1. Patricia Gillis Copper Sensitivity in Glochidia: Assessing the Effect of Water Composition on the Sensitive Larvae of Freshwater Mussels

2. Introduction - Freshwater Mussels 70% of North American freshwater mussels are either endangered, threatened or in decline Their decline is attributed to a number of factors including: - loss of habitat - decline in fish host populations - invasive species - over harvesting - environmental pollution

3. Introduction - Lifecycle of Freshwater Mussels

4. Introduction – Contaminants as Threats to Mussels  Field surveys find that populations are skewed towards older individuals – failed recruitment?  Early life stages most sensitive to contaminants.  Recovery strategies for endangered mussels indicate the need to assess the impact of waterborne contaminants.

5. Introduction – Copper and the BLM  Like all molluscs - freshwater mussels are especially sensitive to copper.  Concerns whether current water quality regulations for copper would protect the sensitive early life stages.  Copper bioavailability is influenced by water composition → Biotic Ligand Model (BLM).

6. Introduction - Biotic Ligand Model chlorides hydroxides carbonates sulphides M n+ M Na + NOM Ca 2+ H+H+ ChemistryPhysiology Toxicology Regulatory needs Gill or Biotic Ligand Na + H+H+ Ca 2+ Cl - HCO 3 - organic complexes inorganic complexes active influx passive efflux renal excretion. Paquin et al., 2002. Comp. Biochem. Physiol.

7. Introduction - Biotic Ligand Model chlorides hydroxides carbonates sulphides M n+ M Na + NOM Ca 2+ H+H+ ChemistryPhysiology Toxicology Regulatory needs Gill or Biotic Ligand Na + H+H+ Ca 2+ Cl - HCO 3 - organic complexes inorganic complexes active influx passive efflux renal excretion. Paquin et al., 2002. Comp. Biochem. Physiol.

8. AB Part I Assessing the Copper Sensitivity of Glochidia

9. AB Part I - Goals  Examine copper sensitivity in glochidia from both common and endangered species of freshwater mussels.  Investigate the influence of water composition (hardness, DOC) on the sensitivity of glochidia to copper.  Determine if current water quality regulations for copper protect glochidia.

10. AB Methods - Collection and Holding of Mussels  Gravid mussels were collected under a Species at Risk Permit (SECT 06 SCI 007).  Mussels were held in the lab at 10 o C (in order to prevent the release of glochidia).  Mussels were fed a commercial shellfish diet (4.6 x 10 6 cells mL -1 day -1 ).

11. AB 1) Collect glochidia from mature mussel 3) Assess glochidia viability→EC50 (if they close - they are viable) + salt = 2) Expose glochidia to a range of copper concentrations (vary water composition) Methods - Conducting Toxicity Tests with Glochidia ASTM 2006. Guidance for Conducting Laboratory Toxicity Tests with Freshwater Mussels 0.5 mm

12. Acute Copper Toxicity in Glochidia Error bars represent 95% confidence intervals around the EC50s Exposures conducted in reconstituted soft water Gillis et al., AQ TOX. In Press Species 0 10 20 30 40 Copper EC50 (  g/L) L. siliquoidea L. recta A. ligamentina

13. Acute Copper Toxicity in Glochidia Endangered (Canadian) species to right of dashed line Error bars represent 95% confidence intervals around the EC50s Exposures conducted in reconstituted soft water Gillis et al., AQ TOX. In Press

14. Water Composition Affects Copper Sensitivity (E. triquetra) Soft Water = 40 mg CaCO 3 /L, Hard Water = 165 mg CaCO 3 /L ‘Low’ Dissolved Organic Carbon (DOC) = 0.3-0.6 mg C/L, ‘High’ DOC = 1.6-1.7 mg C/L *Bars with different letters are significantly different from each other Gillis et al., AQ TOX. In Press

15. Comparison of Water Quality Regulations and Copper Sensitivity of Glochidia Employed the USEPA online version of the copper BLM to derive exposure-specific water quality criteria. 1) Input pH, DOC, Ca, Mg, Na, K, SO 4, Cl, and alkalinity into BLM. 2) When a parameter varied (e.g. pH 7.4-7.8) entered the full range. 3) Criterion Maximum Concentrations (CMC) were derived for each type of exposures. 4) Compare BLM derived criteria to the observed 24 h Cu EC50s.

16. Comparison of Water Quality Regulations and Copper Sensitivity of Glochidia ExposureCMC a (µg/L) Observed EC50 (µg/L) Soft water (n=9) 0.7 - 1.27 – 36 Hard Water (n=2) 2.8 - 3.418, 50 Low DOC (n=2) 1.6 - 4.431, 44 High DOC (n=2) 11.6 - 12.480, >800 a CMC = Critical Mean Concentration as derived from the USEPA’s BLM

17. Comparison of Water Quality Regulations and Copper Sensitivity of Glochidia ExposureCMC a (µg/L) Observed EC50 (µg/L) CCME b (µg/L) Soft water (n=9) 0.7 - 1.27 – 362 Hard Water (n=2) 2.8 - 3.418, 503 Low DOC (n=2) 1.6 - 4.431, 442 High DOC (n=2) 11.6 - 12.480, >8002 a CMC = Critical Mean Concentration as derived from the USEPA’s BLM b CCME = Canadian Council of Ministers of the Environment Copper Guideline for hardness up to 120 mg CaCO 3 /L equivalents = 2 µg/L, for hardness 120-180 mg CaCO 3 /L equivalents = 3 µg/L

18. Part I - Conclusions 1)Glochidia are very sensitive to copper in reconstituted soft water with the EC50s of 2 endangered species <10 µg/L. 2)Copper sensitivity was significantly affected by water composition: increased hardness and additions of Aldrich Humic acid decreased copper toxicity. 3)Glochidia are protected by current copper water quality regulations, but protection may only be marginal in soft waters with very low concentrations of DOC.

19. BLM-style Exposures with Glochidia and Copper Part II

20. Part II - Conclusions 1)Sensitivity to Cu decreases with increasing water hardness. → EC50 3 fold higher in moderately-hard compared to very soft water 2)Natural DOC provides significant protection from Cu toxicity. → EC50 5 fold higher when DOC increased from <1 to 4.4 mg C/L 3)An increase in pH reduces Cu sensitivity. → EC50 >2 fold higher at pH 7.7 compared to pH 6.0 4)Glochidia can only endure limited range of water composition. → Balanced water chemistry appears to be important

21. Acknowledgments Scientific Input and Lab Assistance Josef Ackerman, Gerry Mackie, Chris Wood, Rebecca Mitchell, Astrid Schwalb, Kelly McNichols Research Funding Copper Development Association Department of Fisheries and Oceans (Species at Risk) World Wildlife Fund (Endangered Species Recovery Fund)

22. Thank-you