communication). 4.3.1.2 Depuration Tests The depuration experiments were ca r r i e d out for one week. Leeches exposed i n Fraser River for one week were used for the depuration experiment. At the end of the exposure time, three of the i n i t i a l f i v e leeches were analysed immediately a f t e r c o l l e c t i o n and two underwent depuration. At the end of 7 day exposure i n clean water(dilution water, which was changed every day), the concentration of chlorophenol i n the leech matrix was at the same l e v e l as found i n the leeches analysed r i g h t a f t e r the 7 day exposure i n the r i v e r (see Table 4.3). The depuration rate constant was not determined since more data would have been required along with an extended period of depuration. Depuration rate constant determination was beyond the scope of t h i s research. Personal communication with J.H Carey, - 100 -TABLE 4.3 CHLOROPHENOLS IN LEECHES EXPOSED IN THE FRASER RIVER ESTUARY Sampling Location Date E x p o s u r e ^ TTCP^2) p Cp(2) S i t e (1984) /Depuration *g/g "g/g 2 Kerr St. Oct. 4-11 7/0 0.476 0.226 Oct. 4-11 /Oct. 11-18 7/7 0.540 0.311 5 Mi t c h e l l Is. Oct. 4-11 7/0 2.946 0.846 Oct. 4-11 /Oct. 11-18 7/7 3.733 0.771 (1) Exposure and depuration time i n days. (2) Average values i n wet weight, n=2 f o r leeches that underwent depuration, n=3 for leeches analyzed immediately a f t e r c o l l e c t i o n . - 1 0 1 -confirmed a very slow rate of depuration for chlorophenols i n the case of leeches( i n i t i a l concentration of the chlorophenol i n the leech was reduced by 50 percent i n approximately 6 months). For th i s research which had the goal of using leeches for r e l a t i v e l y short( seven days) exposure experiments as p o l l u t i o n biointegrators, the estimate of depuration for one week was sa t i s f a c t o r y . The determination of the depuration rate constant( K2) would have been necessary i f the 'bioconcentration factor was to be calculated by the r e l a t i o n s h i p : BF = K1/K2 Determination of BF was chosen to be made by d i r e c t measurements of the p a r t i t i o n i n g at the steady state, which conforms to the equation: BF = C(organism)/ C(water) 4.3.1.3 pH Experiments , The l a s t of the preliminary experiments determined the eff e c t s of pH on bioconcentration. As with a l l preliminary experiments, i t served to set the parameters for more detailed experiments. Only the general trend of the bioconcentration process was monitored. The experiment was carr i e d out to validate the theory behind the pH influence upon bioconcentration under our experimental conditions. The re s u l t s are presented i n Table 4.4 - 102 -TABLE 4.4 CONCENTRATION RATIOS AS A FUNCTION OF pH FOR 24 HOUR EXPOSURES Compound pH 9.2 7.5 5.0 2,4-DCP 42 88 73 2,4,6-TCP - 57 90 2,4,5-TCP 13 5.1 . 115 2,3,4,6-TTCP 13 73 115 PCP 29 71 125 Note: experimental temperature= 4C; solution at 10 ppb of each CPs; the m u l t i p l i e r s are calculated from the concentrations found i n leeches at the end of 24 hours, expressed i n ug/g wet weight, divided by the solution concentration taken as constant over the exposure period; average leech weight = 0.07 9 g, standard deviation = 0.0061 g. Except for 2,4-DCP, the trend shows an increase i n bioconcentration with the decrease i n pH. The theory developed by Konemann and Musch (1981) on the pH influence upon the t o x i c i t y of CPs states that the nonionic form i s p r e f e r e n t i a l l y concentrated i n the tiss u e s . Since l i p o p h i l i c i t y i s a c h a r a c t e r i s t i c of the nonionic form , p a r t i t i o n i n g into the biota i s favored under low pH conditions. The nonionic molecules seem to penetrate the e p i t h e l i a l tissues easier than the charged molecules.(see Chapter 2 for more d e t a i l s ) . A l l mathematical relationships between BF and Po/w, developed by various researchers, f i t the general form: log BF = a log Po/w + b where a and b are constant. pH influences the s o l u b i l i t y i n water of the chlorophenols as i t i s known that the nonionic form i s far less soluble than the i o n i c form (NRCC,1981). Increased s o l u b i l i t y i n water w i l l - 103 -adversely a f f e c t the value of Po/w and i n d i r e c t l y change the value of BF since the two parameters are d i r e c t l y proportional to each other. Saarikovski and Viluksela(1982) developed a re l a t i o n s h i p for the chlorophenol series l i n k i n g t o x i c i t y d i r e c t l y to dpKa. log (1/LC50) =0.36 dpKa +0.69 where dpKa = pKa( phenol) - pKa( substituted phenol) . Since the 1/LC50 values can be substituted with BF values(Kobayashi et a l . , 1978) a d i r e c t proportional r e l a t i o n s h i p i s obtained between BF and dpKa of various chlorophenols. dpKa's are higher for TTCP and PCP( 4.59 and 5.34 respectively) than for the other chlorophenols(Saarikovski and Viluksela,1982) and that explains the higher bioconcentration r a t i o s obtained i n t h e i r case. A decrease i n pH af f e c t s the bioconcentration of the higher chlorinated phenols more, since they have lower values of pKa. In the case of 2,4-DCP which has a pKa value of 7.90, our res u l t s were a l i t t l e confusing because bioconcentration was higher at pH 7.5 than at pH 5.0. In t h i s pH range, 2,4-DCP i s mostly undissociated, and one would expect somewhat comparable concentration r a t i o s at the two pHs. What can be concluded from the r e s u l t s i s that t h i s phenol i s not influenced i n the same manner by pH as the other chlorophenols. The unusual behaviour of 2,4-DCP was consistent with other experiments that w i l l be presented. The i n i t i a l pH of the d i l u t i o n water was 7.5. The pH gradually decreased to 6.5 af t e r a day of exposure which was probably a t t r i b u t a b l e to the low buffering capacity( alk. = 50 - 104 -mg/L CaCO^)• For longer experiments the pH varied between 6.0-6.5. Additional buffering capacity was avoided to prevent any stress on the leeches. However, since the median values of pH i n the Fraser River Estuary varies from 7.0 to 8.0 ( Drinnan and Clark, 1980) these experimental conditions were considered s a t i s f a c t o r y to allow comparison to f i e l d conditions. 4.3.1.4 Summary of Results The 24 h exposure experiment gave values for the bioconcentration r a t i o s from 40 to 135. At the same concentration l e v e l , the higher chlorinated phenols exhibited higher r a t i o s than the lower chlorinated phenols( 88 for PCP as compared with 50 for 2,4,6-TCP, at a solution concentration of 20 ppb). Higher concentration of the exposure solution seemed to increase the r a t i o s . This phenomenon was observed however for moderate increases i n concentration( a few times). At a d r a s t i c increase i n concentration( 50 fold) the bioconcentration r a t i o s leveled o f f ( i n the case of TTCP the r a t i o s were 72 at 20 ppb, 100 at 30 ppb and 100 at 1000 ppb). The depuration rate was very slow for the system leech/ chlorophenol. I t was assumed to be n e g l i g i b l e for the duration of a l l the experiments which never exceeded one week. Dead leeches released the accumulated chlorophenols and t h e i r CPs le v e l s were never included i n any experimental r e s u l t s . The change i n pH affected the bioconcentration as the theory predicted. Lower pHs lead to higher bioconcentration r a t i o s and the e f f e c t of pH was more pronounced for the higher - 105 -chlorinated phenols with lower pKa values than for the other chlorophenols. pH should not have a d i r e c t influence on the bioconcentration of CPs during the f i e l d experiments since the Fraser River water pH i s more/or less constant. Any l o c a l i z e d v a r i a t i o n of pH i s ra p i d l y •neutralized by the great d i l u t i o n capacity of the r i v e r . The pH influence must be considered when laboratory experiments are set up. The pH should be kept constant and the f i e l d and laboratory pH should be s i m i l a r i f the r e s u l t s are to be compared. 4.3.2 Bioconcentration Factors Determination As mentioned before, the bioconcentration factor should be calculated by determination of p a r t i t i o n i n g at steady-state. This method i s by far the most accurate of the three methods used (one other method uses relationships between BF and Po/w and one uses the reaction rates constants; see Esser and Moser, 1980). The major shortcoming of the d i r e c t measurement method i s that i t sometimes takes a long time to es t a b l i s h the steady state conditions. 4.3.2.1 Bioconcentration of Chlorophenols i n a 7-Day Laboratory  Experiment The f i r s t laboratory experiment i n which the bioconcentration factors of various chlorophenols were calculated was ca r r i e d out for 7 days. A mixture of 5 chlorophenols was used at a concentration of 10 ppb each. Experimental temperature was - 1 0 6 -22°C and the large black 1eeches(P.marmorata) were used. Average leech weight was 0.332 g with a standard deviation of ±0.257 g. Most of the graphical r e s u l t s ( see Fig.4.9) are averages for n=2 and a few are for n=l, so the s t a t i s t i c a l l y s i g n i f i c a n c e of t h i s test i s very li m i t e d . The purpose of t h i s 7-day exposure experiment was to determine the pattern followed by leeches i n the bioconcentration process as they were intended to be used for monitoring purposes and f i e l d exposure time never exceeded 7 days. The c a l c u l a t i o n of the bioconcentration factors was determined from the concentrations found i n the leeches at the end of the exposure period. Fig.4.9 shows that steady state conditions were not quite reached a f t e r 7 days. Therefore, the values obtained for the bioconcentration factors could be i n error and they were used with caution. Bioconcentration factors were 2155 for PCP, 2111 for TTCP, 1450 for 2,4-DCP and 1340 for 2,4,6-TCP ;2,4,5-TCP had an unexpected value high value of 2280. Lower experimental temperatures used l a t e r lead to more accurate bioconcentration factors since the steady state was reached within the experimental time. The fact that at 22 °C steady state was not reached and that i t was reached at lower temperatures seemed to indicate that the higher temperature delayed the steady state. This i s contradictory to the observation made by Saarikovski and V i l u k s e l a (1982) about the accumulation of phenols by guppies. They stated that steady state was reached more rapidl y at high temperature. If one needs an accelerated test to determine bioconcentration factors, c a l c u l a t i o n using rate constants of - 1 0 7 -Fig.4.9 Bioconcentration of chlorophenols by leeches i n a seven-day laboratory experiment. Experimental temperature was 22 °C. The leech species used was P. marmorata. - 108 -uptake and clearance i s probably a better method(Branson et a l . ,1975) . 