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The effect of copper on the life history stages of the Harpacticoid Copeod TIGRIOPUS CALIFORNICUS O'Brien, Patrick 1987

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THE EFFECT OF COPPER ON THE LIFE HISTORY STAGES OF THE HARPACTICOID COPEPOD Tiqriopus c a l i f o r n i c u s by PATRICK O'BRIEN B.Sc,the University of B r i t i s h Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Oceanography) We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 1987 (c) Patrick O'Brien, 1987  In  presenting  degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  and study.  thesis for scholarly by  this  his  or  her  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  requirements that the  1 further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  DE-6(3/81)  the  for  an  advanced  Library shall make it  that permission for extensive granted  is  by the  understood be  that  allowed without  head  of  my  copying  or  my written  i i  Abstract  Significant tolerance  of  Tiqriopus The  differences  the  response  predominantly  over  using  the  most  life-history  Copper  N-2  was  equilibrated affect  1.0X10"  or m o r t a l i t y  Copper rate  M Cu)  equilibrated  of egg  naupliar  activity  to widely varying have  increased  The of  hold  in  copper  (between  T i q r iopus  solutions.  of  t h e C-6  (between  1.0x10"  d i d not  (between  1.0X10"  1.0xl0"  natural  the t o l e r a n c e  8  was  i n SOW  1.0X10"  was  observed  upon  KOxlO'  and  1.0X10-  M)  b  1.0X10"  the  6  10  and M ) .  5  the  M)  6  M).  or  Exposure  appears  to  to unnatural stress.  o b s e r v e d f o r some pseudonana No  was  significant  addition to  not  alter  1.0X10~  conditions  of T i g r i o p u s  medium.  1.0x10'  and  and  9  ecological  interaction  10  did  significantly  and  toxicity 6  to  6  food  fecundity  (between  for Tiqriopus  1.0X10~ M  and  p h y t o p l a n k t o n such as T h a l a s s i o s i r a  to  medium.  found t o occur  range  SOW  copper-manganese  copepod,  as  sensitive  (between  with  but  was  copper  stage.  adult  survival  the  seawater  to copper  with the  significantly 6  artificial  t h e most  in  of the marine  concentration  The  tolerant  stages  of T i g r i o p u s  1.5X10~ M. s  found t o e x i s t  life-history  californicus,  copper  were  copper  of  species  not  found  reduction manganese  O.OxlO"  6  M)  and  An a t t e m p t  was made t o q u a n t i f y  manganese  in  with or  pass  react cation that  resin this  saturated  biologically through  with metal  failed ions.  proportion  available  biological  technique of Zorkin attempt  the  forms  (i.e.,  membranes)  e_t a l . , ( 1986).  due t o t h e r e s i n  of  copper able to  using  the  I t i s thought  columns b e i n g  super-  iv  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  viii  ACKNOWLEDGEMENTS  ix  INTRODUCTION  1  MATERIALS AND METHODS  7  1 . Organism  7  2. Preparation of SOW  9  3. Natural Seawater  9  4. Trace Metals  10  5. Incubation Chambers  10  6. Bioassay Procedures  11  (i) The Effect of Copper on Egg V i a b i l i t y  11  ( i i ) The Effect of Copper on Naupliar Survival  12  ( i i i ) The Effect of Copper on Copepodite Survival Preparation of Copper Treated Food for Pre-Equilibration Experiments  .  13  (iv) The Effect of Copper and Food on Egg Cluster Production  14  (v) The Effect of Copper and Food on Adult Survival  14  (vi) The Effect of Copper on Naupliar Survival  15  ( v i i ) The Effect of Manganese on Copper-Stressed F i r s t Nauplius Survival  16  ( v i i i ) The Effect of Manganese on Egg V i a b i l i t y  17  S t a t i s t i c s used in Analysis of Data  17  V  RESULTS  20  (i) The Effect of Copper on Egg V i a b i l i t y  20  ( i i ) The Effect of Copper on Naupliar Survival ( i i i ) The Effect of Copper on Copepodite  ... 21  Survival  23  (iv) The Effect of Copper and Food on Egg Cluster Production  25  (v) The Effect of Copper and Food on Adult Survival  25  (vi) The Effect of Copper on Naupliar A c t i v i t y Levels .... 26 ( v i i ) The Effect of Manganese on Copper-Stressed Nauplius Survival  First 27  ( v i i i ) The Effect of Manganese on Egg Survival  27  DISCUSSION  43  CONCLUSION  55  REFERENCES  57  APPENDIX  64  vi  LIST OF TABLES  Table 1. The Effect of Copper on the v i a b i l i t y of Tiqr iopus c a l i f ornicus eggs  59  Table 2. LC50 and LC95 values for each l i f e - h i s t o r y stage of Tiqriopus c a l i f o r n i c u s (including 95% confidence l i m i t s )  60  Table 3. Percent survival of Tiqriopus c a l i f o r n i c u s N-1 nauplii upon exposure to copper solutions. LC50=1.2x10" M Cu. LC95=3.7X10" M Cu 61 6  6  Table 4. Percent survival of Tiqriopus c a l i f o r n i c u s N-2 nauplii upon exposure to copper solutions. LC50=0.28X10" M Cu. LC95=4.72x10" M Cu 62 6  6  Table 5. Percent survival of Tiqriopus c a l i f o r n i c u s N-3 nauplii upon exposure to copper solutions. LC50 = 2.46xlO" M Cu. LC95 = 8.03x10- M Cu 63 6  6  Table 6. Percent survival of Tiqriopus c a l i f o r n i c u s N-4 nauplii upon exposure to copper solutions. LC50=2.92X10" M Cu. LC95=8.69x10~ M Cu 64 6  6  Table 7. Percent survival of Tiqriopus c a l i f o r n i c u s N-5 nauplii upon exposure to copper solutions. LC50=4.12x10" M Cu. LC96=8.79x10" M Cu 65 6  6  Table 8. Percent survival of Tiqriopus c a l i f o r n i c u s N-6 nauplii upon exposure to copper solutions. LC50=4.75x10" M Cu. LC96=9.55x10" M Cu 66 6  6  Table 9. Percent survival of Tiqriopus c a l i f o r n i c u s C-1 copepodites upon exposure to copper solutions. LC50=5.88x10- M Cu. LC95=1.1 Ox 10" M Cu 67 6  5  Table 10. Percent survival of Tiqriopus c a l i f o r n i c u s C-2 copepodites upon exposure to copper solutions. LC50=7.82X10" M Cu. LC95=1.37x10' M Cu 68 6  5  Table 11. Percent survival of Tiqriopus c a l i f o r n i c u s C-3 copepodites upon exposure to copper solutions. LC50=6.54X10" M Cu. LC95=1.12x10" M Cu 69 6  5  Table 12. Percent survival of Tiqriopus c a l i f o r n i c u s C-4 copepodites upon exposure to copper solutions. LC50=6.19X10" M Cu. LC95=1.25x10" M Cu 70 6  5  Table 13. Percent survival of Tiqriopus c a l i f o r n i c u s C-5 copepodites upon exposure to copper solutions. LC50=6.15X10" M Cu. LC95=1.06x10" M Cu 71 6  5  Table 14. Percent survival of Tigriopus c a l i f o r n i c u s C-6 copepodites upon exposure to copper solutions. LC50=1 .20x10" M Cu. LC95=1.51x10" M Cu 72 5  5  Table 15. The effect of copper-equilibrated food on Tigr iopus c a l i f ornicus egg production  73  Table 16. The effect of copper-equilibrated food on Tigr iopus c a l i f ornicus adult mortality  74  Table 17. The effect of copper on a c t i v i t y levels of the N-1 stage of Tigriopus c a l i f o r n i c u s 75 Table 18. The effect of copper and manganese on survival of the N-1 stage of Tigriopus c a l i f o r n i c u s . The copper concentration in a l l tests was 1 .0x1 0- M Cu 76 6  Table 19. The effect of manganese on the v i a b i l i t y of Tigr iopus c a l i f ornicus eggs  77  viii  LIST OF FIGURES  Figure 1. Tigr iopus LC50 Tolerance  26  Figure 2. Tigr iopus LC95 Tolerance  27  Figure 3. Copper Tolerance of the N-1 Nauplius  28  Figure 4. Copper Tolerance of the N-2 Nauplius  29  Figure 5. Copper Tolerance of the N-3 Nauplius  30  Figure 6. Copper Tolerance of the N-4 Nauplius  31  Figure 7. Copper Tolerance of the N-5 Nauplius  32  Figure 8. Copper Tolerance of the N-6 Nauplius  33  Figure 9. Copper Tolerance of the C-1 Copepodite  34  Figure 10. Copper Tolerance of the C-2 Copepodite  35  Figure 11. Copper Tolerance of the C-3 Copepodite  36  Figure 12. Copper Tolerance of the C-4 Copepodite  37  Figure 13. Copper Tolerance of the C-5 Copepodite  38  Figure 14. Copper Tolerance of the C-6 Copepodite  39  ix  ACKNOWLEDGEMENT  I would l i k e to thank Dr.  A.G.  in my undertaking of t h i s endeavor. the Dr.  Lewis for  I would also l i k e to thank  members of my research committee, Dr. T.F.  Pedersen, for their  during the writing  of t h i s t h e s i s .  time  his patience  P.J.  and helpful  Harrison and criticisms  1  Introduction  The Effects of Copper on the L i f e History Stages of  the  marine  copepod, Tigriopus c a l i f o r n i c u s  Copper  is  a  required  trace  metal  which  can  be  both  detrimental and b e n e f i c i a l to plants (Anderson and Morel,  1978,  and  1978;  Stauber  and  Florence, 1985)  Arnott and Ahsanullah, and  Rice, 1981;  and  Truchet,  transition  1984)  at  elevated  which  effect  Engel  1982).  and  (Cherian  Brouwer,  Copper i s  proteins  Harrison  and Bouquegneau, Martoja,  concentrations.  accounts  for  a  major  and Goyer, 1978;  Overnell and Trewhella,  1980;  1981;  It  is  a  capable of forming strong bonds with proteins  and other ligands  1978;  Barber and Trefry,  Lewis and Cave, 1982;  metal  biological  1979;  and animals (Bengtsson,  1979; 1982,  responsible  Fisher, 1984;  for  the  part  of  its  Talbot and Magee, 1980;  Roesijadi,  and White and Rainbow, conformation  of  some  (metalloproteins), the production of organic molecules  such as haemes and chlorophylls, and enzymes (Cherian and Goyer, 1978;  Copper development  has  been  of  plants  shown  the  development  (Anderson  and  Morel,  photosynthesis  1978)  a  component  of  some  Roesijadi, 1980)."  to be important in the growth and  (Hipkins,  affecting  is  of  1983;  some and  and  Hewitt,  phytoplankton reducing  the  1983),  populations rate  of  by blocking electron transport in photosystem II  2  (Hipkins, and  1983) in kelp (Clendenning and North, 1960) and micro-  nano-flagellates  Wafer,  1978).  deleterious to a bacteria  (Rajendran,  Excess  copper  myriad  of  (Sunda  and  Sumitra-Vijayaraghaven, and  has  also  organisms,  Gillespie,  been  (Coombs  to  yeasts  and  oysters  (Engel  and  1979),  Sprague,  1981),  and George, 1978), barnacles (Rainbow,  Wiggins and Jackson,  1980),  and  be  including  Brouwer, 1982, 1984), rainbow trout (Dixon and molluscs  shown  shrimp  (White  and  Scott,  Rainbow,  1982) .  