Ning Wang

Differential exposure, duration, and sensitivity of unionoidean bivalve life stages to environmental contaminants

Abstract Freshwater mussels (superfamily Unionoidea) are in serious global decline and in urgent need of protection and conservation. The declines have been attributed to a wide array of human activities resulting in pollution and water-quality degradation, and habitat destruction and alteration. Linkages among poor water quality, pollutant sources, and mussel decline in rivers and streams have been associated with results of laboratory-based tests of specific pollutants. However, uncertainties remain about the relationship of laboratory data to actual contaminant exposure routes for various mussel species, life stages, and in the habitats occupied during these exposures. We evaluated the pathways of exposure to environmental pollutants for all 4 life stages (free glochidia, encysted glochidia, juveniles, adults) of unionoidean mussels and found that each life stage has both common and unique characteristics that contribute to observed differences in exposure and sensitivity. Free glochidia typically are exposed only briefly (e.g., seconds to days) through surface water, whereas adults sustain exposure over years to decades through surface water, pore water, sediment, and diet. Juveniles live largely burrowed in the sediment for the first 0 to 4 y of life. Thus, sediment, pore water, and diet are the predominant exposure routes for this life stage, but surface water also might contribute to exposure during certain periods and environmental conditions. The obligate parasitic stage (encysted glochidia stage) on a host fish might be exposed from surface water while partially encysted or from toxicants in host-fish tissue while fully encysted. Laboratory methods for testing for acute and chronic exposures in water have advanced, and toxicant-specific information has increased in recent years. However, additional research is needed to understand interactions of life history, habitat, and long-term exposure to contaminants through water, pore water, sediment, and diet so that the risks of environmental exposures can be properly assessed and managed.

Chronic toxicity of copper and ammonia to juvenile freshwater mussels (unionidae)

The objectives of the present study were to develop methods for conducting chronic toxicity tests with juvenile mussels under flow-through conditions and to determine the chronic toxicity of copper and ammonia to juvenile mussels using these methods. In two feeding tests, two-month-old fatmucket (Lampsilis siliquoidea) and rainbow mussel (Villosa iris) were fed various live algae or nonviable algal mixture for 28 d. The algal mixture was the best food resulting in high survival (>or=90%) and growth. Multiple copper and ammonia toxicity tests were conducted for 28 d starting with two-month-old mussels. Six toxicity tests using the algal mixture were successfully completed with a control survival of 88 to 100%. Among copper tests with rainbow mussel, fatmucket, and oyster mussel (Epioblasma capsaeformis), chronic value ([ChV], geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) ranged from 8.5 to 9.8 microg Cu/L for survival and from 4.6 to 8.5 microg Cu/L for growth. Among ammonia tests with rainbow mussel, fatmucket, and wavy-rayed lampmussel (L. fasciola), the ChV ranged from 0.37 to 1.2 mg total ammonia N/L for survival and from 0.37 to 0.67 mg N/L for growth. These ChVs were below the U.S. Environmental Protection Agency 1996 chronic water quality criterion (WQC) for copper (15 microg/L; hardness 170 mg/L) and 1999 WQC for total ammonia (1.26 mg N/L; pH 8.2 and 20 degrees C). Results indicate that toxicity tests with two-month-old mussels can be conducted for 28 d with >80% control survival; growth was frequently a more sensitive endpoint compared to survival; and the 1996 chronic WQC for copper and the 1999 chronic WQC for total ammonia might not be adequately protective of the mussel species tested. However, a recently revised 2007 chronic WQC for copper based on the biotic ligand model may be more protective in the water tested.

Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (unionidae)

The objective of the present study was to determine acute toxicity of copper, ammonia, or chlorine to larval (glochidia) and juvenile mussels using the recently published American Society for Testing and Materials (ASTM) Standard guide for conducting laboratory toxicity tests with freshwater mussels. Toxicity tests were conducted with glochidia (24- to 48-h exposures) and juveniles (96-h exposures) of up to 11 mussel species in reconstituted ASTM hard water using copper, ammonia, or chlorine as a toxicant. Copper and ammonia tests also were conducted with five commonly tested species, including cladocerans (Daphnia magna and Ceriodaphnia dubia; 48-h exposures), amphipod (Hyalella azteca; 48-h exposures), rainbow trout (Oncorhynchus mykiss; 96-h exposures), and fathead minnow (Pimephales promelas; 96-h exposures). Median effective concentrations (EC50s) for commonly tested species were >58 microg Cu/L (except 15 microg Cu/L for C. dubia) and >13 mg total ammonia N/L, whereas the EC50s for mussels in most cases were <45 microg Cu/L or <12 mg N/L and were often at or below the final acute values (FAVs) used to derive the U.S. Environmental Protection Agency 1996 acute water quality criterion (WQC) for copper and 1999 acute WQC for ammonia. However, the chlorine EC50s for mussels generally were >40 microg/L and above the FAV in the WQC for chlorine. The results indicate that the early life stages of mussels generally were more sensitive to copper and ammonia than other organisms and that, including mussel toxicity data in a revision to the WQC, would lower the WQC for copper or ammonia. Furthermore, including additional mussel data in 2007 WQC for copper based on biotic ligand model would further lower the WQC.