4.3.2.2 Bioconcentration of Chlorophenols at Different  Temperatures The data obtained during these temperature experiments are graphically represented i n F i g . 4.10, F i g . 4.11 and F i g . 4.12. The values represented are averages for n = 3. Most standard deviations were i n the range of 10 to 15 % of the average values, however a few reached 20-25 %. The standard deviations were not presented on the graphs i n order to keep the data presentation less c l u t t e r e d . F i g . 4.13 shows the v a r i a b i l i t y ( standard deviation) that occurred i n the case of TTCP. I t i s obvious that the largest variations were obtained at the highest experimental temperature of 22 "C. A s i m i l a r pattern of v a r i a t i o n of the standard deviation occurred for the other chlorophenols. The size of leeches used i n these experiments was kept as uniform as possible. Table 4.5 shows the size d i s t r i b u t i o n used in the three experiments. TABLE 4.5 SIZE OF LEECHES USED IN TEMPERATURE EXPERIMENTS temperature 4 °C 12 °C 22 °C average size(g) 0.826 '0.669 0.845 S D (g) ±0.214 ±0.160 ±0.274 - 1 0 9 -30 \ 25 3 X X D D O - -O 2,4 DCP 2,4,6 TCP 2,4,5 TCP 2,3,4,6 TTCP PCP 4°C 20 CO L L ) X O LU 15 10 6 o 75 100 TIME (hrs.) Fig.4.10 Bioconcentration of chlorophenols by leeches i n a laboratory experiment at 4 °C. Concentration of the exposure solution was 10 ppb of each chlorophenol. The leech species used was N. obscura. - 110 -Fig.4.11 Bioconcentration of chlorophenols by leeches i n a laboratory experiment at 12 °C. Concentration of the exposure solution was 10 ppb of each chlorophenol. The leech species used was N. obscura. - I l l -+-0 25 50 75 100 125 150 175 TIME ( hrsJ Fig.4.12 Bioconcentration of chlorophenols by leeches i n a laboratory experiment at 22 °C. Concentration of the exposure solution was 10 ppb of each chlorophenol. The leech species used was N. obscura. - 112 -Fig.4.13 Bioconcentration of 2,3,4,6-TTCP by leeches at d i f f e r e n t temperatures. The data was compiled from the three laboratory experiments shown on Fig.4.10 to 4.12. The standard deviations are shown on t h i s graph. - 1 1 3 -The temperature related experiments were the only experiments car r i e d out using brown leeches(N. obscura). These leeches were used as substitute for the black leeches which were not available i n s u f f i c i e n t quantity at that time of the year( A p r i l 1985). Subsequent t e s t i n g proved that the difference i n species did not have a major influence on the bioconcentration c a p a b i l i t y ( see section 4.3.2.3); a uniform size was more important. Table 4.6 presents the bioconcentration factors obtained at various experimental temperatures. TABLE 4.6 EFFECT OF TEMPERATURE ON THE BIOCONCENTRATION OF CHLOROPHENOLS BY LEECHES(1) Compound Log Po/w Bioconc. factor Temperature 4°C 12 °C 22 °C 2,4-DCP 3 .08 282 424 980 2,4,5-TCP 3 .80 593 969 1948 2,4,6-TCP 4 .03 524 869 1988 TTCP 4 .45 614 1059 2501 PCP 5 .15 525 935 2508 At 4 °C( see F i g . 4.10) steady state adsorbtion of chlorophenols was reached r e l a t i v e l y f a s t , namely, a f t e r 4 days of exposure. With the exception of 2,4-DCP, the bioconcentration factors were a l l close to 500( Table 4.6). A lower bioconcentration factor for 2,4-DCP was expected considering that the log Po/w of t h i s compound i s 3.08 (Saarikovski and Vi l u k s e l a , 1982). - 114 -Previous equations developed for log BF i n r e l a t i o n with log Po/w gave a higher value for BF for increasing Po/w values( Saarikovski and V i l u k s e l a , 1982). However, t h i s r e l a t i o n s h i p was not followed i n our experiments. The bioconcentration factor was higher for 2,4,5-TCP than for 2,4,6-TCP( i f bioconcentration conforms to t h e i r log Po/w values the BF should be reversed) and the 2,3,4,6-TTCP bioconcentration factor was higher than the PCP bioconcentration factor( which again should be reversed). The graph at 4°C shows that the four CPs bioconcentrate p r a c t i c a l l y at the same l e v e l , regardless of t h e i r Po/w values. It seems that i n the case of leeches the values of Po/w have l i t t l e influence upon the bioconcentration factor. The experiment c a r r i e d at 12°C followed the same general pattern as the one at 4°C. Steady state was reached a f t e r 5 days and with the exception of 2,4-DCP, the bioconcentration factors for a l l the other chlorophenols were about 1000 ( Table 4.6). The same discussion applies for t h i s temperature and same discrepancies were noticed between the experimental values of bioconcentration factors and the values of bioconcentration factors calculated from equations developed by various researchers. As mentioned for the 4 °C experiment, the bioconcentration factors of the chlorophenols were grouped at the same l e v e l , regardless of the corresponding Po/w values( with the exception of 2,4-DCP). Bioconcentration factors for 2,4,5-TCP and 2,4,6-TCP , and for TTCP and PCP were again reversed when compared to t h e i r Log Po/w. Temperature was a very i n f l u e n t i a l parameter. At 12 °C, the bioconcentration factors were almost doubled as compared with the - 115 -4 °C bioconcentration factors. A higher temperature resulted i n higher BFs. At 22 °C(see Fig. 4.12) the bioconcentration factors were more than twice the values measured at 12°C( Table 4.6). Again, the bioconcentration factors did not follow the pattern established by equations r e l a t i n g BF to Po/w. However, the BF values were grouped i n r e l a t i o n to r e l a t i v e Po/w values. 2,4,5 and 2,4,6-TCPs exhibited almost the same values for BF, and TTCP and PCP, with the highest Po/w values, had s i m i l a r BF values. If the equation log BF = 0.85 log P6/w- 0.70, developed by Veith( 1974), using fathead minnows as the experimental organisms i s applied to our data, and the calculated BFs are compared to the experimental BFs , one can see that they do not follow the same pattern. The best f i t to Veith's equation however, i s obtained by the bioconcentration factors obtained at 22°C, which i s close to the experimental temperature ( 2 5°C) used by Veith( see Table 4.7). TABLE 4.7 EXPERIMENTAL AND CALCULATED BFs Compound Log Po/w Log BF BF Exp. BF ( 22 °C) 2,4-DCP 3 .08 1. 918 83 980 2,4,5-TCP 3 .80 2. 530 339 1948 2,4,6-TCP 4 .03 2. 725 531 1988 TTCP 4 .45 3. 080 1208 2501 PCP 5 .15 3. 680 4758 2508 If other equations were considered, they gave d i f f e r e n t absolute values for the bioconcentration factors, but i n a l l the - 1 1 6 -cases, the BF values increased with higher Po/w( Esser and Moser, 1980). We concluded from our data that for leeches, the bioconcentration factors did not follow the pattern developed by various researchers for other experimental organisms. To summarize, the higher experimental temperature( 22°C) had a few noticeable e f f e c t s on the bioconcentration factor: - the steady state took longer( at least 7 days) to be established when compared with 4 and 5 days for 4 °C and 12 °C respectively. - the bioconcentration factors at 22 °C were doubled i f compared with the bioconcentration factors obtained at 12 °C and quadrupled i f compared with BFs at 4°C. - a simple l i n e a r c o r r e l a t i o n between the logaritmic values of BF and Po/w as described by various equations was not followed by the experimentally determined BFs. Bioconcentration factors of various CPs d i f f e r e n t i a t e d only s l i g h t l y on the basis of t h e i r Po/w values. Fig.4.13 presents the TTCP concentrations found i n leeches at the three lev e l s of temperature, 4, 12, 22°C. The graph shows the influence that temperature had upon the bioconcentration factors. If BFs are plotted against temperature the e f f e c t of temperature upon bioconcentration becomes more obvious. Regression equations were calculated for the temperature-BF re l a t i o n s h i p and they could be used when experimental data have to be normalized at a c e r t a i n temperature. This i s e s p e c i a l l y useful when data from d i f f e r e n t laboratories are to be compared.The main l i m i t a t i o n to the application of the equations for i n t e r p r e t a t i o n of f i e l d experiments i s that they can be used - 1 1 7 -to normalize only equilibrium bioconcentrations. Regression equations were calculated for BF and log BF versus temperature( T). A very good c o r r e l a t i o n was obtained 2 between BF and T with R values between 0.9934 and 0.9648 but a better c o r r e l a t i o n was obtained for the log BF and T , with R values higher than 0.9910. The regression equations are l i s t e d as follows: -for 2,4-DCP: BF = 62 + 39.5 T LOG BF = 2.305 + 0.039 T -for 2,4,5-TCP: BF = 274 + 65.5 T LOG BF = 2.650 + 0.029 T -for 2,4,6-TCP: BF = 81 + 82.6 T LOG BF = 2.614 + 0.027 T -for TTCP: BF = 43 + 106.4 T LOG BF = 2.639 + 0.03 4 T -for PCP: BF = -97 + 112 T LOG BF = 2.549 + 0.038 T TTCP and PCP were bioaccumulated at the same le v e l by leeches. Knowing that at the same exposure concentration and under the same experimental conditions PCP i s more toxic to leeches than TTCP, one can a t t r i b u t e the lower LC50 of PCP to i t s i n t r i n s i c t o x i c i t y . This i s contrary to Kobayashi et a l . study( 1978) who attributed the higher t o x i c i t y of chlorinated phenols R = 0.9648 R 2 = 0.9910 R 2 = 0.9934 R 2 = 0.9990 R 2 = 0.9731 R 2 = 0.9990 R 2 = 0.9731 R 2 = 0.9980 R 2 = 0.9732 R 2 = 0.9960 - 118 -i n g o l d f i s h to t h e i r r e l a t i v e accumulation i n f i s h . The experiment presented i n F i g . 