While  copper  has  been  demonstrated  species of phytoplankton, the addition demonstrated  to  of  or  of  manganese  the  chelating agents  Additions  of  of  Chaetoceros  socialis  and  t o x i c i t y (either t o t a l l y or p a r t i a l l y ) Copper  toxicity  reversed  by the  1983) .  This  to  the  diatom  addition  of  been (Sunda FeC12,  ethylenediaminetetraacetic  acid (EDTA) or n i t r i l o t r i a c e t i c acid (NTA) stimulated cultures  has  reverse copper-induced growth reduction  and Huntsman, 1983; Sunda et a l . , 1981). MnC12,  to be toxic to most  also  (Sunda  reduced et  Thalassiosira  manganese  growth  (Sunda  of  copper  a l . , 1981). pseudonana  and  was  Huntsman,  was interpreted to be a physiological interaction  between copper and manganese with copper competing for manganese uptake sites which would upset c e l l u l a r manganese metabolism.  Work on the diatom that  culture  growth  Thalassiosira was  dependent  pseudonana  demonstrated  on the c e l l u l a r manganese  3  concentration (Sunda and Huntsman, 1983)  which was  function of manganese ion a c t i v i t y and an  inverse  the  and  cupric  ion  concentration.  Sunda  developed a competitive binding model either  cellular  manganese  uptake  in  found to be a function  of  Huntsman  (1983)  copper  blocks  which  or the binding of manganese  within the c e l l to explain the t o x i c i t y of copper.  In contrast, i t has been proposed that manganese hydroxides attached to the c e l l wall of the markedly This  reduced  mechanism  competition effect may uptake  binding  no  sites  in  the  Thus,  the  cell  (Sunda et §_1., 1981)  Thalassiosira  the  cell  (1983)  surface  reduction  was  pseudonana.  in  at  manganese  or sorption of (Stauber  and  mechanism.  of copper and manganese reported by Sunda verified  by  Kazumi  (1985)  with  Using AQUIL (Morel et a l . , 1979), a  well defined medium, and water from a B r i t i s h the  or  copper-manganese  or to some as of yet undiscovered  interaction  Huntsman  closterium,  interaction  be due to manganese-copper competition  Florence, 1985)  and  physiological  sites.  copper by manganese oxides on  The  Nitzschia  copper t o x i c i t y (Stauber and Florence, 1985). involves  for  diatom  Columbian  fjord,  c e l l d i v i s i o n rate produced by excess copper  was ameliorated by high levels of manganese.  While the copper-manganese interaction has been studied for some plants i t has not been  explored  for  any  animals.  This  4  study  includes  a  test  of  the  hypothesis that manganese may  reduce copper t o x i c i t y to animals. effect  Determining i f there  an  of manganese on the copper t o x i c i t y to an aquatic animal  would be a natural extension of the phytoplankton work help  is  to  determine  the  mechanism  and  may  of metal t o x i c i t y in marine  animals.  I n t e r t i d a l marine animals are subjected to a great of  conditions because of tidally-induced changes in water level  combined with varying atmospheric conditions. in  variety  the  intertidal  and  splashpool  tolerate a wider range of would  planktonic  zones  Organisms may  living  be expected to  environmentally-induced stress  organisms.  Tiqriopus  californicus  than is  i n t e r t i d a l harpacticoid copepod common to splash and tide from  C a l i f o r n i a to Alaska.  pools  It has a discontinuous l i f e history  (Feldman, 1985), and i s readily cultured in the laboratory. copper tolerance of Tiqriopus was examined not only its  hardiness  because  naturally-occurring  tolerance  to  stress  would  increase  Cherian  1980) and i f tolerance  of  and  there  Goyer, was  organism  a  marked  difference  any of i t s l i f e history stages.  has  been  an  non-toxic  1978; Fisher, 1980; and Roesijadi, in  reached,  i t may  the  copper  Roesijadi (1980)  suggested that once some c r i t i c a l metal-complexing an  of  unnatural stress (perhaps through the  production of metallothioneins which bind metals into forms:  The  as an i n t e r t i d a l organism, but also to determine  i f exposure to organism's  an  capacity  of  no longer be able to  process or store metal into non-toxic forms; increased mortality  5  would be expected.  Copper  and  concentrations volumes  of  manganese  used  "total"  metal  where known amounts of metal were added to known  synthetic  experimental  experiments  medium.  and  natural  seawater  to  produce  an  However, both copper and manganese can be  involved in a complex series of reactions  with  seawater  ions.  Only some metal species are b i o l o g i c a l l y "available" and able to react  with or pass through membranes of l i v i n g c e l l s (Cross and  Sunda, 1977). available  While elevated  form  concentrations of  metal  in an  can be toxic, metal in an unavailable form w i l l  not be harmful to organisms.  The  proportion  of  total  metal  which i s in a b i o l o g i c a l l y - a v a i l a b l e form can be quantified with the  resin  column  technique  of  Zorkin  et. a l . , (1986) which  retains weakly charged metal species.  The medium used in the majority of solution  (SOW)  from  AQUIL.  Similar  natural seawater to allow comparison of  bioassays  was  bioassays  were  results  in a  a  salt run in  defined  medium, SOW, with those in natural seawater.  While  metal  speciation  biological  effect  organism.  Tolerance  confers  i s determined  by  metal b i o a v a i l a b i l i t y , a the  tolerance  of  the  tends to vary both with organism type and  with i t s l i f e - h i s t o r y stage.  The response of  the  life-history  6  stages  of T i g r i o p u s  hypothesis increase stress.  that an  c a l i f o r n i c u s t o c o p p e r was  exposure  organism's  to  used  naturally-occurring  tolerance  to both n a t u r a l  to test  stress and  the  would  unnatural  7  Materials and Methods  Organism  Laboratory cultures of the harpacticoid californicus  8.0.  Tigriopus  were maintained in 2.8 L Pyrex® Fernbach flasks in  the chelexed salt medium  copepod  (AQUIL;  solution Morel  et  (SOW)  of  an  artificial  a l . , 1979) at 35 ppt s a l i n i t y and pH  Temperature was maintained at 16 + 1 C on 16:8  cycle.  seawater  light:dark  Culture medium was replaced every 96-120 h.  Animals  to  be  used  in  bioassays were isolated from the  laboratory cultures at the egg stage.  Females with  mature  egg  clusters were collected on a glass s l i d e , in a drop of water and egg  sacs  were removed with stainless steel dissecting tweezers  using a Bausch washed  three  and  Lomb®  times  in  dissecting SOW  before  incubation chambers containing SOW  microscope. their  Eggs  transfer  to 20 ml  (this washing was intended to  remove particulate and soluble materials  which  may  associated with the adult female or the egg c l u s t e r ) . and  were  have  been  Between 4  8 clusters were kept in each chamber u n t i l the nauplii were  released;  approximately  24  clusters  were  used  in  each  exper iment.  Hatched  nauplii  were  maintained  Pyrex® evaporating dishes containing SOW life  history  stage  to  be  tested.  in acid-washed (1N HCL) u n t i l they were at Specimens  the  to be used in  8  bioassays were transferred through three rinses of SOW micro-pipette  and  a  dissecting  microscope.  using  The animals were  then isolated in SOW for 6 to 8 h prior to their use, to any  excreted  materials  which  could  affect  Organisms were f i n a l l y isolated in solutions experimental bioassays. copepods  solutions  immediately  Any solution would  thus  carried  be  prior  into  the  to  remove  experiment.  identical  the  a  being  chamber  to the used  in  with  the  very similar to the test solution and  medium carry-over would be expected to have  minimal  effect  on  the composition of the test solutions.  Cultures  and  batches  of Tigr iopus were fed f i n e l y ground  f i s h food (Wardley's® basic food flakes) every 24 h.  There were  two advantages  fish  instead  of  to  using  a  phytoplankton  commercially-available for feeding  the  copepod cultures.  F i r s t , Tiqriopus i s a detritus-feeding benthic copepod variable  isolated  and the  size of the available food p a r t i c l e s was more suitable  for a l l of the feeding stages than was Second,  food  increased from  cultured  phytoplankton.  survival was obtained when the copepods were  the  food  flakes  by  micro-pipette  than  when  isolated from phytoplankton-containing medium by f i l t r a t i o n .  A  second  source  of  nourishment  for the  bacteria which accumulated in the culture comm).  The  at t h i s time. growing  flasks  copepods  was  (Lewis,  pers  importance of the b a c t e r i a l food source i s unknown Tigriopus  in culture  may  medium,  graze:  ( i ) solely  ( i i ) solely  on  on bacteria  the ground food  9  flakes, or on a combination of ( i ) and  (ii).  No  attempt  was  made to keep the culture axenic.  Preparation of  The  major  dissolved Pyrex®  SOW  seawater  in 12-L of  vessel.  salts  (except  glass-distilled  for  water  MgC12.6H20) were (GDW)  in  to  40-L.  40-L  Once a l l of the major s a l t s were dissolved the  previously-dried MgC12 was added and the f i n a l volume up  a  The  SOW  was  was then bubbled with f i l t e r e d  made  (0.4 urn  Nuclepore® a i r passed through 1N H2S04 u n t i l the solution pH 8.0 + 0.05, then passed through an ion-exchange column  was  (Chelex-  100, 100-200 mesh, Bio-Rad® Laboratories) to reduce the l e v e l of trace metal contamination. and  A l l transfers, including trace metal  chemical solutions and organism transfers, were c a r r i e d out  in a class 100 laminar flow hood with a l l possible replaced  with polypropylene.  