Patricia Gillis

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

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. H+ Gill or Biotic Ligand Ca2+ Na+ Na+ organic complexes H+ Mn+ NOM M . inorganic complexes Ca2+ HCO3- active influx Cl- renal excretion chlorides hydroxides carbonates sulphides passive efflux Chemistry Physiology Toxicology Regulatory needs Introduction - Biotic Ligand Model Paquin et al., 2002. Comp. Biochem. Physiol.

7. H+ Gill or Biotic Ligand Ca2+ Na+ Na+ organic complexes H+ Mn+ NOM M . inorganic complexes Ca2+ HCO3- active influx Cl- renal excretion chlorides hydroxides carbonates sulphides passive efflux Chemistry Physiology Toxicology Regulatory needs Introduction - Biotic Ligand Model Paquin et al., 2002. Comp. Biochem. Physiol.

8. Part I Assessing the Copper Sensitivity of Glochidia A B

9. 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. A B

10. 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 10oC (in order to prevent the release of glochidia). • Mussels were fed a commercial shellfish diet (4.6x106 cells mL-1 day-1). A B

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

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

13. A. ligamentina 40 L. siliquoidea L. recta E. torulosa rangiana E. triquetra L. fasciola 30 O. subrotunda P. fasciolaris V. fabalis Copper EC50 (mg/L) 20 10 0 Species Acute Copper Toxicity in Glochidia Endangered (Canadian)species to right of dashed line Exposures conducted in reconstituted soft water Error bars represent 95% confidence intervals around the EC50s Gillis et al., AQ TOX. In Press

14. Water Composition Affects Copper Sensitivity (E.triquetra) Soft Water = 40 mg CaCO3/L, Hard Water = 165 mg CaCO3/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. • Input pH, DOC, Ca, Mg, Na, K, SO4, Cl, and alkalinity into BLM. • When a parameter varied (e.g. pH 7.4-7.8) entered the full range. • Criterion Maximum Concentrations (CMC) were derived for each type of exposures. • Compare BLM derived criteria to the observed 24 h Cu EC50s.

16. Comparison of Water Quality Regulations and Copper Sensitivity of Glochidia aCMC = Critical Mean Concentration as derived from the USEPA’s BLM

17. Comparison of Water Quality Regulations and Copper Sensitivity of Glochidia aCMC = Critical Mean Concentration as derived from the USEPA’s BLM bCCME = Canadian Council of Ministers of the Environment Copper Guideline for hardness up to 120 mg CaCO3/L equivalents = 2 µg/L, for hardness 120-180 mg CaCO3/L equivalents = 3 µg/L

18. Part I - Conclusions • Glochidia are very sensitive to copper in reconstituted soft water with the EC50s of 2 endangered species <10 µg/L. • Copper sensitivity was significantly affected by water composition: increased hardness and additions of Aldrich Humic acid decreased copper toxicity. • 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. Part II BLM-style Exposures with Glochidia and Copper

20. Part II - Conclusions • Sensitivity to Cu decreases with increasing water hardness. →EC50 3 fold higher in moderately-hard compared to very soft water • Natural DOC provides significant protection from Cu toxicity. →EC50 5 fold higher when DOC increased from <1 to 4.4 mg C/L • An increase in pH reduces Cu sensitivity. →EC50 >2 fold higher at pH 7.7 compared to pH 6.0 • 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