4.12 was carr i e d out under very s i m i l a r experimental conditions as the experiment presented in Fig.4.9( the length of both experiments was 7 days, experimental temperature was 22 C i n both, and the experimental exposure solution was made up of the same chlorophenols at 10 ppb each). The two experiments lead to sim i l a r r e s u l t s . In both cases the equilibrium was not quite reached a f t e r 7 days and the bioconcentration factors did not seem to depend too much on the Po/w. The main difference between the experimental conditions was that they used d i f f e r e n t species of leeches, namely the black leech(P. marmorata) and the brown leech(N.obscura). This difference might be enough to account for the differences between the experimental bioconcentration factors obtained i n the two experiments. The brown leech exhibited a somewhat higher bioconcentration c a p a b i l i t y than the black leech since the bioconcentration factors were 2 0 % higher for the brown leech(in the case of TTCP and PCP). The data i n the experiment that used the black leech could also be s l i g h t l y biased by the fact that they represent single experimental points. Therefore , the black leech experiment has a greater experimental error than the experiment using brown leeches where data are obtained by averaging 3 points. The influence of the leech species upon the bioconcentration factor was studied i n more d e t a i l i n separate experiments presented i n the following section. - 119 -4.3.2.3 Species Influence on the Bioconcentration Since previous experiments indicated that the two leech species bioconcentrated at s l i g h t l y d i f f e r e n t l e v e l s , a s p e c i f i c t e s t to determine speciation differences was performed. The experiment was ca r r i e d out at 4°C. The exposure solution was made of the same CPs used throughout a l l the experimental work, at a concentration of 10 ppb each. A t o t a l of 9 leeches of each species(P. marmorata and N.obscura) were subjected to the exposure solution for 6 days. Three leeches of each species were analysed for chlorophenols a f t e r 24, 96 and 168 hours. At the end of the six days(168 hours) the concentration of TTCP found i n the black leech was 3.52 ug/g wet weight(average of 3 leeches) and 4.92 ug/g wet weight i n the brown leeches(average of 3 leeches).In the case of PCP, the concentration i n the black leeches was 3.16 ug/g wet weight (average of 3 leeches) and 4.3 3 u<3/g i n the brown leeches (average of 3 leeches). The r e s u l t s showed once again that the brown leech had a higher bioconcentration c a p a b i l i t y than the black leech. According to the experimental data, bioconcentration was 40% higher i n the case of TTCP and 37% higher i n the case of PCP. Since at 22°C the difference i n bioconcentration between the two species was only 2 0% i n the favor of the brown leech, one can conclude that the higher temperature made the absorptive performance of the two leech species more uniform. The size of leeches used i n the two experiments were quite d i f f e r e n t and t h i s could have contributed to the v a r i a t i o n i n the bioconcentration factors; the bioconcentration c a p a b i l i t y of the brown leech could - 1 2 0 -have been even higher than the reported value i f they had similar weight/ surface area r a t i o to the black leech. Average weight for the black leech was 0.239 g and for the brown leech was 0.683 g( the black leech i s usually larger than the brown leech , but at the time of that experiment the black leeches were very small). 4 . 3 . 2 . 4 Weight of Leech Specimens and Its Influence on the  Bioconcentration The s i z e of leech was o r i g i n a l l y not selected as a variable parameter i n any of the experiments. Only observations made during experimental work used to study other variables led to a few conclusions on the e f f e c t s of the size of the leeches, on bioconcentration. Theor e t i c a l l y , bioconcentration i s a surface process achieved by r e s p i r a t i o n . The organisms pass water containing contaminants through the e p i t h e l i a l tissue and r e t a i n and bioconcentrate those contaminants i n t h e i r body. If they have a l i p o p h i l i c structure, the contaminants w i l l be p r e f e r e n t i a l l y deposited i n the f a t tissues of the biota. The concentration of chlorophenol i n leeches was expressed as ug/g wet weight and not g/unit of surface area. That means that smaller the leech, the higher the r a t i o of surface area to weight. Therefore, a small leech i s expected to exhibit a higher bioconcentration factor. Also, as bioconcentration was correlated to organism bioenergetics(Norstrom et a l . , 1976 ) , a higher bioconcentration factor was expected from the younger organisms since they have a higher metabolic rate than the older, larger, - 121 -ones. These considerations made i t necessary to t r y and keep the size range of leeches as narrow as possible. In selecting leeches, a maximum standard deviation of 40% was set as the desired c r i t e r i o n . In some cases, due to the limited supply of leeches, i t was necessary to use leeches from a wider weight range. A few observations were made i n respect with the bioconcentration calculated for various size ranges used i n the same experiment: -when a very small weight was used along with larger weights, the small leeches had occasionally very high concentrations of chlorophenol. A viable explanation for t h i s could be based either on the higher surface area per unit weight for smaller leeches or on extraction and preparation techniques: a very minor error or contamination could have been a r t i f i c i a l l y magnified when the amount found was calculated on the basis of a smaller weight. the extracts of control(blank) leeches of larger specimens were more complex( the chromatograms exhibited more peaks) than the extracts of control leech of smaller specimens. This observation made us use large control leeches for large experimental leeches and small control leeches for small experimental leeches. - 122 -4.3.2.5 Summary of Results Experimental temperature was the most important parameter when determining bioconcentration factors. No bioconcentration factor should be considered unless the experimental temperature at which i t was determined i s given. Temperature affected the length of time required to reach the steady state and the magnitude of the bioconcentration factor. Steady state was reached i n 4 days at 4°C, i n 5 days at 12°C and i n 7+ days at 22°C. TTCP and PCP had bioconcentration factors of approximately 500 at 4 °C, of about 1000 at 12°C and of about 2500 at 22°C. Regression equations were established and 2 the R values obtained were a l l higher than 0.96 which showed a good f i t of the equations to the data. The equations could be used to normalize bioconcentration factors to a preset temperature.Their use i s limited to equilibrium bioconcentrations and to a concentration of the exposure solution i n the range of 10 ppb. I t has been previously shown ( see section 4.3.1.1) that bioconcentration c a p a b i l i t y i s influenced by the exposure solution concentration. The assumption that steady state condition was reached i n the 4 and 12°C experiments was based on the fact that the CP concentrations found i n leeches were not s i g n i f i c a n t l y d i f f e r e n t a f t e r 4 and 5 days respectively. However, a s o l i d confirmation of the steady state condition can be only given by a longer exposure experiment, of 30 to 45 days. Log Po/w values of the chlorophenols used i n the exposure experiments were not well correlated to the le v e l of bioconcentration i n leeches. The general equation: Log BF = a Log Po/w + b , was not followed i n - 1 2 3 -the case of leeches exposed to chlorophenol. Higher experimental temperatures seemed to enhance the influence of the Po/w upon the bioconcentration factor . The two species used i n the experiments exhibited d i f f e r e n t bioconcentration factors which were approximately 40% higher, at 4 C, for the brown leech than for the black leech. Higher experimental temperature( 22 C) seemed to lower the difference i n bioconcentration between the two species. The brown leech exhibited a bioconcentration factor only 20 % higher than the black leech when the experimental temperature was 22 C. Size of leech should be seriously considered for the se l e c t i o n of specimens for various experiments. A narrow size range should be kept for more reproducible r e s u l t s . Empirically, i t was decided to work with a maximum of 4 0% standard v a r i a t i o n and to keep the leech weight i n the same order of magnitude. 4.4 F i e l d Experiments F i e l d experiments were conducted i n the North Arm of the Fraser River to determine the lev e l s of chlorophenol p o l l u t i o n . Water samples were c o l l e c t e d and analyzed for chlorophenols and exposure t r i a l s were made with leeches to evaluate t h e i r potential as bioassay organisms for chlorophenol bioconcentration. Water sampling was ca r r i e d out independently, to evaluate the temporal and s p a t i a l d i s t r i b u t i o n of chlorophenols i n the r i v e r , and i n synchrony with leech bioassays to v a l i d a t e the a b i l i t y of the leeches to integrate chlorophenol p o l l u t i o n over time. 4.4.1 Water Sampling Water sampling was performed i n two ways: grab sampling and - 124 -automatic sampling. The sampling s i t e s are located i n F i g . 3.1. They were numbered from 1 to 7 and are i d e n t i f i e d by geographical landmarks( Table 4.8). 