metal  parts  Solutions were equilibrated for 2  h before they were used.  Natural Seawater  Seawater was Fisheries  collected  Research  Branch  from  the  Fisheries  in West Vancouver.  through a Gelman Metricel® membrane f i l t e r shortly  after  collection  polypropylene bottles. adjusted to 8.0 + 0.05.  and  and  Oceans'  It was  filtered  (0.45 urn  stored  Water s a l i n i t y was 33  at  16 ppt  pore C and  size)  in pH  20-L was  10  Trace Metals  Copper  stocks  reagent grade 1N adjust  the  were prepared by dissolving copper metal in  HN03.  final  Glass-distilled  copper  water  concentration  polypropylene  bottles  3  with  Immediately with  GDW  and  then  Volumetric  replacement.  A l l bottles were rinsed  soaked  in  1N  p r i o r to use, the bottles were  GDW.  medium  Fresh  and volumetric flasks were used in  the preparation of metal stocks. times  added to  to 1.0x10" M.  solutions were prepared daily for bioassay Only  was  three  HCl for seven days. rinsed  three  times  flasks were handled in a similar manner  except that they were soaked in 3N HCl for 24 h.  Incubation Chambers  Polystyrene Nunclon® soaked  in  1N  HCL  Prior to subsequent a  minimum  incubation  chambers  were  for 7 d and then rinsed three times in GDW. use, the chambers were soaked in 1N HCl for  of 4 d and then rinsed 3 times in GDW.  solutions were allowed to equilibrate for at least use.  Ten  ml  initially  Experimental 2  h  before  of the appropriate metal-containing solution was  pipetted into the incubation chambers and allowed to equilibrate with the chamber surface for 2 replaced  with  fresh  h;  experimental  these  solutions  solutions  were  then  and the bioassay  organisms added.  Changes  in metal  chemistry  or  concentration  may  have  11  occurred  in the experimental solutions during a bioassay due to  a number of factors, copepod  cuticle,  including:  ( i ) adsorption  of  metal  to  ( i i ) complexation of metal ions with organic  contaminants, or ( i i i ) continuing, non-equilibrium adsorption of metal to the walls of the incubation chambers. metal  Relative to  concentration used, adsorption i s not considered to have  caused a major change in the metal concentration or The  the  daily  chemistry.  changing of experimental solutions would remove most  of the organic contaminants capable of binding metals, and would reduce the effect of metal loss through adsorption.  Bioassay Procedure  Tigriopus was studied copper  on  survival, (iv)  the  egg v i a b i l i t y ,  to  ( i ) the  copper  on  copepodite  of  of  survival,  of copper and food on egg cluster production,  (v) the effect of copper and food on adult effect  effect  ( i i ) the effect of copper on naupliar  ( i i i ) the effect of effect  determine:  copper  on  naupliar  survival,  activity,  (vi) the  ( v i i ) the effect of  manganese on copper-stressed f i r s t naupliar survival, and the effect of manganese  on  egg  viability.  The  (viii)  experimental  procedure used for each section w i l l be discussed.  (i) The Effect of Copper on Egg V i a b i l i t y  Eggs  were  collected  48  +  1 h after their appearance on  previously-berried females, rinsed in SOW,  then exposed  to  SOW  12  solutions spiked with copper (from preliminary work i t was found that  egg  clusters  less  than 48 h old suffered high mortality  when removed from the donor females). each  One cluster was used for  of three replicates for each concentration.  Collecting an  adequate number of clusters at the 48 h stage of development was d i f f i c u l t and limited the number of clusters to three  for each  concentration  the  eggs that  into nauplii at 96 h was calculated for each  replicate  hatched for  each  of  copper.  concentration  survival value.  The  and  percentage  averaged  to  of  produce  a  Concentrations of copper used in this series of  experiments ranged from 1.0x10"  10  to 1.0x10"" M.  Unless stated otherwise, the following conditions effect  percent  for t h i s  and  every  bioassay:  were in  ( i ) a l l experimental  solutions were replaced every 24 h, ( i i ) a l l bioassays were for  96  h,  ( i i i ) three  replicates  were  run  for  concentration used, (iv) a l l bioassays were run in a environment,  (v)  run  every  food-free  sample size was 20 individuals per replicate,  and (vi) observations were made and dead organisms were  removed  at 24, 48, 72, and at 96 h.  ( i i ) The Effect of Copper on Naupliar Survival  Naupliar conditions  stages  identical  from to  N-1  those  to of  N-6 the  were  maintained  laboratory  in  cultures.  Nauplii to be used in bioassays were isolated in SOW three times over and f i n a l l y isolated in solutions identical to experimental  13  solutions  and counted into pre-equilibrated incubation chambers  containing 10 ml of experimental solution.  LC50 and LC95 values  were calculated and survival data were  plotted  concentration  stage  for  each  naupliar  concentration of a material which was found mortality  of  48 h, 96 h.  against (an  to  copper  LC50  result  is a in 50%  a group of organisms within a specified time e.g.  S i m i l a r l y , an LC95 i s a concentration which results  in 95% mortality within the specified time).  ( i i i ) The Effect of Copper on Copepodite  An  identical  mortality  studies  procedure was  to  Survival  that  employed  in  used the  in  the naupliar  copepodite mortality  i  studies.  Each copepodite stage was  bioassay  usage.  Bioassays  were  grown run  and  as  maintained  they  had  been for  naupliar bioassays and LC50 and LC95 values were calculated each  life-history  stage.  for  for  The percentage survival was plotted  against copper concentration for each copepodite stage. Preparat ion  of  Copper-Treated  Food  for  Pre-Equilibrat ion  Exper iments  Wardley's® f i s h food flakes (0.1 g) were f i n e l y ground with a  mortar  and  copper solution bottles.  pestle and allowed to equilibrate with 250 ml of overnight  in  500  ml  Nalgene®  polypropylene  The "equilibrated" food flakes were then removed from  suspension by centrifugation for  10  min  in an  International  14  Equipment  Company® centrifuge (model PR-J).  decanted  and  equilibrated"  the food food  flakes  flakes  were  in fresh  The supernatant was  re-suspended  as "pre-  experimental  solutions  (control and copper-enriched solutions) immediately prior to the start of bioassays.  Two series of bioassays were run using this procedure. series  was  concerned  with  One  egg cluster production and one was  concerned with adult tolerance to copper.  (iv) The Ef fect of Copper and Food on Egg Cluster Product ion  Previously-berried  adults  were  placed  in  incubation  chambers containing experimental solutions with pre-equilibrated food  (Tigriopus  w i l l not produce egg clusters when food i s not  available).  Copper  concentrations used  ranged  1.0x10"  10  from  to  produced between replicates value.  1.0xl0" was  5  M.  averaged  in these  bioassays  The number of clusters to produce  a  mean  Four replicates of f i f t e e n organisms were used for each  concentration.  (v) The Effect of Copper and Food on Adult Survival  The method from section (iv) was used for a of  second  series  bioassays to determine the tolerance of the adult copepod to  copper-labelled food.  Animals were collected from cultures with  a pipette and placed in SOW for 6 to 8 h before the start  of a  15  bioassay.  This  isolation  was  intended to remove  such as phytoplankton and bacteria which may have culture  medium.  To  experimental solutions,  minimize  carry-over  copepods  were  contaminants been  and  isolated  in  the  dilution in  of  solutions  i d e n t i c a l to the experimental solution prior to their being used in  bioassays.  Solution  carry-over to the incubation chambers  would thus be expected to have minimal effect on the composition of experimental solutions.  Bioassays were run with the pre-equilibrated food daily.  The  percent adult mortality at 96 h was determined for  each of the copper concentrations used. was  replaced  Percent  survival  data  transformed into arcsin (Zar, 1984) values to generate LC50  and LC95 values.  (vi)  The Effect of Copper on Naupliar A c t i v i t y  Observations made during i n i t i a l experiments  suggested that  the a c t i v i t y of the early naupliar stages (especially of the 1)  decreased with increasing copper concentration ( a c t i v i t y  defined  as  a  voluntary  motion  elicited  in  response  to  Nwas a  stimulus: in this case, the stimulus was a jet of water from the t i p of a micro-pipette).  The  first  naupliar stage was selected for this  because i t was  found to be active and sensitive to  and  isolate.  easy  to  Mature  experiment  stimulation,  egg clusters were removed from  16  adults and placed in SOW u n t i l release of the nauplii Nauplii  were  0-2  h  old when they were collected from the SOW  with a micro-pipette and placed in 20 ml chambers  occurred.  containing  experimental  multi-well  solutions.  incubation  (The  dilution  effect of adding 50 u l of SOW to 10 ml of solution i s « Copper  1.0%).  concentrations in the culture medium were varied between  I.OxlO"  10  and 1.0X10" M Cu. 5  A c t i v i t y was quantified by counting the number of seconds a nauplius was active once stimulated; a test time found that  to  stimulation). in  10  s  was  be ideal (from preliminary observations, i t was found  control  times  of  nauplii  would  often  stop  moving  15  s  after  Organisms used in bioassays were stimulated three  one minute.  If no response was noted from any of the  stimulations, the organism was termed dead and removed from  the  medium.  If a response was observed, the three time-values were  recorded  and  averaged  to  Activity  was  followed  every  produce 24  a h  mean  response  value.  