4.4.1.1. Grab Sampling The main objective of the grab sampling was to investigate the general d i s t r i b u t i o n of chlorophenol p o l l u t i o n i n the area of study and help select an area for the detailed sampling and leech studies. Table 4.8 presents the data from the grab sampling t r i p s . The only two chlorophenols detected i n the grab samples were TTCP and PCP. This i s consistent with the main source of chlorophenol p o l l u t i o n to the North Arm of the Fraser River which i s the forest industry. Other reported sources are the sewage treatment plants but our sampling s i t e s were not i n the v i c i n i t y of sewage o u t f a l l s . The grab samples, which were taken either from the shore or from a boat, represented reasonable well-mixed water samples. The values ranged from nd( not detected; detection l i m i t f or TTCP and PCP i s 0.002 ppb) to high values recorded at sampling s i t e 5, Mi t c h e l l Island location, namely, 11 ppb TTCP and 2.25 ppb PCP. On the basis of these high values and the known busy and active forest industry i n t h i s area, the M i t c h e l l Island area was chosen for a permanent s i t e for the detailed sampling and leech exposure program. The i n t e r p r e t a t i o n of the grab sampling data lead to the following conclusions: - A low background l e v e l of 2,3,4,6-TTCP,at concentrations ( - 1 2 5 -TABLE 4.8 GRAB SAMPLING RESULTS (1) Sampling Site No. Location Date (1984) 2,3,4,6-TTCP (ppb) PCP (ppb) 1 Scott Paper Sept. Oct. Oct. 11 4 11 nd 0.480 nd nd nd nd 2 Kerr St. June June Sept. Oct. Oct. 28 28 11 4 11 0.470 0.580 nd nd nd 0.040 0.003 nd 0.120 nd 3 Fraser St. Aug. 1 0.060 0.010 4 Oak St. Aug. 1 0.440 0.110 5 Mitchell Is. Sept. Oct. Oct. 10 4 11 0.614 11.00 0.180 nd 2.25 nd 6 Arthur Laing Bridge Oct. Oct. 4 11 1.560 nd 0.140 nd 7 Wood Is. Sept. 10 0.132 nd See Fig.3.1 for location of stations. nd = not detected. Detection l i m i t for TTCP and PCP i s 0.002 ppb. Detection l i m i t for the other chlorophenols i s 0.010 ppb but they were not observed at t h i s l e v e l . - 1 2 6 -between 0.1 and 0.4 ppb was almost always present i n the study area. PCP was present at a lower concentration level( 0.03 to 0.1 ppb). - Stations i n the upper reaches of the North Arm, such as Scott Paper and Mac M i l l a n Bloedel, exhibited lower levels of CPs( more values close to the detection l i m i t and blank values). This could be due to a series of factors such as: cleaner or ceased operations, a lower frequency of grab samples which missed any sporadic discharges that occurred, and the fact that water from the lower stations could be subjected to multiple dosing a t t r i b u t a b l e to a greater t i d a l influence i n t h i s area( Joy, 1975). - Higher chlorophenol levels were found but they were sporadic. The grab sampling technique could have missed other high l e v e l s of CPs and the need for a more integrated sampling technique became obvious. The high l e v e l of TTCP found at sta t i o n 5 provided a guideline for s e l e c t i o n of the concentration to use i n the laboratory experiments: 10 ppb concentration was used to make up the exposure solutions. This l e v e l of chlorophenol was not acutely toxic to leeches and since i t was the highest l e v e l that could be found i n the r i v e r , t h i s l e v e l led us to believe that the leeches would survive the f i e l d exposures. During the subsequent automatic sampling, concentrations of TTCP at the same l e v e l were recorded. - 1 2 7 -4.4.1.2 Automatic Sampling The automatic sampling used a 2 hour sampling frequency. Depending on the t o t a l length of the sampling period the water samples were analysed as discrete or composite samples. In January 1985, the automatic sampler was placed on the loading dock of the West Coast C e l l u f i b r e Industries Ltd.( near station # 5 ) . A l l the automatic sampling was done at t h i s location. One has to bear i n mind that the data regarding the chlorophenol lev e l s at t h i s location are representative for that p a r t i c u l a r location and not for the l e v e l of chlorophenol i n the Fraser River Estuary. This p a r t i c u l a r sampling station was selected with the expectation that the water would contain some high concentrations of chlorophenol. The presence of contaminant in water was needed i n order to be able to demonstrate the ca p a b i l i t y of leeches to bioconcentrate i t . The presence of chlorophenol i n that location was probably due to either surface runoff containing the contaminant, improper disposal of the rinse waters from the clean-up operations, or to leaching from the treated lumber. The data of a two-day sampling experiment are represented in Fig.4.14. A l l the graphed points are a n a l y t i c a l r e s u l t s for discrete samples taken every two hours. This two-day experiment demonstrated how i n e f f e c t i v e single grab samples were i n documenting chlorophenol contamination. Two peaks of high concentrations of TTCP and PCP occurred over a period of 10 hours and, as the graph shows, a grab sampling method could have missed them both i f sampling was done at the wrong time. The m i l l - 128 -TIME (HRS) Fig.4.14 Chlorophenol concentration at M i t c h e l l Island over a two-day period. Average concentrations were 1.250 ppb TTCP and 0.167 ppb PCP. Water temperature was 3-4°C. Daily average water flow for the sampling period was 1330 to 1410 m3/s( Water Survey of Canada, 1985 data). - 1 2 9 -adjacent to the s i t e of sampling, was i n operation 24 h/day, 5 days a week and the high plumes of chlorophenol could have been due to a s p i l l or midnight clean-up operation. If the t i d a l e f f e c t was considered, the two peaks measured at that p a r t i c u l a r location could have been the r e s u l t of just one discharge of contaminant which took place at some other location and which appeared as two separate peaks due to the ebb and flood of the t i d e . The Fraser River water temperature and the average d a i l y flow range for the sampling periods are given on a l l the figures pertinent to water sampling. The r i v e r flow i s measured at the Port Mann pumping sta t i o n since no recording i s done at the North Arm( Water Survey of Canada, 1985 data). The North Arm flow i s estimated! personal communication) at 10 -14 % of the flow at Port Mann. The r i v e r flows are to be used as r e l a t i v e values to compare the flows i n the study area at various times of the year. In January, the Fraser River flow was at i t s lowest point and d i l u t i o n was minimal. Any spike of contaminant was present i n the r i v e r at i t s maximum concentration. Low r i v e r flows were also recorded i n February and March. This i s the most c r i t i c a l time for the Fraser River to receive any toxic discharge i n terms of d i l u t i o n capacity. Plumes of contaminants are also easier to detect by sampling the r i v e r at short i n t e r v a l s . At periods of high flow, the contaminant spikes are di l u t e d more rapidl y and t h e i r presence i s detected more as a plateau determined by the average concentration. During periods of high flow, the t u r b i d i t y of water increases and t h i s can a f f e c t the a v a i l a b i l i t y of the contaminants to the biota as the suspended p a r t i c l e s compete for - 1 3 0 -the contaminants. Also, recovery of those contaminants from the water matrix i s more d i f f i c u l t due to the high load of suspended sol i d s and special recovery tests must accompany the sample analysis. T i d a l height was represented on a l l the graphs( Canadian Tide and Current Tables, 1985). Again, only the r e l a t i v e values of the t i d e height must be considered since the t i d a l height that i s represented i s taken at Point Atkinson. The reason for including the t i d a l height i n the graphical representation of the water sampling r e s u l t s was to show the estuarine c h a r a c t e r i s t i c s of the study area. The t i d a l cycle influences the d i l u t i o n factor and the d i r e c t i o n of movement of the contaminant plume. The r e s u l t s were presented d i f f e r e n t l y when longer sampling periods were involved. Since composite samples were used i n order to keep the number of analyses reasonable and to maintain the same sampling frequency( every two hours),the r e s u l t s were represented as histograms covering a s ix hour period. To f a c i l i t a t e compound i d e n t i f i c a t i o n on the graph, PCP histograms were represented as triang u l a r instead of rectangular areas. The composite samples were obtained by manually mixing three consecutive samples taken at two hour i n t e r v a l s . The concentration of the composite samples represented the average chlorophenol concentration for the six hours. High concentrations were measured, but t h e i r l e v e l s were lower than found during the two day experiment since high concentrations were dil u t e d by mixing with more d i l u t e samples co l l e c t e d during t h i s period. The automatic sampling experiment car r i e d out between March 31 and A p r i l 6, 1985( see F i g . 4.15), gave an average - 1 3 1 -Fig.4.15 Chlorophenol concentrations at M i t c h e l l Island over a six-day period. Average concentrations were 1.058 ppb TTCP and 0.175 ppb PCP. Water temperature was 4°C. Between A p r i l 1 and A p r i l 6, 1985, the d a i l y average water flow was 1790 to 2020 m3/s( Water Survey of Canada, 1985 data). - 1 3 2 -concentration of chlorophenol over the entire sampling period of 1.058 ppb TTCP and 0.175 ppb PCP. Concentrations as high as 3.678 ppb TTCP were reported. This high value for a composite sample could mask an even higher peak which was averaged by mixing with two other less contaminated samples. What was c h a r a c t e r i s t i c of t h i s experimental period was the repeated occurrence of high lev e l s of chlorophenol between the same hours of each day: 11 p.m. and 5 a.m. on A p r i l 3, 4, and 5.This could not be related to the t i d a l o s c i l l a t i o n since s i m i l a r t i d a l v a r i a t i o n was experienced during the daylight period. The regular frequency of the high lev e l s of chlorophenol could more r e a l i s t i c a l l y be related to a midnight clean up operation by the lumber treating industries adjacent to the sampling s i t e . Again, because of the current reversal over the t i d a l period i t was quite impossible to pinpoint the exact location where the discharge took place. The purpose of the water sampling was not to i d e n t i f y the g u i l t y party, but to est a b l i s h the l e v e l and pattern of v a r i a t i o n of chlorophenol contamination in water. The comparison of the average d a i l y flows at the beginning of A p r i l with the average d a i l y flows during the January experiment showed that a greater d i l u t i o n occurred i n A p r i l . This could be one of the possible explanations for the lower average concentration of chlorophenol i n early A p r i l than i n January( 1.058 ppb of TTCP i n A p r i l as compared with 1.250 ppm of TTCP i n January). The seven day experiment between A p r i l 18 and A p r i l 25, 1985( see Fig.4.16) showed an even lower average concentration of - 1 3 3 -TIME ( H R S . ) Fig.4.16 Chlorophenol concentrations at M i t c h e l l Island over a seven-day period. Average concentrations were 0.430ppb TTCP and 0.080 ppb PCP. Water temperature was 5-7°C. Between A p r i l 18 and 24, 1985, the d a i l y average water flow was 3490 to 3840 m3/s( Water Survey of Canada, 1985 data). - 1 3 ^ -chlorophenol i n water: 0.430 ppb for TTCP and 0.080 ppb for PCP. This could be again due to the higher d i l u t i o n capacity of the r i v e r at that time of the year. Cleaner operations and less lumber processed i n t h i s time could also be a viable explanation for the decreased l e v e l of pollutant. Due to the high content of suspended so l i d s i n the r i v e r water at the time of increasing flow, recovery values had to be applied to the a n a l y t i c a l r e s u l t s ( 52% recovery for TTCP and 44 % recovery for PCP). The l a s t of the series of automatic water sampling experiments was ca r r i e d out i n September 1985( see Fig.4.17). The average concentration of chlorophenol at t h i s time of the year was found to be 1.680 ppb for TTCP and 0.428 ppb for PCP. The r i v e r flow was decreasing i n early f a l l but the change i n flow was not as dramatic as occurred during the spring. Average d a i l y flows for the duration of the September experiment were a great deal lower than during the l a s t A p r i l experiment. A general c h a r a c t e r i s t i c of the chlorophenol l e v e l i n the September experiment was a more uniform concentration over the sampling period. Two higher values were found, but i n general the levels of chlorophenol exhibited l i t t l e v a r i a t i o n from the mean concentration. 4.4.1.3 Summary of Results The automatic sampling experiments showed how i n e f f e c t i v e grab samples could be and how e a s i l y high spikes of contaminants can be missed i f the sampling time did not coincide with t h e i r occurrence. The CPs lev e l s showed a large v a r i a t i o n with time. Peaks as high as 14 ppb TTCP were reduced to the background le v e l - 1 3 5 -T I M E ( H R S ) Fig.4.17 Chlorophenol concentrations at M i t c h e l l Island over a six-day period. Average concentrations were 1.680 ppb TTCP and 0.42 8 ppb PCP. Water temperature was 14°C. Between September 12 and 18, 1985, the d a i l y average water flow was 1760 to 2740 m3/s( Water Survey of Canada, 1985 data). - 1 3 6 -i n only two hours. The r i v e r flow seemed to play an important role for the contaminant leve l s found i n the water at various times of the year. The water q u a l i t y such as r e f l e c t e d by the suspended s o l i d s content affected chlorophenol recovery from the water matrix. At times of high loads of suspended s o l i d s , the recovery values had to be included i n c a l c u l a t i o n s . High levels of chlorophenol, found during the automatic water sampling experiments, were very comparable to high levels reported by previous studies conducted i n areas impacted by the forest industry operations( EPS, 1979). The background levels for the area of study estimated at 0.1 ppb to 0.4 ppb for TTCP and 0.03 ppb to 0.1 ppb for PCP were also confirmed by Carey (unpublished data) who sampled the same area i n 1984. 4.4.2 F i e l d Leech Experiments Table 4.3 contains the r e s u l t s of the f i r s t leech exposure experiment i n which leeches were exposed for seven days to the chlorophenol levels i n the Fraser River water. The experiment used 5 leeches at each of the two locations. After 7 days of exposure, three leeches were analysed for chlorophenol and two underwent depuration. The leeches exposed at the Mi t c h e l l Island location exhibited a high chlorophenol concentration( 2.946 ug/g TTCP and 0.846 ug/g PCP). This indicated that higher levels of chlorophenol should be encountered i n the water co l l e c t e d at that location.This was confirmed by the high concentration of chlorophenol( 11 ppb TTCP and 2.2 2 ppb PCP) detected at that - 1 3 7 -location on October 4. Considering that no chlorophenols were reported i n the grab samples c o l l e c t e d on October 4 and 11 at the Kerr station, the le v e l of chlorophenol found i n leeches aft e r 7 days exposure at that s i t e , indicated that chlorophenols were present i n water at some periods during t h i s 7 days exposure. A l l the other leech exposure experiments were carried out at one location:the loading dock of the West Coast C e l l u f i b r e Ind., where the automatic sampler was located. Two of the leech exposure experiments were accompanied by water sampling. For these experiments, the pattern of chlorophenol v a r i a t i o n was available and the average concentration of chlorophenol during the exposure could be calculated( A p r i l 18-25, 1985, and September 12-18, 1985). The leeches exposed for 7 days, between A p r i l 18 and 25, 1985, contained a chlorophenol concentration of 124 ppb for TTCP and 55 ppb for PCP. The average chlorophenol concentration i n the water was 0.430 ppb TTCP and 0.080 ppb PCP(see footnotes Fig.4.16). The leeches exposed for 6 days between September 12 and 18, 1985, contained a chlorophenol concentration of 273 ppb TTCP and 196 ppb PCP. The average chlorophenol concentration i n the water was 1.680 ppb TTCP and 0.428 ppb PCP(see footnotes F i g 4.17) . Comparing the chlorophenol levels found i n the leeches at the end of the two exposure experiments i t i s clear that leeches exposed to a higher average chlorophenol concentration at a higher temperature accumulated more contaminant i n t h e i r t i s s u e s . The f a c t that one experiment was car r i e d out for 6 days and the other for 7 days should not influence the f i n a l bioconcentration - 1 3 8 -l e v e l very much since i t was shown before that a steady state condition i s reached i n 5 days at 12°C temperature and 4 days at 4°C. During the two leech experiments presented above, the water temperature was i n the neighbourhood of these values(see footnotes of F i g . 4.16 and 4.17). The chlorophenol concentration during the experiment from September 1985 was quite constant and i t could be possible that a steady state was reached. For the experiment from A p r i l 1985 the steady state was not achieved because of the high v a r i a t i o n of the chlorophenol concentration. There i s considerable v a r i a t i o n i n chlorophenol lev e l s i n the Fraser River Estuary, t h u s t h e leeches are subjected to a wide dynamic range that they must integrate over an exposure period. Only estimates of the average chlorophenol concentration i n water can be made on the basis of the leech chlorophenol content at the end of the exposure experiments. Metcalfe et al.(1984) reported chlorophenol levels i n leeches at 40,000 to 140,000 times the average chlorophenol concentration i n water. This does not mean that the bioconcentration factor of those leeches i s i n t h i s high range of values. The leeches sampled must have been exposed to a high concentration of chlorophenol for some time p r i o r to t h e i r c o l l e c t i o n . Since the depuration rate of chlorophenol from the leech matrix i s a very slow process, the leeches s t i l l contained the accumulated contaminants i n t h e i r t i s s u e s . They exhibited an a r t i f i c i a l l y high bioconcentration factor because of the low chlorophenol concentration i n water at the time of sampling. Two more leech f i e l d experiments were carr i e d out between May - 1 3 9 -31- June 7, 1985, and September 27- October 3, 1985. They were not accompanied by automatic water sampling and the interp r e t a t i o n of the CPs level s i n the water from levels i n leeches was done s o l e l y by using the laboratory data. The leeches exposed between May 31-June 7, 1985, had TTCP at 235 ppb and PCP at 57 ppb . The leeches exposed between September 27-October 3,1985 had TTCP at 28 ppb and PCP at 29 ppb. Table 4.91 contains a summarized presentation of the f i e l d leech experiments carried out at the West Coast C e l l u f i b r e location . Pertinent information, such as size of leeches, water temperature and concentration of CPs i n the water ( i f available) are l i s t e d for easy reference. Interpretation of the leech f i e l d r e s u l t s w i l l be done i n section 4.6. 4.5 Specially Designed Laboratory Exposure Experiments The bioconcentration c a p a b i l i t y of leeches and the slow depuration rate of contaminants from t h e i r tissues make the use of leeches an a t t r a t i v e proposition for monitoring purposes. They cannot be used as t i t r a n t s of the chemicals present i n the water column since i t i s v i r t u a l l y impossible to account for a l l the variables i n a natural system. However they seem to be r e l i a b l e indicators of past and present p o l l u t i o n and estimates of the p o l l u t i o n l e v e l can be made on the basis of t h e i r pollutant content. Implanted for various lengths of time i n the region of study , they w i l l integrate the concentration of contaminant and should TABLE 4.9 FIELD LEECH EXPERIMENTS Exp. Duration Date (1985) Water Temp Leech Weight CPs in Leech CPs in Water Observations No. TTCP PCP TTCP PCP 1 7 days April 18-25 5-7 C 0 .4 g 124 ppb 55 ppb 0.430 ppb 0.080 ppb Low recovery values 2 7 days May 31-June 7 8 C 0 .6--1. 0 g 235 ppb 57 ppb 3 6 days Sept 12-18 14 C 1 .2--1. 3 g 273 ppb 196 ppb 1.680 ppb 0.428 ppb 4 6 days Sept 27-Oct 3 12 C 1 .0--1. 