for the  duration of the  ( v i i ) The Effect of Manganese on Copper-Stressed  First-Naupliar  bioassay.  Survival  The  first  naupliar  stage was chosen for t h i s metal-metal  study because i t had a low copper tolerance.  N-1  nauplii  were  collected  and isolated as previously discussed in section ( i i ) .  Manganese  concentrations  used  in  experiments  varied  from  17  1.Ox 10~  to  6  1.0x10""  M;  in preliminary  concentrations of 1.0x10" to 1.0X10" 9  7  experiments  M, i t was found that  effect on naupliar mortality was s t a t i s t i c a l l y from  control mortality.  1.Ox 10"  6  using the  indistinguishable  Copper concentration was maintained at  M.  ( v i i i ) The Effect of Manganese on Egg V i a b i l i t y  Mature egg clusters were females  when  removed  from  the clusters were 48 + 1 h old.  with SOW and isolated in experimental discussed.  previously-berried  The  percentage  of  They were rinsed  solution  as  previously  eggs that hatched into nauplii  within 96 h was averaged to produce a  percent  survival  The manganese concentration was varied from 1.0x10~  9  value.  to 1.0X10"  5  M.  S t a t i s t i c s used in Analysis of Data  Both  Anova  and  experimental groups. analyze  multigroup  Tukey tests were used to compare means of Anova i s a s t a t i s t i c a l technique  experiments.  to  The test provided one overall  comparison to determine i f there was between  used  a  significant  difference  the means of the groups due to the independent variable  (whenever the term s i g n i f i c a n t i s used, i t i s to  be  understood  that the l e v e l of significance intended i s alpha = 0.05).  The  Tukey test compared a l l pairs of means in a multigroup  18  experiment to determine greater  than  a  i f any  critical  one  value.  pair  was  significantly  If the mean of any group was  s i g n i f i c a n t l y different than the mean of any other group, i t was readily i d e n t i f i e d .  If differences existed, i t was of  interest  to know which of the conditions d i f f e r e d from the others.  In  cases  where  the mean  of  a  group was s i g n i f i c a n t l y  greater than some c r i t i c a l value, the independent plotted  variable  was  against the dependent variable and the closeness of f i t  to a straight l i n e mortality  was  bioassays  One  example  of  this  was  where percent survival was plotted against  copper concentration. manipulation  examined.  These  were  non-linear  plots  and  data  was required since i t was of interest to determine  LC50 and LC95 values from these p l o t s .  Two transformations which were found to be applicable the  Probit  and  the Arcsine  transformations.  The  were  Probit  transformation has been used to straighten cumulative curves changing  the  ordinate  probability scale graduated 1962;  Sokal and Rolf,  variability  of  normal  into  a  in standard deviation units (Finney,  1981).  for the LC50  the cumulative  by  This values  test but  provided  acceptable  the data for LC95 data  contained too much uncertainty to be useable.  One transformation which i s especially appropriate to work with  percentages  i s the arcsine transformation (Zar, 1984). It  stretches out both t a i l s of a d i s t r i b u t i o n  of  percentages  and  19  compresses  the middle.  This manipulation provided improved f i t  of the data to a straight line with acceptable  variability  both LC50 and LC95 values.  i  for  20  Results  The experimental series has been broken up into 8 sections, each. of  which  explore  a  facet  of the response of Tiqriopus  c a l i f o r n i c u s to copper and to manganese.  (i) The Effect of Copper on Egg V i a b i l i t y  In this study the term egg was used for the embryonic of  the  organism,  Copper  after  concentrations  fertilization  ranged  from  and  before  1.0X10"  to  9  Results are presented in Table 1 ( a l l Tables  phase  hatching.  1.0X10"  M.  contained  in  5  are  the appendix).  The  percent of eggs in a cluster which were found to hatch  was high (78.8 to 81.5%) 1.0x10"  M.  6  over  the  copper  range  1.0X10"  There was a marked decrease in the number of eggs  that hatched at the 1.0x10" M l e v e l ; egg survival plunged 5  over  80%  at  1.0X10"  6  M  to  Because of v a r i a b i l i t y in differences  in  the  the  6  and  copper  that  percentage  the was  less data,  than  5% at 1.0X10" M Cu. 5  specific  comments  about 9  Using the Tukey HSD  test  (alpha  =  One-way Anova (alpha = 0.05), the only l e v e l of found  to  significantly  of eggs that hatched was  for the egg stage was 3.6x10" M Cu, 6  affect  1.0X10" M. 5  and  the  9.6X10" M Cu (Table 2, Figure 1 and Figure 2). 6  from  copper response over the range 1.0X10" to  1.0X10" M cannot be made. 0.05)  to  9  the  mean  The LC50 value LC95  value  was  21  ( i i ) The E f f e c t of Copper on Naupliar Survival  Ninety-six  hour LC 50 and LC 95 values are l i s t e d for each  stage in Table 2. is  given  The percent survival of each  naupliar  in Figures 3 to 8 and in Tables 3 to 8.  concentrations ranged from 1.0x10"  10  was  a  suggested  that  s i g n i f i c a n t difference in the effect of copper on  the survival of n a u p l i i over Between  Added copper  to 1.0x10"" M.  Both one way Anova and the Tukey HSD tests there  stage  1.0X10"  and 4.0X10"  6  the  concentration  range  tested.  M Cu the survival of the N-1  6  f i r s t nauplius) dropped from 53.3 to 3.3%.  (the  The rate of survival  decreased rapidly between 1.0X10"  and 2.0x10" M (from 53.3  33.3%),  4.0X10"  6  then  more  linearly  to  6  1.2X10" M Cu and the LC95 was 3.7X10" 6  6  M Cu.  6  The LC50 was  M Cu.  Over the same concentration range, the N-2 appeared more  sensitive  to  from  1.0X10" M to 6  to  be  the lower concentrations of copper and less  sensitive to the higher concentrations than the declined  to  31.7  to  4.0X10"  6  11.7%  M Cu.  over  the  N-1.  Survival  concentration  The LC50 for the N-2 was  range  0.3X10"  6  M Cu and the LC95 value was found to be 4.7x10" M Cu. 6  The the N-1 21.7%  N-3  had  and N-2. over  the  an increased tolerance to copper r e l a t i v e to The rate of survival decreased range 1.0X10"  6  to 6.0X10" M Cu.  rate dropped off rapidly at 7.0X10"  6  6  from  65.0  to  The survival  M (to 15.0% + 5.0) and  was  22  1.7%  +2.9  at 8.0X10'  6  M.  The LC50 and LC95 values were 2.5X10"  6  M Cu and 8.0X10" M Cu. 6  The N-4 of  copper  appeared to be more tolerant to high concentrations than  were the f i r s t three naupliar stages.  survival was high (61.7 to 55.0%) over 3.0X10'  M  6  Cu  but  between  survival dropped from 45.0  the  4.0X10~  to 8.3%.  range  and  6  The  N-4  1.0X10"  to  8.0x10-6  6  M Cu the  The LC50 value was  2.9X10  -6  M Cu and the LC95 was 8.7x10" M Cu. 6  The N-5 stage. 85.0  appeared to be more copper-tolerant  There was a regular decrease  the  N-4  in naupliar survival (from  to 13.3%) over the range 1.0X10"  6  LC50  than  to 8.0X10  M  -6  value  was  4.1X10" M Cu and the LC95 value was  The N-6  was  the most copper-tolerant  6  Cu.  The  8.8X10"  6  M  Cu.  possessed  the  naupliar  stage,  highest naupliar LC50 and LC95 values (Figures 1  and 2).  Survival remained r e l a t i v e l y high (86.7 to 61.7%)  I. 0  4.0X10"  to  II. 7%)  with  M  6  Cu  increasing  8.0X10' M).  but  from  dropped off rapidly (from 53.3  copper  The LC50 value was  6  and  concentration 4.8X10"  6  (5.0x10  -6  to to  M Cu, and the LC95 was  9.6x10" M Cu. 6  There from the N-1 M Cu).  was  a  to N-6  progressive increase in the 96 h LC95 values stage (3.7, 4.7,  8.0,  8.7,  8.8,  and 9.6X10"  There was a similar trend in the LC50 values,  with  6  the  23  exception (1.2,  of  the the N-2  which was lower than the N-1 value  0.3, 2.5, 2.9, 4.1, and 4.8X10~ M Cu). 6  ( i i i ) The Effect of Copper on Copepodite Survival  The survival of each copepodite stage (including the adult) i s given in Figures 9-14 and in Tables 9 to 14. LC95  values are given in Table 2.  varied from 1.0X10"  10  than  the N-6.  Copper concentrations tested  appeared  to be  more  copper-  The LC50 for the C-1 was 5.9x10" M Cu 6  and the LC95 was 1.1x10" M Cu 5  values  LC50 and  to 1.0x10~@ 4 M.  The C-1 ( f i r s t copepodite) tolerant  The  while  were s l i g h t l y lower (4.8X10~  6  the N-6  LC50  M and 9.6x10~  and LC95  M Cu). The  6  C-1 survival remained high (96.7 to 88.3%) at the lower concentrations (from  75.0  (3.0xl0"  6  (1.0X10"  to 8.3%)  to 2.0X10~ M) but decreased steadily  6  6  at  the higher  Over 5  concentrations  5  of the C-2 remained high (95.0 to 71.6%) over the  lower copper concentrations (1.0X10"  l.lxlO"  copper  to 1.0X10" M) (Figure 9).  Survival  10).  copper  the higher  copper  6  to 7.0X10"  concentrations  M)  6  (Figure  (8.0X10~  6  to  M) the C-2 survival dropped markedly from 60.0 to 5.0%.  The LC50 was 7.8X10" M Cu and the LC95 was 1.4X10" M Cu 6  5  which  made the C-2 more tolerant than the C-1.  The  C-3  survival  rate  was high (96.7 to 65.0%) over the  24  copper concentration range I.OxlO" dropped  off  rapidly  (from  concentrations (8.0xl0" 6.5X10"  6  M  Cu  to  6  33.3  to I.OxlO"  s  7.0X10"  to M).  5  M.  6  8.3%)  at  The  LC50  and the LC95 value was 1 .1x10  _5  Survival the  higher  value  M Cu.  was  Both the  LC50 and the LC95 values were less than the C-2 values (7.8x10"  6  M and 1 .4x10" M Cu). 5  The C-4 survival rate was r e l a t i v e l y stable (93.3 to 68.3%) from 1.Ox 10"  6  to 4.0X10" M Cu.  From 5.0xl0"  6  to 1 .1x10" M Cu,  6  5  the rate of survival dropped from 48.3 to 11.7%. C-4  copepodites  Cu.  The LC50 value for this stage was s l i g h t l y  LC50  was  The  LC50 for  6.2X10" M Cu and the LC95 was 1.3X10" M 6  5  value for the C-3 stage (at 6.5x10"  6  less  than  the  M Cu), while the LC95  value was s l i g h t l y greater than the C-3 LC95 (1.1x10" M Cu). 5  The C-5 survival was high (96.7 to 70.0%) over range  1.0X10"  6  to  5.0xl0"  M.  6  There  the  copper  was an abrupt drop in  survival from 50.0 to 11.7% between 7.0X10" and 9.0X10" M 6  The  LC50  value  for this stage was 6.2x10" 5  M Cu.  The LC50 value was close  C-4 LC50 values (6.5X10  LC95 value was less than through  C-4 at I.1x10 ~  5  -6  the  Cu.  M Cu, and the LC95  6  value was 1.1x10" and  6  to  the  C-3  M and 6.2X10" M Cu), whereas the 6  four  previous  stages  (the C-1  M, 1.4X10" , 1.1x10" M, and 1.3xl0" 5  5  5  M  Cu) .  The  survival  plot  of  the  C-6  (adult  copepods)  was  distinguished from a l l others by containing the highest LC50 and  25  LC95  values  as  well  as  by  a  very abrupt decrease in adult  survival (from 33.3 to 0.0%) between Survival I.OxlO to  -5  remained M Cu.  1.5X10"  high  (98.3 to  1.4  and  1.5X10"  M  5  Cu.  80.0%) between 8.0x10" and 6  With increasing copper concentrations  (1.1x10"  5  M) the percent survival dropped from 68.3 to 0.0%.  5  The LC50 value was 1.2X10 significantly  higher  M Cu and the LC95 was 1.5X10"  -5  M Cu,  5  than  the C-5  values  (6.2x10~  6  M and  1.1x10~ M Cu). 5  (iv) The Effect of Copper and Food on Egg Cluster Production  The  percent of previously-berried females which produced a  second cluster when exposed to was determined. the  females  1.0x10"  and I.OxlO"  10  M  8  Cu.  There  was  a  in cluster production below 80% in females exposed to  between  1.0x10"  and  7  females were berried. production  solutions  produced a second cluster when placed in solutions  copper concentrations between 4.0 and 8.0X10" increase  food  Results are presented in Table 15. Over 80% of  containing between decrease  copper-labelled  between  There  then  an  6  and  6  Cu,  I.OxlO" M Cu where 87% of the  was  1.0X10"  M  8  an  abrupt  1.Ox 10"  5  decrease M  in egg  Cu (none of the  copepods in the 1.0x10" M group produced a c l u s t e r ) . 5  (v) The Effect of Copper and Food on Adult Survival  The percent survival for adults exposed to food  (1.0x10"  1 0  to  I.OxlO"  5  M)  was  copper-labelled  determined  using  the  26  technique discussed in section ( i v ) .  The  for  was averaged and the mean  each  of  the three  replicates  percentage  survival  percent survival for each concentration was compared (Table 16). One-way Anova and the Tukey HSD test f a i l e d any  significant  to  establish  that  difference existed between the mean percentage  survival rates for adult copepods at copper concentrations than  I.OxlO'  mortality  M.  5  rates  There between  was  no  significant  copper-labelled  equilibrated in SOW alone between 1.0X10  -7  less  difference in  food  and  and I.OxlO  copper M Cu.  -5  (vi) The Effect of Copper on Naupliar A c t i v i t y Levels  A  series  of  experiments  was  run to determine  i f the  i  a c t i v i t y of the N-1  stage  decreased  concentration.  level  of a c t i v i t y observed was assigned a  The  with  increasing  value between 0 and 10. One-way Anova and the • Tukey were  used  between  copper  HSD  test  to determine i f there was any s i g n i f i c a n t difference  the a c t i v i t y  levels  of  nauplii  exposed  to  the  experimental solutions. Nauplii  exposed  to copper concentrations between 1.0X10'  b  and 1.0X10~ M Cu were active for an average 6  17).  At  1.0X10~  s  (there was  only  M  one  Cu,  of  7.0  s  (Table  the nauplius was active for 0.41 s  nauplius  alive  in the  I.OxlO  -5  M  Cu  bioassay).  The  only  statistically  significant  effect  of copper on  27  naupliar a c t i v i t y levels was found at I.OxlO" M Cu. 5  no  significant  difference  in a c t i v i t y  experimental groups between I.OxlO"  8  There  was  values for any of the  and 1.0x10" M Cu. 6  ( v i i ) The Effect of Manganese on Copper-Stressed  First  Naupliar  Survival  The  f i r s t naupliar stage was used to e s t a b l i s h a manganese  tolerance for one l i f e - h i s t o r y stage of Tigriopus and to examine the hypothesised manganese-copper interaction in a series of 72h  bioassays.  (1.0x10"  6  The  M)  copper  concentration  in experimental  was  solutions  and  concentration was varied between 1.0x10" to 6  rate  of  N-1  survival  held  constant  the manganese  1.0x10""  M.  The  lay between 35.0% +28.2 and 50.0% +14.1  over t h i s concentration range (Table 18).  Concentrations of manganese less than I.OxlO" M 6  used  in preliminary  studies  but  the percent  had been  survival  was  s t a t i s t i c a l l y indistinguishable from control survival (95% +5.0 and  100% +0.0).  The mean percent survival values were compared  using One-way Anova (alpha = 0.05) and the Tukey HSD test (alpha = 0.05).  There was no apparent  s t a t i s t i c a l l y s i g n i f i c a n t effect  of manganese on the copper t o x i c i t y for the N-1 naupliar stage.  ( v i i i ) The Effect of Manganese on Egg Survival  Tigriopus eggs were  exposed  to manganese  concentrations  28  between 1.0X10'  and I.OxlO' M Mn (Table 19). Between 1.0X10'  to  Cu,  9  1.0X10~  consistently  6  M  5  the  rate  survival  high (77.0 to 84.0%); at 1.0X10  rate has dropped to 54.0% +41.3. +17.9,  of  and 74.0% +18.83.  HSD tests, there was  no  Control  to -5  hatching  9  was  M Mn the survival  survival  was  85.0%  Using the One-way Anova and the Tukey statistically  significant  manganese on egg survival at any of the concentrations  effect  of  tested.  T i g r i o p u s LC50 T o l e r a n c e  i  1  egg  N-l  1  N-2  1  N-3  1  N-4  i  i  i  i  i  i  i  i  N-5  N-6  C-i  C-2  C-3  C-4  C-5  C-6  L i f e History Stage  Figure  1. LC50 v a l u e s f o r t h e l i f e - h i s t o r y s t a g e s o f T i g r i o p u s c a l i f o r n i c u s u p o n e x p o s u r e t o c o p p e r s o l u t i o n s (* s i g n i f i e s t h e mean v a l u e and t h e v e r t i c a l b a r r e p r e s e n t s t h e 95% c o n f i d e n c e i n t e r v a l f o r t h i s a n d e v e r y f i g u r e ) .  Tigriopus  • egg  i  «  N-l  N-2  i N-3  i N-4  LC95  i  t  i  i  N-5  N-6  L i f a History  Tolerance  i C-i  i  i  C-2  C-3  1  1  C-4  C-5  1 C-6  Stags  LC95 v a l u e s f o r t h e l i f e - h i s t o r y s t a g e s o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  Copper Tolerance of the N - l  Nauplius  too -  >  c *»  C u c o  c u  L.  4  6  Copper Concentration  8  10  12  (X10/-6M)  F i g u r e 3. A r c s i n e o f percent s u r v i v a l o f the f i r s t n a u p l i a r s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  u>  Copper Tolerance of  the N - 2 N a u p l i u s  100 -  >  c  3  01  u t •  a »• o  • c u c  4  6  Copper C o n c e n t r a t i o n  F i g u r e 4.  8  10  12  (X10/-6M)  A r c s i n e o f percent s u r v i v a l o f the second n a u p l i a r s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  OJ  Copper  Figure  5.  T o l e r a n c e of  the  N-3  Nauplius  A r c s i n e of percent s u r v i v a l o f the t h i r d n a u p l i a r s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  Copper  6.  Tolerance  of  the  N-4  Nauplius  A r c s i n e o f percent s u r v i v a l o f the f o u r t h n a u p l i a r s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  Copper T o l e r a n c e  of  the  N-5  Nauplius  100 -  >  c3  cn +»  c o c  u c  <  4  6  Copper C o n c e n t r a t i o n  8  10  (X10/-6M)  F i g u r e 7. A r c s i n e o f percent s u r v i v a l o f the f i f t h n a u p l i a r stage o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  12  Copper  Tolerance  of  the  N-6  Nauplius  Copper  Tolerance  of  t h e C-1 C o p e p o d i t e  100  >  c 3  01  *J c • uc o •  c o  c <  4  6  Copper C o n c e n t r a t i o n  8  10  12  (x!0/-6M)  F i g u r e 9. A r c s i n e o f percent s u r v i v a l o f the f i r s t c o p e p o d i t e c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  stage o f T i g r i o p u s  CO  Copper  Tolerance  of  the C-2 C o p e p o d i t e  100 -  > > 3  in u  o  a cu c  4  6  Copper C o n c e n t r a t i o n  8  10  12  (X10/-6M)  F i g u r e 10. A r c s i n e o f percent s u r v i v a l o f the second c o p e p o d i t e s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure to copper s o l u t i o n s .  oo  Copper T o l e r a n c e  of  the C-3 C o p e p o d i t e  11. A r c s i n e o f percent s u r v i v a l o f the t h i r d c o p e p o d i t e s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  Copper T o l e r a n c e  of  the C-4  Copepodite  gure 12. A r c s i n e o f percent s u r v i v a l o f the f o u r t h c o p e p o d i t e s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  Copper  Tolerance  of  the C-5 Copepodite  fc  i  i  i  1  1  u  O  2  4  6  8  10  12  Copper C o n c e n t r a t i o n  (X10/-6M)  13. A r c s i n e o f percent s u r v i v a l o f the f i f t h c o p e p o d i t e s t a g e o f T i g r i o p u s c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  Copper  T o l e r a n c e of  the c - 6 Copepodite  100 -  c  at  uc a  »o  c  uc  8  10  Copper C o n c e n t r a t i o n  12  14  16  (xl0/-6M)  F i g u r e 14. A r c s i n e of percent s u r v i v a l o f the s i x t h copepodite c a l i f o r n i c u s upon exposure t o copper s o l u t i o n s .  stage o f T i g r i o p u s  N)  43  Discussion  As  a  splash  pool  inhabitant, Tigriopus c a l i f o r n i c u s i s  subjected to marked changes in water temperature, organic  matter  content.  other organisms (1980),  can  salinity,  and  It survives in part because very few  exist  under  these  conditions.  Dethier  suggests that Tigriopus i s a a poor competitor and, for  this reason, has been ecosystems.  excluded  Although  harsh i t i s usually  from  a l l except  splash  pool  the natural habitat of Tigriopus i s very  pristine;  splash  pools  are  infrequently  subjected to contamination.  This varying  study but  tolerance  natural  to  the hypothesis that exposure to widely would  increase  unnatural as well as natural stresses.  