9 g 28 ppb 29 ppb Values close to the leech blank - 141 -signal unusual high levels of contaminant by unusual high contaminant concentration i n t h e i r tissues. For a proper i n t e r p r e t a t i o n of the f i e l d r e s u l t s one has to consider the major parameters that influence bioconcentration. It has been shown that some important parameters that regulate bioconcentration are temperature, suspended s o l i d s , hardness, pH of water and leech s i z e . These parameters can be r e l a t i v e l y e a s i l y monitored and t h e i r influence upon v a r i a b i l i t y of the res u l t s reduced to the minimum. River pH varies l i t t l e during the year and can be e a s i l y matched i n a laboratory experiment. Size of leeches can be controlled by c a r e f u l l y s e l e c t i n g the specimens. Temperature of the r i v e r water does change during the year but i t stays r e l a t i v e l y constant for short periods such as 6-7 days, and again can be reproduced i n a. laboratory experiment. Temperature, pH, a l k a l i n i t y and hardness are water c h a r a c t e r i s t i c s easy to reproduce. Other c h a r a c t e r i s t i c s such as suspended s o l i d s are harder to match because not only the load of suspended s o l i d s influences the bioconcentration process (Muir et a l . , 1980) but the composition of those s o l i d s as well ( i . e . a higher organic load w i l l have more impact on the a v a i l a b i l i t y of chlorophenol to the aquatic b i o t a ) . A laboratory controlled experiment i n which a l l these parameters are matched with the r i v e r water c h a r a c t e r i s t i c s has to be run i f an accurate estimation of the contaminant l e v e l i n the water i s to be made on the basis of the contaminant l e v e l found i n the leeches. For a f i e l d t e s t to be successful one of i t s main c h a r a c t e r i s t i c s i s for i t to be simple. If a laboratory controlled experiment i s to be run concomitant with the leech - 142 -f i e l d t e s t , i t has to be kept uncomplicated as well. Special laboratory experiments were designed with a l l the above considerations i n mind and t h e i r main purpose-was to help i n estimating the f i e l d r e s u l t s . For p r a c t i c a l reasons, the concentration of the exposure solution must be chosen somewhere i n the range considered to be safe for a natural environment such as the Fraser River Estuary. At present, there are no such regulatory l i m i t s for chlorophenol. Fox( 1980) suggested 0.4 ppb PCP as the safe concentration for the protection of aquatic l i f e . Concentrations i n the range of 1 ppb have been chosen for the laboratory experiments. On the basis of the r e s u l t s obtained i n these experiments, the l e v e l of CPs found i n leeches was used to estimate the average chlorophenol concentration i n water during the exposure. 4.6 Interpretation of the F i e l d Results on the Basis of the  Laboratory Experiments Laboratory experiments were set up to reproduce at best the f i e l d conditions: water c h a r a c t e r i s t i c s ( temperature, pH, hardness, a l k a l i n i t y ) and exposure time. Since no regulatory values are imposed for CPs, a r b i t r a r y concentrations of 0.5 ppb, 1 ppb, and 2 ppb were used. The r e s u l t s of the s p e c i a l l y designed laboratory experiments are shown i n Table 4.10. They represent averages of three leeches . Only the levels of TTCP and PCP are presented since these are the only two CPs found i n the r i v e r . Experiments 1 and 2 were TABLE 4.10 SPECIAL LABORATORY CONTROLLED LEECH EXPERIMENTS Experiment No. Duration Water Temperature Exposure Solution CPs CPs in Leech Leech Weight Concentration3 TTCP PCP 7 days 7 days 4°C 4"C 6 days 14 C 6 days 14 C 0.5 ppb 1.0 ppb 1.0 ppb 2.0 ppb 84 ppb 27 ppb 1.7-2.8 g 231 ppb 157 ppb 1.0-2.7 g 121 ppb 191 ppb 2.2-2.8 g 238 ppb 383 ppb 2.4-2.9 g The exposure solution was made of five chlorophenols (2,4-DCP, 2,4,6-TCP, 2,4,5-TCP, 2,3,4,6-TTCP, PCP), each at the indicated concentration. Dilution water was made after EPA formulation for medium hard water ( see section 3.3.2). Only the concentration of TTCP and PCP are given because only they are to be used to estimate the average concentration of CPs in water during the leech field experiments; only TTCP and PCP were found in the Fraser River water. - 144 -c a r r i e d out at 4 C to reproduce the best winter conditions and experiments 3 and 4 were carr i e d out at 14 C to reproduce the summer and early f a l l conditions of the Fraser River water. The higher CPs concentration exhibited by leeches i n experiment 2 compared with experiment 1 was expected since the CPs concentration i n the exposure solution was twicw as high. However, i f one compared r e s u l t s of experiment 2 and 3, both carr i e d out at the same CPs concentration of 1 ppb, one could see that the r e s u l t s of experiment 2 were unusually high. The two experiments d i f f e r e d by the experimental temperature, and i t was expected that the higher temperature would r e s u l t i n a higher l e v e l of CPs i n the leech tissue( see section 4.3.2.1). Experiment 4, ca r r i e d out at the same temperature as experiment 3, but at a twice concentration i n the exposure solution, showed higher bioconcentration l e v e l s . They r e f l e c t e d the increased dosage of CPs. No viable explanation was found for the high r e s u l t s obtained i n experiment 2 and a r e p l i c a t e experiment was not ca r r i e d out to confirm or r e j e c t the values. Its data were not used i n the in t e r p r e t a t i o n of the f i e l d r e s u l t s . The f a c t that the experiments were carr i e d out for various lengths of time(6 or 7 days) should not influence the f i n a l r e s u l t s . I t was shown before that the steady state condition was established i n 4 or 5 days for the experimental temperatures used( see section 4.3.2.1) . This means that the bioconcentration factor should not change a f t e r 5 days of exposure. Since TTCP was the main chlorophenol found i n the Fraser River water the estimation was done only for t h i s compound.The - 1 4 5 -excercise consisted i n comparing the CPs levels i n the f i e l d leeches with the CPs levels i n the leeches exposed under laboratory conditions to known concentrations of chlorophenol solutions. The f i e l d conditions were matched by the laboratory conditions and the chlorophenol concentration was considered to be the only var i a b l e . Matching of f i e l d and laboratory conditions was not perfect as f i e l d leeches were exposed to dynamic changes of chlorophenol levels and other water parameters( suspended s o l i d content, s a l i n i t y , etc.) over the bioassay period. Temperatures were not normalized to an i d e n t i c a l value since equilibrium conditions were not considered to be reached under f i e l d conditions. Only the general trend of v a r i a t i o n of bioconcentration with temperature was considered when making the estimate. F i e l d experiment 2( see Table 4.8 for a l l the f i e l d experiments c a r r i e d out at West Coast C e l l u f i b r e location) showed CPs levels i n leeches of 235 ppb for TTCP and 57 ppb for PCP. The lev e l of TTCP was si m i l a r to the le v e l reached i n laboratory experiment 4. The CPs concentration used i n that experiment was 2 ppb. The temperature at which these f i e l d and laboratory experiments were done d i f f e r e d from each other. With the knowledge that at higher temperatures the bioconcentration i s higher, an average value of 2 ppb TTCP i n water during f i e l d experiment 2 was very conservative estimate. F i e l d experiment 4 showed very low level s of CPs i n leeches. A resonable estimation would conclude that no measurable levels of CPs were present at t h i s location during that i n t e r v a l of time, since the levels found were very close to the control - 146 -leech CPs l e v e l s . For the leech experiment c a r r i e d out i n October 1984 at the Kerr St. location! see Table 4.3) when TTCP was found at 476 ppb and PCP at 226 ppb, one can estimate the concentration of TTCP i n water as being higher than 2 ppb for the entire period of exposure(field water temperature was si m i l a r with the laboratory-experiment 4 temperature). It was shown by the grab sample taken at the beginning and end of the experimental period that TTCP was below the detection l e v e l . This means that high concentrations of TTCP occurred at various times during the exposure period and they brought the average TTCP concentration at over 2 ppb for the entire length of the exposure. The leech experiment car r i e d at the same time at the M i t c h e l l Island location! see Table 4.3) showed levels of CPs i n leeches even higher: 2946 ppb for TTCP and 846 ppb for PCP. The average TTCP concentration was d e f i n i t e l y higher than 2 ppb. The grab sample taken at the beginning of the experiment indicated 11 ppb TTCP and 2.25 ppb PCP. The leeches exposed at t h i s location exhibited the same le v e l of TTCP concentration with leeches exposed for 3 5 hours to a solution of 10 ppb TTCP(comparison was made with the temperature controlled experiment car r i e d out at 12 °C, a temperature s i m i l a r to the water temperature during the f i e l d exposure). This made us estimate that a high concentration of TTCP was present at that p a r t i c u l a r location for an extended period of time or that the frequency of high levels of TTCP i n water was very high. Another p o s s i b i l i t y i s that a very l o c a l i z e d s p i l l at even higher concentration of TTCP occurred at that location for a shorter time. A very high concentration for a - 1 4 7 -short time would have led to the same bioconcentration l e v e l as lower concentration for a longer time. F i e l d leech experiments 1 and 3 were accompanied by automatic water sampling. For these experiments the average CPs concentration i n water was known. If one t r i e s to estimate the concentration of TTCP during f i e l d leech experiment 1 using the laboratory experiments, a concentration higher than 0.5 ppb, lower than 2 ppb and about 1 ppb would be estimated. Since the measured average TTCP concentration was 0.43 ppb th i s made the estimate approximately twice as high as the re a l value. If the r e s u l t s of f i e l d experiment 3 were used to estimate the average concentration of TTCP on the basis of the laboratory experiments, a concentration of 2 ppb TTCP was estimated for the r i v e r water at that loca t i o n . The measured value was 1.68 ppb TTCP and that made the estimated value within + 15% of the real value. Considering that those estimates are made using a l i v i n g organism as the integrator of the contaminant l e v e l , the acquired accuracy i s well within expected values. Experiments using biota are considered successful i f the order of magnitude can be accurately predicted. - 148 -5. CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions As shown by the grab sampling program r e s u l t s and by the extreme variati o n s of chlorophenols levels during the high frequency automatic sampling periods, the actual system of monitoring p o l l u t i o n levels i s t o t a l l y inadequate. The use of periodic grab samples to monitor pollutant levels i s not representative for the pollutant presence i n the area under survey. Since a high frequency sampling program would run into enormous costs, the use of a bioind i c a t o r organism i s the only viable proposition. Leeches proved to be a very suitable organism for monitoring of chlorophenol p o l l u t i o n and only more research can prove i f they can be used to estimate the time averaged concentration of other contaminants. Preliminary laboratory controlled leech experiments indicated that the leeches have great bioconcentration c a p a b i l i t i e s for chlorophenols and set the main parameters for further laboratory controlled experiments. During f i e l d experiments, only two chlorophenols were detected i n the water samples: 2,3,4,6-TTCP and PCP. The average concentration for TTCP determined during the various sampling periods, c a r r i e d out between January 1985 and September 1985 at the automatic sampling location, was always around the 1 ppb l e v e l . PCP concentrations were about 4 to 5 times lower. This indicated that the main source of chlorophenol p o l l u t i o n i n the - 149 -study area was the forest industry( West Coast C e l l u f i b r e Indutries Ltd) which uses formulations of the two chlorophenols in r a t i o of 4 to 1, TTCP to PCP. Temperature proved to be a major parameter influencing the bioconcentration factor of chlorophenols i n leeches. Bioconcentration increased with temperature. A l l chlorophenols exhibited the same bioconcentration factors with the exception of the 2,4 DCP. The relationships established by various researchers between the Po/w and BF were not followed by laboratory exposure experiments with leeches. For the purpose of using leeches as bioindicators of p o l l u t i o n i n a r i v e r environment, absolute values of the bioconcentration factors have l i t t l e importance since equilibrium state i s never reached under environmental conditions. What i s more relevant for the use of leeches as bioindicators i s t h e i r a b i l i t y to concentrate chlorophenols to high lev e l s and show a low depuration rate from t h e i r matrix. Leeches are capable of r e f l e c t i n g high concentrations of pollutants that have occurred at various times over a long period of exposure. pH influence on the bioconcentration of chlorophenols followed the expected trend for ionizable compounds: bioconcentration increased at lower pH. pH i s not as important as temperature, since i n a r i v e r environment pH does not change much. Any d r a s t i c changes of pH are usually l o c a l i z e d and r a p i d l y neutralized by d i l u t i o n . pH has to be considered when d i l u t i o n water c h a r a c t e r i s t i c s are matched to the c h a r a c t e r i s t i c s of the body of water monitored by the use of leeches. Other water c h a r a c t e r i s t i c s , such as suspended s o l i d s content and - 1 5 0 -composition, have an impact on the accumulation of chlorophenols i n biota t i s s u e s . This variable was not studied, but higher loads of suspended s o l i d s were r e f l e c t e d i n the re s u l t s of chlorophenol analysis by low recovery values. When recovery values were too low, corrections had to be made to the r e s u l t s . Estimation of the average concentration of chlorophenols i n the water on the basis of the accumulated chlorophenols by the leeches gave values within the same order of magnitude with the rea l chlorophenol concentration i n water monitored by automatic sampling. The worst estimate was twice as high as the actual monitored chlorophenol concentration. For a b i o l o g i c a l sample th i s estimation i s very good. Laboratory controlled experiments, using a series of concentrations of the exposure solution, had to be run i n order to compare the bioconcentration of chlorophenols i n leeches, at various lev e l s of p o l l u t i o n . They proved to be an e f f e c t i v e aid to the in t e r p r e t a t i o n of the f i e l d r e s u l t s . 5.1 Recommendations Recommendations under t h i s section w i l l cover two main aspects: the use of the r e s u l t s of t h i s research i n setting up the methodology for a monitoring program using leeches, and future research needed for better understanding the bioconcentration c a p a b i l i t y of leeches. A basic t e s t using leeches as bioindicators of chlorophenol p o l l u t i o n i n an aquatic environment should consist of two tes t s : a laboratory controlled exposure experiment and a f i e l d leech - 1 5 1 -exposure t e s t . Both exposure tests should be run for the same duration, using the same number and size range of leeches. The exposure solution used i n the laboratory controlled experiment should match cl o s e l y the c h a r a c t e r i s t i c s of the water where levels of p o l l u t i o n are surveyed. Temperature and pH should be i d e n t i c a l with the f i e l d values. The chlorophenol concentration selected for the laboratory co n t r o l l e d exposure should be sim i l a r to l e v e l s set by regulations or other considerations. At the end of the exposure time, the levels of the bioconcentrated chlorophenols i n the f i e l d and l a b o r a t o r y leeches are t o be compared. I f a s i g n i f i c a n t l y higher value was obtained i n the f i e l d leeches than i n the laboratory leeches, t h i s would indicate a p o l l u t i o n problem i n the surveyed environment. Since the maximum acceptable levels were exceeded, a more intense monitoring a c t i v i t y f o r that location roust be i n i t i a t e d to pinpoint the source and the extent of the p o l l u t i o n . An * estimation of the average chlorophenol concentration i n water could also be made. This estimation would define the range of contamination and determine the severity of the p o l l u t i o n problem. If the f i e l d leeches exhibited lower lev e l s of chlorophenols than the laboratory leeches t h i s would indicate chlorophenol levels within the l i m i t s established by regulations. This kind of monitoring program w i l l e f f e c t i v e l y reveal the areas of serious contamination and signal occasional s p i l l s or malpractices of waste disposal. As a need for further research on bioconcentration a c t i v i t y of leeches the following areas should be investigated: the implementation of a program of growing leeches under - 1 5 2 -laboratory conditions: t h i s would assure a supply of leeches a l l year round, a uniformity i n s i z e , and more consistent and lower background levels of contamination. the study of the bioconcentration process k i n e t i c s : determination of the uptake and depuration rates; determination of the bioconcentration factors using the k i n e t i c approach. - the study of the influence of the load and composition of the suspended s o l i d s upon the a v a i l a b i l i t y of the chlorophenols to the leeches. Assessment of the impact t h i s delayed load of chlorophenols might have upon other aquatic organisms i n the region of study. In order to have such a monitoring program i n place, a more serious involvement of the regulatory agencies i s required. 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Peaks: 1 = 2-chlorophenol acetate( 2.21); 2 = 3-chlorophenol acetate( 2.22); 3 = 4-chlorophenol acetate( 2.12); 4 = 2,6-dichlorophenol acetatet 0.090); 5 = 2,5-dichlorophenol acetate( 0.081); 6 = 2.4- dichlorophenol acetate( 0.086); 7 = 3,4-dichlorophenol acetate( 0.084); 8 = 2,3-dichlorophenol acetate( 0.092); 9 = 3.5- dichlorophenol acetate( 0.084); 10 = 2,4,6-trichlorophenol acetate( 0.049); 11 = 2,4,5-trichlorophenolacetate( 0.059); 12 = 2,3,4,6-tetrachlorophenol acetate( 0.049); 13 = pentachlorophenolacetate( 0.018), afte r Krijgsman and Van de Kamp,1977. - 164 -APPENDIX II - 165 -TABLE II.1 SINGLE ION MONITORING OF THE CHLORINATED PHENOLS Parameters Group 1: Group 2: Group 3: Group 4 2,4,5-TCP 2,4-DCP 2,4,6-TCP 2,3,4,6-TTCP PCP Start Time(min) 16.5 19.0 23.0 26.0 Run Time(miri) 2.5 4.0 3.0 3.0 Mass 1 162 196 230 264 (chlorine isotope pattern) Mass 2 164 198 232 266 (chlorine isotope pattern) Mass 3 204 238 272 306 (molecular ion) - 166 -HVDPOCAREOM I STD . 2cHLOF:Or-H£ttOL DERIVATIVE 2 ,4-DICLOROFHENOL 1 DERIVATIVE 2,4,6-TR I CHLOROPHENOL DERIVATIVE 2,3,4,6 -TETRACHLORO PHENOL DERIVATIVE HVROCORBOH I STD X PENTACHLORO PHENOL DERIVATIVE .^.»'Vs..''*»s'-W''-V.,\».^ . ^ w - A ^ ~ i i i T i 1 1 r 1 1 1 1 1 1 1 1 r 11 12 13 14 15 16 17 13 1? 20 21 22 2 3 24 25 26 27 23 Time(min) F i g . I I . 1 . T o t a l i o n c h r o m a t o g r a m o f a s t a n d a r d m i x t u r e o f c h l o r o p h e n o l s u s e d f o r m a s s s c a n n i n g b y e l e c t r o n i m p a c t . S t a n d a r d c o m p o n e n t s a t 100 p p m c o n c e n t r a t i o n e a c h . - 16? -WORK AREA SPECTRUM f-'RM 114*6 LARGST 4 i 123.2,103 .6 130.9, 34.1 LOST 4: 3 3 5 . 3 , .1 3 6 7 . 5 , .1 -295 + 299 PAGE 1 Y - = 1.ee 4 3 . 9 , 17.4 6 3 . 0 , 11.9 3 7 6 . 9 , .1 4 2 3 . 2 , .1 P •H W -p 100 30. 69 40^ 29. 0' iee. 80! 40. 29 9 2-CHL0R0PHEN0L D E R I V A T I V E ( 179 M.U. ) 1 1 1 1 • 1 r• 1 1 1 1 1 i n i | i " i 1 : n n 111 • 1; 11,11! 1 • • I'!I:r. 1 n i i l l l l n i i i i i i l l M i i i i i M r i i i i i i l i i nilllm i n i l l m u n i m 20 40 ' 60 80 ' 1019 120 11n•<11111111 1 1 1 1 1 1 1 1 1 • r 1 1 1 1 •"•B 160 14 2 9 0 '22b ' ' 24'Q ' "'' '269"' "" T > 23'0 I" I" 300" 2b m/ e F i g . II.2..Mass s p e c t r a of 2-CP a c e t a t e , o b t a i n e d by e l e c t r o n impact. Values f o r peak i n t e n s i t y are measured r e l a t i v e t o the most i n t e n s e i o n which i s gi v e n a val u e of 100. UORK AREA SPECTRUM FRN 11496 : PAGE 1 Y - » HW LARGST 4 i 162.1,100.9 164.1, 63.7 4 3 . 9 . 34.5 6 2 . 