Very high  levels of copper life-history  tested  ecological  in experimental  stages  of  conditions  solutions  Tigriopus  were  containing  various  used as the unnatural  stress with which to test this hypothesis.  The egg stage of Tigriopus i s deposited attached  to  the  into  not  survive.  The  values  were  The adult stage 1.5X10"  5  M  Cu.  sac  3.6X10"  LC50 The  and  to, or  they  egg stage was found to be tolerant to  t o t a l copper levels approaching LC95  egg  mother and must be able to withstand the same  environmental stress which the adults are exposed will  an  6  1.0x10" M  (the LC50  5  M and 9.6x10"  6  LC95  embryonic  values stage  and  the  M Cu: see Table 1).  were  1.2X10"  5  M  and  i s c a r r i e d by, and i s  44  exposed to the same conditions, as the mother u n t i l the eggs are released or hatch.  It i s not known i f the egg sac increases  an  egg's tolerance to the environment.  It  was expected that either the N-1  or the embryonic stage  would have the lowest LC50 and LC95 values since least  developed  they  The N-1  embryonic  l i f e - h i s t o r y stage and would  reserves  and  strength  is the f i r s t post-  have  the  environment  egg  has,  however,  been  to  energy  the  same  that the mother copepod experienced.  sedimentary  debris  and  swim  they  aggregate  a c t i v e l y about.  tolerance of each naupliar stage was determined in a bioassays.  energy  smaller  exposed  Once the eggs are released as n a u p l i i , the  least  of any of the naupliar stages while the  egg stage would be even less developed and have The  the  stages and would be expected to have the least  resistance to environmental changes.  pools.  are  The  in  The copper series  of  N-2 stage appeared to be the most sensitive to  copper with each succeeding stage being more tolerant (Table 2). Each stage past N-1 was physically larger, further developed and more mobile than the previous stages.  It i s interesting to note  that the egg stage was found to be more tolerant of copper the  than  f i r s t four naupliar stages based on LC95 values.  Natural  perturbation  of  the  environment  (such  as  dessication of a tidepool or the introduction of a predator to a community) may cause mortality approaching  50  to  95%  of  the  45  population.  The  probability of population recovery after half  (or 95%) of a population i s lost due likely  be  greater  than  if  the  to  natural  causes  would  cause was unnatural.  A 96 h  bioassay i s a r t i f i c i a l in that i t determines the added  material  hours. the  on  the  affect  of  survival of a group of organisms at 96  It does not take into account the longer term impact  chemical  test period. would  or  an  of  the metal on the bioassay organisms after the  It would be expected that an LC50  or  LC95  value  underestimate the true mortaltiy; an LC50 value would not  mean that half of the population would survive.  The sequence of increasing metal tolerance with developmental  state  hold for  the  copepodite  suggests  the  following  2>C-3>C-1>C-5. copepodite  observed  The  stage  for the naupliar stages does not  stages.  The  LC95  copepodite  appeared  to  be  the  most  copper  than  any  tolerant  C-5 the most sensitive copepodite stage.  The adult (C-6) copepod appeared able to tolerate more  data  trends in copper tolerance: C-6>C-4>C-  C-6  and  increasing  other  stage  significantly  of Tigriopus.  developed and having survived longer than previous  Being more stages,  the  C-6 could be expected to have developed more tolerance to stress than the other stages.  In tolerance copepodite  contrast with  to  this  increasing  which  appears  predicted life-history to  be  the  regime  of  stage  is  increasing the  C-5  most copper-sensitive  copepodite stage, tolerating the least copper of any  copepodite  46  stage  (see Table 2). An explanation of this may be found upon  examination of the LC50 and LC95 values in Table 2 and in Figure 1 and 2. the  There i s no s t a t i s t i c a l l y  significant  difference  tolerance of any copepodite stage from the others.  obtain  only  an  estimate  of  the tolerance  of  in  One may  the various  copepodite stages from these data.  The  LC50  data suggests a d i f f e r e n t sequence than the LC95  data: C-6>C-2>C-3>C-4>C-5>C-1. values  preclude  Again, large standard  the sequencing of the copepodite stages into a  copper-tolerance series of s i g n i f i c a n c e . values 2).  The majority  of  LC50  (C-1 to C-5) l i e between 5.9 (C-1) and 7.8X10" M Cu (C6  As in the LC95 series, the C-6 stage appears to be the most  tolerant copepodite stage (rather than least  deviation  tolerant stage.  the C-5)  and  C-1 the  The C-2 LC50 and LC95 values (7.8x10" M 6  and 1.4X10" . M Cu) are both higher than one might expect  ifit  5  is assumed that each successive l i f e - h i s t o r y stage would be more tolerant  than the previous stage.  The C-2 LC50 and LC95 values  were greater than the C-3, C-4, and C-5 values (6.5X10~ 1.1x10"  5  1.1x10"  5  M and  6  M Cu, 6.2X10" M and 1.3X10- M Cu, and 6.2x10" 6  5  6  M and  M Cu, respectively).  The survival plot of the adult stage was distinguished by a marked jump in both the LC50 and the LC95 values (1.2x10" 1.5X10" M C U ) over the C-5 values (6.2X10" 5  6  M  and  5  M and  1.1x10"  5  M  Cu) as well as by a very abrupt decrease in adult survival (from 33.3  to  00.0%)  between  1.4X10" and 1.5X10" M Cu. 5  5  Survival  47  remained high Cu,  but  to  (98.3  between  1.1X10~ M 5  dropped from  68.3  reached,  further  a  to  between  80.0%)  and  8.0X10"  1.5X10~  5  M  and  6  1.0X10~  M  Cu, percent survival  Once a c r u c i a l level of copper  00.0%.  5  was  increase produced a more pronounced effect  than was expected.  It appears that once the f i r s t copepodite reached  factors  other  stage  has  than the organism's developmental stage  affect i t s a b i l i t y to tolerate copper stress.  These factors may  be associated with or be part of the organism's biology and include  been  metal-complexing  organics  may  such as metallothionein, or  they may be as simple as adsorption of metal  onto  the  copepod  cut i c l e .  Metal  uptake  may  be  active  The  fate  of  metal  californicus. determined,  although  i t has  been  or in  passive this  in  copepod  Tigriopus was  suggested that the copepod  c u t i c l e has been found to be a sink for metal (pers comm. Lewis).  not  A.G.  Once metal i s in the gut of the copepod, i t i s subject  to change in speciation since the low pH of the gut (relative to seawater pH) w i l l cause metals to be  freed  from  chelators  as  ionic species).  Metallothioneins  (low  molecular weight proteins with high  t h i o l content and a high a f f i n i t y for Hg, Cd, initially  found  in  vertebrates:  Cherian  Zn, and  Ag,  and  Cu  Goyer, 1 9 7 8 ;  Roesijadi, 1 9 8 0 ; Overnell and Trewhella, 1 9 7 9 ; Talbot and Magee,  48  1978)  play an important  role in the metabolism and regulation of  essential metals (such as Zn and metalloenzymes,  membranes,  Cu)  which  are  and nucleic acids.  important  in  Metallothionein  production has been found to be induced by the presence of toxic metals ( p a r t i c u l a r l y Cd and Hg), suggesting that the of  this  protein  will  occurrence  increase during periods of metal stress  (metallothioneins contain cysteinyl residues which are e f f i c i e n t metal binding residues, forming three mercaptide bonds per metal atom).  Metallothioneins, or very similar proteins, have also found  in several invertebrate species (such as mussels Mytilus  edulis (Talbot and Magee, 1978), crab Cancer and  been  Trewhella,  paqurus  (Overnell  1979) and S c y l l a serrata (Fisher, 1980), oyster  Crassostrea v i r g i n i c a (Engel and Brouwer, 1982), clam Protothaca staminea (Roesijadi, 1980), blue-green  algae Anacystis  nidulans  and yeast Candida u t i l i s (Fisher, 1980; Lerch, 1980)) suggesting that these groups have the a b i l i t y to bind metals into non-toxic forms.  It copepod  would  thus  Tigriopus  metallothioneins,  be e n t i r e l y possible that the harpacticoid californicus  would  be  able  to  produce  or similar proteins, capable of binding toxic  metals into non-available forms.  Roesijadi (1980) suggests that may  exist  for  metals  below  a  threshold  which  the  concentration production  of  49  metallothionein i s non-detectable and that once is  passed  protein  production  i s increased.  Table 2, there does appear to be a threshold Tigriopus  at  there is an  approximately increase  increasing  the  copper  LC50  stage  There  tolerance  copper  not be present  copper.  with  M  6  developmental  metallothionein may bind  in  6.0x10"  this  threshold  From the data in concentration  Cu; below t h i s l e v e l , tolerance  values  with  suggesting  that  the  in s i g n i f i c a n t quantities  i s , however,  increasing  There  is  developmental  stage  also  thought  to  when  Tigr iopus  1.5x10" M Cu producing  exceeded,  will  allow  at  concentrations  metal  to  Spillover may  be  between  1.4  and  the observed increase in adult mortality  5  (going from 41.7%  that the  be a "saturation capacity" of  over as b i o l o g i c a l l y available metal.  occurring with  at  be functioning to bind metals.  metallothionein, which, "spill"  to  very l i t t l e change in  concentrations above this "threshold" value, suggesting metallothionein may  for  +5.8  survival at 1.3x10 ~  5  to 00.0%  survival at  1.5x10" M Cu). 5  It bioassay  is  not  known  solutions  Experiments  were  seawater and SOW unsuccessful  was run  in  a  form  available  to  Tigriopus.  to determine the a b i l i t y of materials in  to bind  attempt  what proportion of t o t a l metal added to  metals  into  unavailable  forms.  was made to determine the amount of metal  in an available state with the resin column technique of et a l . , (1986).  An  Zorkin  It is believed that the lack of success was  due  50  to  insufficient  resin being used in the columns which may have  lead to the columns becoming  supersaturated with  copper.  As  such, there was no s i g n i f i c a n t difference found in the amount of biologically  available  copper  in  any  of  the  natural  and  synthetic seawater tests.  While i t was not possible to determine the amount of  metal  which was b i o l o g i c a l l y available, known concentrations of copper were equilibrated with ground f i s h food and added to cultures of Tigriopus  in an attempt to determine i f the ingestion of copper  would affect egg production (without a  food  source,  Tigriopus  w i l l not produce eggs).  There was no s i g n i f i c a n t effect of the copper-enriched food on  adult  15).  fecundity  between 1.0x10'  10  and 1.0x10" M Cu (Table 6  The percentage of berried females ranged from  (8.0x10- )  to  8  85.0%  s i g n i f i c a n t effect 1.0x10" M. 5  Between  +10.0  of  (1.0X10" ° 1  copper  on  egg  M  Cu).  cluster  65.0% There  +16.4  The  At t h i s concentration, there was no egg production.  I.OxlO"  9  percentage  at 1.0X10"  6  whether  egg  and 1.0X10  M Cu, there was no discrete  -6  case  of  berried  to zero at I.OxlO" production  past  96  hours,  produce  females dropped from 86.5% 5  M Cu.  It  is  not  known  the f i r s t c l u s t e r was nclu  affected by the copper solutions since the bioassay only  was a  production at  concentration threshold above which Tigriopus would not eggs.  +5.8  lasted  for  not enough time for the release of a second or  51  t h i r d egg sac.  The effect of copper-labelled food on was  Tigriopus  mortality  determined as i t was thought that food containing copper in  a SOW  solution may be more toxic to Tigriopus than copper in SOW  alone.  Survival  compared  to  in  medium  containing  survival in SOW  exposed  to  and  metal  was  solutions containing copper alone.  There was no s i g n i f i c a n t difference in organisms  food  copper  adult  solutions  survival  between  containing  metal-  equilibrated food and those exposed to copper solely in solution over the range 1.0x10"  10  to KOxlO'  5  M Cu (Table 15).  This result i s surprising since the pH of the is  lower  result in released  than  the  metal  pH of SOW  bound  to  the  copepod  or natural seawater. organic  food  gut  This would  material  being  in the lower pH environment: this would be expected to  increase adult mortality.  An apparent r e l a t i o n s h i p between copper naupliar  a c t i v i t y was studied with N-1  concentration  nauplii being exposed to  copper over the range 1.0x10~ to I.OxlO"  5  copper  reduction  8  which  a c t i v i t y was  produced  a  significant  and  M.  The only l e v e l of in  naupliar  1.0X10~ M Cu. 5  While manganese has been found to reduce copper t o x i c i t y to some  species  of  phytoplankton, (Sunda et a_l., 1981; Sunda and  Huntsman, 1983; Stauber and Florence,  1985)  i t has  not  been  52  documented  to  produce the same result in aquatic animals.  addition of manganese to copper solutions did not reduce  copper  toxicity  to  Tiqriopus  copper  toxicity  to  an  aquatic  significantly  N-1 nauplii (Table 18).  There was some indication, however, that the  The  manganese  may  reduce  animal such as Tigriopus  c a l i f o r n i c u s (at a non-significant l e v e l of confidence).  Although ameliorate  the  copper  time, i t i s thought manganese  uptake  mechanism  by  which  manganese  acts  to  t o x i c i t y in phytoplankton i s unknown at t h i s to site  hydroxide sorption of  be  either  (Sunda  copper  (Stauber and Florence, 1985).  through  et  onto  competition  a l . , 1981) the  surface  at a  or  manganese  of  the  cell  The l a t t e r mechanism could reduce i  t o t a l copper levels in both plants and animals.  The  addition of manganese to bioassay solutions containing  eggs of Tigriopus did not reveal the existence of a threshold of response over the manganese concentration range of 1.0x10"  5  which  I.OxlO"  9  to  M (Table 19). There was no concentration of manganese  produced  a  statistically  significant  change  in  the  percentage of eggs found to be v i a b l e .  It  i s interesting to compare the tolerance of Tiqriopus, a  splashpool inhabitant, to a planktonic calanoid copepod such  as  Euchaeta japonica whose metal tolerance has been studied in some detail  (Lewis et a_l. , 1973).  This would allow one to determine  i f exposure to fluctuating environmental conditions  may  affect  53  the  response of an organism's l i f e - h i s t o r y stages r e l a t i v e to an  organism that does not know a harsh environment. specialist  at  coping  with  stress  Tigriopus i s a  in i t s natural  environment  while Euchaeta i s not exposed to the same environmental as  open  marine  waters  do  not  undergo the rapid and extreme  changes in conditions found in i n t e r t i d a l waters. not  changes  Euchaeta does  have to cope with major changes in i t s environment since i t s  environment  i s stable.  One would thus not expect Eucheata to be  as stress tolerant as Tigriopus.  This work with Tigriopus c a l i f o r n i c u s has shown that i t can tolerate more copper than Euchaeta tolerance  of  demonstrated naupliar  each that  (with  a  life-history the  most  copper  japonica. stage  sensitive  Calibrating  the  of Tiqriopus to copper stage  was  the  LC50 of 1.1x10" M Cu).  first  Although LC  6  values were not calculated for Euchaeta the copper tolerance its  egg  stage  Physiological splashpools;  was  adaptations the  The  allow  production  organism's response to forms.  1.0xl0"  bind  physiological  of  B  M  (Lewis  et  Tigriopus  to  a_l., 1973). survive  metallothioneins may  available  metals  adaptations  are  evolutionary adaptations and the metallothionein  of  into  in  be  an  non-toxic  thought  to  be  production  is  believed to be a response to anthropogenic influences.  The  tolerance to copper observed in the egg, naupliar, and  copepodite l i f e - h i s t o r y stages of exposure  Tigriopus  californicus  upon  to copper solutions, together with the adult tolerance  54  to  copper-labelled  food  experiments, and the N-1 experiments,  may  be  in  the  fecundity  and  mortality  tolerance in the a c t i v i t y and manganese interpreted  as support of the hypothesis  that exposure to stress increases tolerance to stress.  The with  fact that Tigriopus survives in  inhospitable  conditions  tolerant organism. weaker, or less  It may  tolerant,  in  niche  and  copes  nature suggests that i t i s a  be that natural selection removes the organisms  and  stress-resistant individuals survive. natural  its  only  the  flexible,  This a b i l i t y to cope with  stress does not suggest that i t would be at a l l able to  tolerate  anthropogenic  demonstrated,  sources  of  stress.  It  has  been  however, that Tigriopus can tolerate severe metal  stress in the form of copper concentrations  far beyond those  it  would be l i k e l y to encounter in nature.  Since Tigriopus has been demonstrated to be stress-tolerant and  is  i t may  be  organisms in  the  however,  readily cultured in the laboratory, i t was useful are  for  bioassay  applications.  thought that  Most  bioassay  tolerant to stress or they could not be cultured  laboratory. revealed  Preliminary  experiments  that Tigriopus was  in most bioassay work.  with  copper,  too tolerant to be useful  55  Conclusion  This work attempted to v e r i f y the hypothesis that to  widely  varying  but  increase an organism's to  natural  ecological  exposure  conditions  tolerance to unnatural stress.  would  Exposure  high concentrations of copper was selected as the agent with  which to induce a stress response in Tigriopus c a l i f o r n i c u s .  The response of each l i f e history copper  stage  of  was determined and LC50 and LC95 values were calculated.  The egg stage i s the f i r s t l i f e - h i s t o r y stage and to  be  Tigriopus to  one  of  the  was  expected  most sensitive stages; i t i s also the only  l i f e - h i s t o r y stage which i s exposed to  the  same  conditions as the adult u n t i l i t i s released.  environmental  The egg stage was  approximately as copper-tolerant as the N-4.  The  egg sac i s actually a gel or coat of chitinous protein  which binds the eggs together and t h i s material i s thought to be highly selective for metals.  When egg clusters are  exposed  to  metals, the metal solution may wash through the gel with some of the metal being adsorbed or absorbed into the coating.  The adult (LC95 = 1.5x10" M Cu) was approximately 50% more 5  tolerant than the egg stage (LC95 = 9.6x10" general  6  M Cu). There was a  increase in the a b i l i t y of Tigriopus to tolerate copper  with increasing developmental the last copepodite.  stage from the f i r s t  naupliar to  56  It with  was  thought  ground  fish  that  copper  food  might  copper e q u i l i b r a t e d with both  with  its  food  significant  effect  production,  except  difference food, in  adult  was that  the  copper  the  naupliar  upon  with  suggested the  M.  5  and  activity  from  nor  of  a  tidepool  toxicity thought  masked some of  those  was  the  some  the  has  be  food  was  no  egg  any  difference  exposed  to  copper  copper-labelled  was  a  of  to vary I.OxlO  function  the  of  However,  significantly - 8  and  stress associated increased  food.  Tigriopus  were e x p o s e d .  between  case  copper-labelled  observations  found  no  significant  significant  animals  ingested  T h e r e was  on  there  than  I.OxlO"  with  6  life  tolerance  of  stress.  suggestion  to T i g r i o p u s , that  not  natural  environment  would  exhibited  solutions  to T i g r i o p u s  solution.  