7 , 16.9 LAST 4» 416 . 2 , .9 4 2 5 . 3 , .9 4 3 9 . 7 , .9 4 4 4 . 7 , .9 -492 + 497 P •H W CD -p n 109. 39. 60 40^ 20' 0' 190. 30' 60^ 49. m. 0 2 ,4-DICHL0P.0PHEM0L D E R I V A T I V E ( 204 M.U. ) I I I I I M I I I I I M n i l i i i n u 1111 1 11 n n I IT 11 1111111 r: •, 20 4b -till! llll|ll!rinl|llllVll|llll'lHli:ill lllllllll'lllllt 60 89 ' 100 111111111 • 11 i 11 n 11 * H 1 1 1 • • 11' 11111 < 1 1 11 1 1 1 1 1 120 140 160 tS^a'"' ' 200'' 2 2 ' e i 2 4 0 " ' 26'9 ' 2 3 0 ' 300"' 320 m/ e F i g . I I . 3 . Mass s p e c t r a of 2,4-DCP a c e t a t e , o b t a i n e d by e l e c t r o n impact. - 168 -WORK AREA SPECTRUM FRN 1149T LARGST 4 i 196.9,100.0 LPST 4 I 393.1 , -617 + 623 PAGE 1 1 .00 42.8, 99.1 193.0, 94.7 97.3, 44.3 409.0, .3 420.3, .3 441.7, .3 -P •i-t W CD H 100. '90. 60 40^  20' 0^  100 80 60 40 20 0 20 4^1U 2 4 6 '-TRICHLOROPHENOL DERIVATIVE ( 238 M.U. . - - -» . -*ti. I -.-•„ J|h„ 1 . ^ . 1 . ....... M j j j l . ......... ^ „ ^ . . l ......... „g,^  m/e Fig.II.4. Mass spectra of 2,4,6-TCP acetate, obtained by impact. electron -p W CD -P WORK AREB SPECTRUM FRN U406 Ptttl 1 — — f T W LARGST 4i 43.1,100.0 231.9, 53.5 229.9, 41.8 234.0, 26.9 LAST 4: 387.7, .3 390.6, ..3 419.3, .3 446.0, .3 -849 + 854 100. 80 6 8 40 20 e 100 . 8 0 ' 68^ 40' 20' 0 TI r i i i i n r r T T i r i i i i i itrrttln*ttu:if 1 2B 1 40 TETRACHLORGPHENOL DERIVATIVE t 272 M .U. ) I»111111 i l i l J H l i ' M M i l l l l ' l l n ^ l l I ' l l l l i l l i l ^ 60 10' I l i t 11 l I l l l l l M l l l l l 10 120 l l t ' . i i . i l t l ' " I I I I I ' H H I M I I l u l l 140 16? 18o"' ' 20 •-4J-U " ' ' 2 2 0 ' 1 2 4 V 320 A m/e Fig.II.5. Mass spectra of 2,3,4,6-TTCP acetate, obtained by electron impact. - 169 -UORK AREA SPECTRUM FRN 11406 PAGE i 7~S i .00 LARGST 4 i 4 3 . 1 , 1 0 0 . 0 2 6 5 . 8 , 40 .7 2 6 7 . 9 , 25 .9 2 6 4 . 0 , 23.2 LAST 4 : 3 7 1 . 0 , .4 3 9 5 . 4 , .4 4 0 5 . 3 , .4 4 1 8 . 4 , .4 1055 +1060 >5 P •H W p H 100. 80^  60^  48 20^  0 III! 111111J VI I PEMTACHLOROFHENOL D E R I V A T I V E ( 306 M.U. ) IM^III »ri i j 1111 * 11 M ^1 I i l i i jMh' i lH^l i ' l in i l i l l I IM|HIHI I I |MI I ' | IMHJII illl^lil un llll 'Mll^lll 'ilil|iili Mtt; 100. 80' 60' 4e' 20' 0 JL '200 '"22ft" ' I- g4p" ' 26B ' 2 8 0 ' ' ' " 3 0 0 " 1 320 A m/ e F i g . I I . 6 . Mass s p e c t r a o f PCP a c e t a t e , o b t a i n e d by e l e c t r o n i m p a c t . - 1 7 0 -Time(rain) F i g . II.7. Total ion current( TIC) chromatogram of a leech extract. The leech was subjected to 7 day exposure to a standard CP sol u t i o n . The standard contained 2-CP, 2,4-DCP, 2,4,6-TCP, 2,3,4,6-TTCP and PCP which are i d e n t i f i e d as peaks 2, 3, 4, 7, and 10, respectively. Peaks 6, 8, and 9 are i d e n t i f i e d as dibromophenol acetate , tribromophenol acetate, and underivatized PCP, respectively. - 171 -o H PHENOL DER ' v o t i v e ; 2-CHL0P0PHEHCL 2,4 , 6-TRICHLORO PhEHOL 2 ,4 - iaC HLCiftOFMEitOL 11 15 TETRflCHLOROPHEHOL PENTRCHLOR0PHENOL 12 13 14 16 i 17 T 18 r i ? 29 Time(min) F i g . II.8. Chromatogram of a standard mixture of CPs used for pos i t i v e ion chemical i o n i z a t i o n . Ion source pressure = 0.2 x 10 . Methane used as i o n i z i n g gas. - 1 7 2 -WORK AREA SPECTRUM FRN' 1140 3 LARGST 4: 129.2,188.0 171.2, 77.6 LAST 4: 171.2, 77.6 172.0, 11.9 -167 + 172 PAGE 1 V = 1.OO 173.1 , 32.13 131 .2, 31 .3 173.1, 32.8 135.6, 6.0 P •H 01 C CD -P. C 100. 30. 68 40 20 8^  100. SO' 60 40. 20 2-CHLOR0F'HEHOL DERI V0T I VE ' 1111111 r • > i > 11 T i p ii!i|iir-TTTT'Trn IMTTI iTt~iiii(ni) i: nil HI IT'IIIIII iiiri:.n iiiinin !tn nil iiuillil l i l l | l :n ' i ' i i inn innlin ihh 20 . 40 6 0 30 100 120 140 160 248 ' 3 0 T T ^ 3 i , 0 l m/ e F i g . II.9. Mass spectra of 2-CP acetate, obtained by chemical i o n i z a t i o n . MORK AREA SPECTRUM FRN U 4 0 3 LARGST 4: 205.1 ,108.0 207 . 1 , 73.3 LAST 4: 205.1,100.0 206.0, 15.1 -393 + 400 PAGE 1 Y = 1 .00 163.1 , 36.4 16S . 1 , 21.2 207.1. 73.3 209.2, 13.2 >5 -P •H W CD 100 38. (JO 4 0 100 30 60 40 2,4-DICHLOROPHENOL DERIVATIVE f f n i r - T T T T l K T 40 inrjnii-TTi.-inu : i: i ri i n inni.r 60 30 " i'olj" islg" 1 4 W " j'ljig "'"" is'o 240 26 ' 6 ' "~^ ' " 2 3 0 ' " " 300" ^ 3 2 \ j | m/ e F i g . 11.10. Mass spectra of 2,4-DCP acetate, obtained by chemical i o n i z a t i o n . - 1 7 3 -IjnRK AREA SPECTRUM FR'M 11403 PAGE 1 Y = 1.00 LARGST 4: 2 3 9 . 0 , 1 0 9 . 0 2 4 1 . 0 , 93.2 2 4 3 . 0 , 36.2 196.0, 14.1 LAST 4: 2 6 8 . 9 , 3.4 2 7 1 . 0 . 2.3 2 7 9 . 0 , 2.3 2 8 1 . 0 , 2.8 -537 + 544 P •H 03 C CD P c 100. so. HQ, 40 20' O 1 00 88 60 40 , 4 - T R IC H L O R O F ' H E M O L D E R I V A T I V E inn ni) I I T T H minni i innin niilini nninii ininni nirrrni iniiini mliltn nniiin i l inii l . iininil tmiiin 11 n i 1 28 40 68 80 180 120 140 160 I,I,-,I, • I.I... nu- • M l f M i l ' . l . l . | I I IJ I I ' " . . M l Jll l I 'M . M i l l I I I | 220 248 260 280 300 320 1 3 0 200 m/e F i g . I I 1 1 . M a s s s p e c t r a o f 2 , 4 , 6 - T C P a c e t a t e , o b t a i n e d c h e m i c a l i o n i z a t i o n . >> •H K) CD -P WORK AREA SPECTRUM FRN 1140:; LARGST 4: 2 7 4 . 9 , 1 0 0 . 0 27 LAST 4: 2 7 6 . 9 . 52.3 £7 -793 + S03 PAGE 1 Y = 1.00 •, 73.3 2 7 6 . 9 , 52.8 2 3 1 . 9 , 22.6 », 6.6 2 7 3 . 9 , 12.3 3 0 2 . 3 . 5.7 100. S8. 40 2o! 8' 1 OO. 80' 60 40 TETRACHLOROPHEMOL D E R I V A T I V E 130 200 220 24 0 26.) 300 320 m, / e F i g . 1 1 . 1 2 . M a s s s p e c t r a o f 2 , 3 , 4 , 6 - T T C P a c e t a t e , o b t a i n e d c h e m i c a l i o n i z a t i o n . - 17^ -WORK AREA SPECTRUM FRN 11483 LflRGST 4: 303.3,130.0 310.7, 76. UOST 4: 309.7, 17.2 310.7, 76. 1027 +1033 PAGE 1 Y = 1 .00 306.3, 75.0 265.8, 26.6 311.3, 7.3 312.7, 20.3 >5 -P •H CO CD P 100. 30. 60^  4o' 20 0 100. S9 60 40 PENTACHLQROPHEHUL DER IVATIVE ^ l s'a Z 0 9 2 2 0 M i l ] , , , , I . . . ; , , . , MMI. | I H . M l M i l | I . U MM| ^49 260 239 399 320 m/ e F i g . 11.13. Mass spectra of PCP acetate, obtained by chemical i o n i z a t i o n . - 1 7 5 -Knna**LEECH 6 DRY EXPOSURE - CL-PHEHOLS S.I.M. •HHS3UL 3 3 ( 1 ) - 2 8 0 3C/-M THEN SC/MIH 18 MIN •dflil 10008 112 2,4-DCP V 2,4,6-TCP 2,4,5-TCP 2 , 3 , 4 , 6 - T T C P PCP — r — 20 17 18 19 21 22 23 —r— 24 —r~ 25 26 —r~ 27 28 Time(min) F i g . II.14a. Total ion chromatogram of a leech extract. The leech was exposed to the Fraser River water for 6 days. The chlorophenols were further i d e n t i f i e d and semiquantitated by single ion monitoring. 0 tr w-rt-iQ P> • H-3 H CD H & • tr *>. •< O • 01 H-3 fD CD O rt H- fD 0 Q> 3 3 H-0 O 3 H-rt 0 O 3* H- H 3 0 3 • 0) rt O n 3 0 H i M | 1 n •ti n> o fD rt Q) rt (D Selected Ion Intensity i-9 H" 3 CD 3 -a 3" f <: 5 o ^ tr H-r t i Q 0) • H-3 CD H Selected Ion Intensity tr tr cn 3 i - " fD (D >-• fD H- O O rt 3 fD 3 O 3 H-H- O rt 3 O H 1 3 O 3* i-i O 3 Cu rt O iQ 3 o i-h I D n O CD rt 0) rt CD CD • 3 2 ^ **]— \ . — J ^ _ r -fe s , • 1 ? c_ *~jif.^ r 1 <: 1 J urn o x H C I >^ m m 3 i Z I - 1 7 7 -• M a i l * - * L E E C H 6 DRY EXPCOJPE - CL-PHEMOLJ S . I . M . HUIiaO*? • T l 11** JUL 3e<l)-2S8 3C'M THEM SC/MIN 19 MIH ,-24 25 Time(min) F i g . II.14d. Selected ion chromatogram of 2,3,4,6-TTCP acetate, obtained by single ion monitoring. -p •H Kl CU P M C o H T j <D -P O 0) H 0 fciaiig-fLEECH 6 IlfiY EXPOSURE - CL-PHEMOLS S.I.M. K l M H 30(1)-289 3CM THEN SCvMIM 10 MIH 35 •139 l e e w 52 PCP Time(min) Fi g . II.14e. Selected ion chromatogram of PCP acetate, obtained by singl e ion monitoring. - 1 7 8 -•IEEH-"*WATER SBMPLE - CL-PHENOLJ S.I.M. SC-'MS •;iK>13LIL 30CD-28O 3C-^ M THEN SC'MIN 10 MIN UJi l 10007 171 TRACE 2,4-DCP \ 2 ,4 ,6-TCP 2 ,4 ,S-TCP 2,3,4,6-TTCP —1 1 1 1 r— 17 18 19 20 21 —I 1 1 I 1— 23 24 2S 26 27 — I — 28 Time(min) F i g . 11.15a. Total ion chromatogram of a Fraser River water sample. The CPs were i d e n t i f i e d and semiquantitated by single ion monitoring. - 1 7 9 -KEHHI-'MOTEP SAMPLE - CL-PHEHOLS S . I . M . SC/MS KlHSlJ'.IL 30(11-230 3C/-M THEN SC-MIN 10 Mill D33 10097 P •H W 0 -P o CD -P o CD H CD in 12 •* TRACE 2,4-DCP 17 — I — 18 Time(min) Fig.II.15b. Selected ion chromatogram of 2,4-DCP acetate from a derivatized Fraser River water sample. •JEGIgrn.lflTER SAMPLE - CL-PHEIIDLS S.I.M. SCMS BQ3 1O00' BHEHS'JL 30(i>-28e 3C/M THEM SC/MIN 10 MIN 18 >5 p •H m c o •p o c u •p o CD I—I CD C/5 2 ,4,6-TCP 2,4,5-TCP (TRACE) 1 L 1 L 1 111111  11 11 rin im 1 linn)MI I 1W1 ni/nii L iiiii'/.nn, 111 iinn'mmiiniH .W.M A ,'rt IJU'lifiJU 1.11 Pi.Hl.  urnluiui'.  iiuiLjiui^1 IUIIIUI 11 tuuu IIIUUUUIUI IUIII i|dliuiiiiutit UUUII iiiiiuji IUIyniijiiiu uiLAjiultriuiyli LAJI IUIII Time(min,) Fig.II.15c. Selected ion chromatogram of 2,4,6 and 2,4,5-TCP acetates from a derivatized Fraser River water sample. - 180 -• I C l i l * - * WATER SAMPLE - CL-PHEMOLI S.I.M. GC'MS •HlJMSIJL 30<l>-289 3C"M THEM SC-MIN 16 MIM "P •H to P o •p O <D H CD to 2,3,4 ,6-TT:P Time(min) F i g . I I . 1 5 d . S e l e c t e d i o n chromatogram of 2,3,4,6-TTCP a c e t a t e from a d e r i v a t i z e d F r a s e r R i v e r water sample. >> -P •H tn 0) P H o H -d Q) P O 0) i—I <D CO •aailji-'-'WBTER SAMPLE - CL-PHENOLS S.I.M. GOMS EEHsi iUL 3 e m - 2 s o 3C^M THEN ECMIN i e MIN 2S 23 Time(min) F i g . I I . 1 5 e . S e l e c t e d i o n chromatogram of PCP a c e t a t e from a d e r i v a t i z e d F r a s e r R i v e r water sample. - 181 -APPENDIX III - 182 -SYMBOLS BF : bioconcentration factor CF : concentration of chemical i n f i s h CW : concentration of chemical i n water CP : chlorophenol DCP : dichlorophenol GC : gas chromatograph, or gas chromatography GC/MS : GC-mass spectrometer or GC mass-spectrometry Kl : uptake rate constant K2 : depuration rate constant Ka : acid d i s s o c i a t i o n constant Koc : organic carbonrwater p a r t i t i o n c o e f f i c i e n t Kom : organic matter:water p a r t i t i o n c o e f f i c i e n t Kow : octanol:water p a r t i t i o n c o e f f i c i e n t Ks : s o i l p a r t i t i o n c o e f f i c i e n t not detected pentachlorophenol octanol:water p a r t i t i o n c o e f f i c i e n t quantitative s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p temperature trichlorophenol t o t a l ion current tetrachlorophenol Note: TTCP symbol refers always to the 2,3,4,6-TTCP isomer, unless mentioned-otherwise i n text. PCP Po/w QSAR T TCP TIC TTCP 

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