to which they  level  that  to unnatural  T h e r e was  T h e r e was  activity  to e q u i l i b r a t e  containing  from p r e l i m i n a r y  to copper  in  metal  those c o n t a i n i n g  concentration  i s apparent  is  M,  - 5  between  level  It  It  as  absorbed  I.OxlO  at  SOW  M.  copper  alone  more t o x i c  copper-labelled  I.OxlO""  at  exposure  Tigriopus  of  mortality  equilibrated  nauplii  and  be  in s u r v i v a l in solutions  except  It  SOW  solutions allowed  the  but  copper  manganese  that not  manganese t o any  tolerance  effect.  of  could  reduce  significant  degree.  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An ion-  exchange procedure for quantifying b i o l o g i c a l l y active copper in sea water.  Anal.  Chim.  Acta, 183: 163-177.  64  APPENDIX  Table 1.  The effect of copper on  the  viability  of  c a l i f o r n i c u s eggs.  Total Added Copper  Average  Standard  Concentration  Survival  Deviat ion  (M)  (%)  I.OxlO-  9  78.8  13.0  I.OxlO"  8  86.8  8.5  1.0x10"  7  74.0  22.1  1.0X10"  6  81.5  18.7  1.0X10"  5  3.0  7.0  Control  85.0  17.9  Tigriopus  65  Table  2.  LC50  and LC95 values for each l i f e - h i s t o r y stage of  Tigriopus c a l i f o r n i c u s (including  L i f e History Stage  LC50 U 1 0 " M Cu) 6  95% confidence l i m i t s ) .  LC95 (X10'  6  M Cu)  N-1  1.15 (0. 62-1 .60)  3.72 (3.28-4.24)  N-2  0.28 (-1 .92-1 .95)  4.72 (2.96-7.95)  N-3  2.46 (0. 88-3. 87)  8.03 (6.57-9.80)  N-4  2.92 (1. 49-4.23)  8.69 (7.28-10.43)  N-5  4.12 (3. 28-4.96)  8.79 (7.87-9.85)  N-6  4.75 (3. 48-6. 05)  9.'55 (8.12-11.33)  C-1  5.88 (4. 57-7. 20)  11.0 (9.60-12.63)  C-2  7.82 (4. 52-1 1.45)  13.7 (10.23-18.81 )  C-3  6.54 (4. 96-8. 16)  11.2 (9.52-13.26)  C-4  6.19 (3. 94-8. 50)  12.5 (10.13-15.64)  C-5  6.15 (4. 50-7. 88)  10.6 (8.82-12.94)  C-6  12.00 (1 .00-1 .40)  15.1 (1.32-1.78)  egg  3.63 (1 .28-6. 17)  9.60 (6.97-13.1)  66  Table 3.  Percent survival of Tiqriopus c a l i f o r n i c u s N-1 nauplii  upon exposure to copper  solutions.  LC50 = 1.2xl0"  6  = 3.7x10" M Cu. 6  Total Added Copper Concentration (X10-  6  M)  Percent Range Survival  Average  Standard  Value  Deviation  (Percent)  1.0  45-60  53.3  7.6  2.0  25-45  33.3  10.4  3.0  5-15  11.7  5.8  4.0  0-10  3.3  5.8  90-100  95.0  14.1  Control  M Cu.  LC95  67  Table 4.  Percent survival of Tigriopus c a l i f o r n i c u s N-2 nauplii  upon exposure to copper solutions.  LC50 = 0.28X10  = 4.72X10" M Cu. 6  Total Added Copper  Percent  Concentration (X10"  6  M)  Range Survival  Average Value  Standard Deviation  (Percent)  0.2  50-70  61 .7  10.4  0.4  50-60  55.0  5.0  0.6  40-55  48.3  7.6  0.8  25-45  33.3  10.4  1 .0  30-35  31.7  2.9  2.0  1 5-30  23.3  2.6  3.0  20-25  21.7  2.9  4.0  5-20  11.7  7.6  Control  85-95  90.0  14. 1  -6  M Cu.  LC95  68  Table 5.  Percent survival of Tigriopus c a l i f o r n i c u s N-3 nauplii  upon exposure to copper solutions. = 8.03X10"  6  M  LC50 = 2.46x10  _6  Cu.  Total Added Copper Concentration (X10'  s  M)  Percent  Average  Range  Value  Survival  Standard Deviation  (Percent)  1.0  50-85  65.0  10.0  2.0  45-55  50.0  5.0  3.0  40-50  45.0  5.0  4.0  30-40  35.0  5.0  5.0  20-35  26.7  2.9  6.0  10-30  21.7  2.9  7.0  10-20  15.0  5.0  8.0  0-5  1.7  2.9  Control  80-85  82.5  7.1  M Cu.  LC95  69  Table 6.  Percent survival of Tigriopus c a l i f o r n i c u s N-4 nauplii  upon exposure to copper solutions.  LC50 = 2.92x10" M Cu. 6  = 8.69X10" M Cu. 6  Total Added Copper Concentration (x10"  6  M)  Percent Range Survival  Average  Standard  Value  Deviat ion  (Percent)  1.0  60-65  61.7  18.3  2.0  55-60  58.3  5.0  3.0  50-60  55.0  5.0  4.0  40-50  45.0  5.0  5.0  25-40  31.7  2.9  6.0  15-30  23.3  2.9  7.0  0-15  10.0  5.0  8.0  5-15  8.3  2.9  Control  85-90  87.5  7.1  LC95  70  Table 7.  Percent survival of Tigriopus c a l i f o r n i c u s n-5 nauplii  upon exposure to copper solutions. = 8.79X10"  6  M  LC50 = 4.12x10" M Cu. 6  Cu.  Total Added Copper Concentration (X10-  6  M)  Percent  Average  Range  Value  Survival  Standard Deviation  (Percent)  1.0  75-95  85.0  10.0  2.0  60-85  75.0  13.2  3.0  45-75  60.0  15.0  4.0  40-75  56.7  17.7  5.0  30-45  36.7  7.6  6.0  20-35  26.7  7.6  7.0  10-25  15.0  8.7  8.0  5-15  13.3  7.6  Control  85-95  90.0  14.1  LC95  71  Table 8.  Percent survival of Tigriopus c a l i f o r n i c u s N-6 nauplii  upon exposure to copper solutions.  LC50 = 4.75X10' M Cu. 6  = 9.55X10" M Cu. 6  Total Added Copper Concentration (X10-  6  M)  Percent  Average  Range  Value  Survival  Standard Deviation  (Percent)  1.0  80-95  86.7  7.6  2.0  70-75  73.3  2.9  3.0  70-85  76.7  7.6  4.0  55-70  61.7  7.6  5.0  50-55  53.3  2.9  6.0  25-45  33.3  10.3  7.0  20-35  26.7  7.6  8.0  10-15  11.7  2.9  Control  80-85  82.5  4.5  LC95  Table  9.  Percent  survival  of  Tigriopus  copepodites upon exposure to copper solutions. M Cu.  californicus  LC50 = 5.88x10  LC95 = 1.10x10" M Cu. 5  Total Added Copper Concentration (X10"  6  M)  Percent Range Survival  C  Average  Standard  Value  Deviation  (Percent)  1.0  95-100  96.7  2.9  2.0  85-90  88.3  2.9  3.0  70-80  75.0  5.0  4.0  55-75  65.0  5.0  5.0  40-65  53.3  12.6  6.0  40-50  43.3  5.8  7.0  30-50  36.7  11.6  8.0  30-45  36.7  7.6  9.0  15-25  20.0  5.0  10.0  5-10  8.3  2.9  Control  85-95  90.0  14.1  73  Table  10.  copepodites M Cu.  Total  Percent  survival  upon e x p o s u r e  LC95 = 1.37x10"  5  of  Tigriopus  t o copper  solutions.  californicus  LC50 = 7.82X10"  M Cu.  Added  Copper Concentration (X10"  6  M)  Percent  Average  Range  Value  Survival  Standard Deviat ion  (Percent)  1.0  85-100  95.0  8.7  2.5  85-100  91.7  7.6  4.0  75-85  80.0 -  5.0  5.0  65-75  70.0  5.0  6.0  65-85  76.7  10.4  7.0  65-75  71.7  5.7  8.0  55-65  60.0  5.0  9.0  45-55  50.0  5.0  10.0  25-35  28.3  5.8  1 1 .0  0-1 0  5.0  5.0  90-100  95.0  14.1  Control  C-2 6  Table  11.  Percent  survival  of  Tigriopus  copepodites upon exposure to copper solutions. M Cu.  LC95 = 1.12X10-  5  californicus  LC50 = 6.54x10  M Cu.  Total Added Copper Concentration (X10"  6  M)  Percent  Average  Range  Value  Survival  Standard Deviation  (Percent)  1.0  90-100  96.7  5.8  3.0  85-90  86.7  2.9  5.0  70-80  75.0  5.0  7.0  50-60  65.0  5.0  8.0  30-35  33.3  2.9  9.0  15-25  20.0  5.0  10.0  5-10  8.3  2.9  85-100  92.5  21.2  Control  C  Table  12.  Percent  survival  of  Tigriopus  copepodites upon exposure to copper solutions. M Cu.  LC95 = 1.25X10"  5  californicus  LC50 = 6.19x10  M Cu.  Total Added Copper  Percent  Average  Concentration  Range  Value  (xl0'  6  M)  Survival  Standard Deviat ion  (Percent)  1.0  85-100  93.3  7.6  2.5  70-90  81.6  10.4  4.0  60-80  68.3  10.4  5.0  40-55  48.3  7.6  7.5  30-40  35.0  5.0  10.0  25-30  26.7  2.9  11.0  0-20  11.7  10.4  100  100.0  00.0  Control  C  Table  13.  Percent  survival  of  Tigriopus  copepodites upon exposure to copper solutions. M Cu.  LC95 =  1.06X10"  5  californicus  LC50 = 6.15x10  M Cu.  Total Added Copper Concentrat ion (X10"  6  M)  Percent  Average  Standard  Range  Value  Deviation  Survival  C  (Percent)  1.0  95-100  96.7  2.9  3.0  80-90  85.0  5.0  5.0  65-75  70.0  5.0  7.0  45-55  50.0  5.0  8.0  20-30  25.0  5.0  9.0  5-20  11.7  7.6  Control  90-95  92.5  7.1  Table  14.  Percent  survival  of  Tigriopus  copepodites upon exposure to copper solutions. M Cu.  californicus  LC50 = 1.20x10  LC95 =1.51x 1 0" M Cu. 5  Total Added Copper Concentration (X10"  6  M)  Percent Range Survival  Average  Standard  Value  Deviation  (Percent)  8.0  95-100  98.3  2.9  9.0  90-95  93.3  2.9  10.0  75-85  80.0  5.0  11.0  60-75  68.3  7.6  12.0  55-60  56.7  2.9  13.0  35-45  41.7  5.8  14.0  25-40  33.3  7.6  15.0  00-00  00.0  0.0  100.0  100.0  0.0  Control  C  78 Table  15.  The e f f e c t s of c o p p e r - e q u i l i b r a t e d food on Tiqriopus  c a l i f o r n i c u s egg production.  Copper  Percent  Concentration (M)  Deviation  Berried  1.0x10" ° 1  1.0x10"  Females  Standard  9  85.0 81.5  10.0 17.1  3.0X10"  9  80.0  5.9  6.0X10"  9  82.5  15.2  I.OxlO"  8  84.0  10.0  4.0X10"  8  76.5  17.1  8.0X10"  8  65.0  5.8  80.0  14.1  1.0x10"  7  3.0X10"  7  77.5  12.6  6.0X10"  7  83.5  7.0  I.OxlO"  6  86.5  16.4  I.OxlO"  5  00.0  00.0  81.3  15.5  Control  79  Table  16.  The effect of copper-equilibrated food  on  c a l i f o r n i c u s adult mortality.  Copper  Percent  Standard  Concentration  Survival  Deviation  (M)  (%)  I.OxlO-  85.0  10.0  9  82.5  17.0  3.0x10"  9  82.5  5.0  6.0xl0'  9  95.0  10.0  8  95.0  5.8  8  87.5  9.6  8.0x10-8  95.0  5.8  I.OxlO"  7  100.0  0.0  3.0X10-  7  100.0  0.0  6.0x10"  7  92.5  9.6  1.0x10"  6  95.0  5.8  1.0x10"  82.5  24.5  Control  96.3  5.2  I.OxlO-  I.OxlO" 4.0xl0-  5  10  Tigriopus  80  Table  17.  The  effect of copper on a c t i v i t y levels of the N-1  stage of Tigriopus c a l i f o r n i c u s .  Copper  Time  Standard  Concentrat ion  Act ive  Deviation  (M)  (s)  I.OxlO" 1.0X10" 1.0X10" 1 . 0 X 1 0 "  Control  8  6.87  3.7  7  7.75  1.6  6  6.33  3.3  5  0.41  2.0  9.66  4.8  Table 18.  The Effects of Copper and Manganese  the N-1 of Tigriopus c a l i f o r n i c u s . a l l tests was 1.0x10  -6  on  Survival  The copper concentration  M.  Manganese  Percent  Standard  Concentration  Naupliar  Deviation  (M)  Survival  1.0x10"  6  35.0  28.2  1.0x10-=  50.0  14.1  1.0x10-*  40.0  28.2  Control  97.5  7.1  82  Table  19.  The Effect of Manganese on The V i a b i l i t y of Tigriopus  c a l i f o r n i c u s Eggs (no copper added).  Total Added Manganese Concentration (M)  Average  Standard  Survival  Deviation  (%)  I.OxlO-  9  84.0  8.4  I.OxlO"  8  77.0  17.8  7  77.0  15.6  6  82.0  10.7  1.0x10"  5  54.0  41.3  Control  85.0  17.9  1.0X10"  I.OxlO-  

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