Use of reconstituted waters to evaluate effects of elevated major ions associated with mountaintop coal mining on freshwater invertebrates

In previous laboratory chronic 7-d toxicity tests conducted with the cladoceran Ceriodaphnia dubia, surface waters collected from Appalachian sites impacted by coal mining have shown toxic effects associated with elevated total dissolved solids (TDS). The objective of the present study was to evaluate the effects of elevated major ions in chronic laboratory tests with C. dubia (7-d exposure), a unionid mussel (Lampsilis siliquoidea; 28-d exposure), an amphipod (Hyalella azteca; 28-d exposure), and a mayfly (Centroptilum triangulifer; 35-d exposure) in 3 reconstituted waters designed to be representative of 3 Appalachian sites impacted by coal mining. Two of the reconstituted waters had ionic compositions representative of alkaline mine drainage associated with mountaintop removal and valley fill-impacted streams (Winding Shoals and Boardtree, with elevated Mg, Ca, K, SO₄, HCO₃), and a third reconstituted water had an ionic composition representative of neutralized mine drainage (Upper Dempsey, with elevated Na, K, SO₄, and HCO₃). The waters with similar conductivities but, with different ionic compositions had different effects on the test organisms. The Winding Shoals and Boardtree reconstituted waters were consistently toxic to the mussel, the amphipod, and the mayfly. In contrast, the Upper Dempsey reconstituted water was toxic to the mussel, the amphipod, and the cladoceran but was not toxic to the mayfly. These results indicate that, although elevated TDS can be correlated with toxicity, the specific major ion composition of the water is important. Moreover, the choice of test organism is critical, particularly if a test species is to be used as a surrogate for a range of faunal groups.

Sensitivity of early life stages of freshwater mussels (Unionidae) to acute and chronic toxicity of lead, cadmium, and zinc in water.

Toxicity of lead, cadmium, or zinc to early life stages of freshwater mussels (fatmucket, Lampsilis siliquoidea; Neosho mucket, L. rafinesqueana) was evaluated in 48-h exposures with mussel larvae (glochidia), in 96-h exposures with newly transformed (5-d-old) and two- or six-month-old juvenile mussels, or in 28-d exposures with two- or four-month-old mussels in reconstituted soft water. The 24-h median effect concentrations (EC50s) for fatmucket glochidia (>299 microg Pb/L, >227 microg Cd/L, 2,685 microg Zn/L) and 96-h EC50s for two- or six-month-old fatmucket (>426 microg Pb/L, 199 microg Cd/L, 1,700 microg Zn/L) were much higher than 96-h EC50s for newly transformed fatmucket (142 and 298 microg Pb/L, 16 microg Cd/L, 151 and 175 microg Zn/L) and Neosho mucket (188 microg Pb/L, 20 microg Cd/L, 145 microg Zn/L). Chronic values for fatmucket were 10 microg Pb/L, 6.0 microg Cd/L, and 63 and 68 microg Zn/L. When mussel data from the present study and the literature were included in updated databases for deriving U.S. Environmental Protection Agency water quality criteria, mussel genus mean acute values were in the lower percentiles of the sensitivity distribution of all freshwater species for Pb (the 26th percentile), Cd (the 15th to 29th percentile), or Zn (the 12th to 21st percentile). The mussel (Lampsilis) genus mean chronic value was the lowest value ever reported for Pb (the 9th percentile) but was near the middle of the sensitivity distribution for Cd (the 61st percentile) or Zn (the 44th percentile). These results indicate that mussels were relatively sensitive to the acute toxicity of these three metals and to the chronic toxicity of Pb, but were moderately sensitive to the chronic toxicity of Cd or Zn compared to other freshwater species.

An evaluation of freshwater mussel toxicity data in the derivation of water quality guidance and standards for copper

The state of Oklahoma has designated several areas as freshwater mussel sanctuaries in an attempt to provide freshwater mussel species a degree of protection and to facilitate their reproduction. We evaluated the protection afforded freshwater mussels by the U.S. Environmental Protection Agency (U.S. EPA) hardness-based 1996 ambient copper water quality criteria, the 2007 U.S. EPA water quality criteria based on the biotic ligand model and the 2005 state of Oklahoma copper water quality standards. Both the criterion maximum concentration and criterion continuous concentration were evaluated. Published acute and chronic copper toxicity data that met American Society for Testing and Materials guidance for test acceptability were obtained for exposures conducted with glochidia or juvenile freshwater mussels. We tabulated toxicity data for glochidia and juveniles to calculate 20 species mean acute values for freshwater mussels. Generally, freshwater mussel species mean acute values were similar to those of the more sensitive species included in the U.S. EPA water quality derivation database. When added to the database of genus mean acute values used in deriving 1996 copper water quality criteria, 14 freshwater mussel genus mean acute values included 10 of the lowest 15 genus mean acute values, with three mussel species having the lowest values. Chronic exposure and sublethal effects freshwater mussel data available for four species and acute to chronic ratios were used to evaluate the criterion continuous concentration. On the basis of the freshwater mussel toxicity data used in this assessment, the hardness-based 1996 U.S. EPA water quality criteria, the 2005 Oklahoma water quality standards, and the 2007 U.S. EPA water quality criteria based on the biotic ligand model might need to be revised to afford protection to freshwater mussels.

Intra‐ and interlaboratory variability in acute toxicity tests with glochidia and juveniles of freshwater mussels (unionidae)

The present study evaluated the performance and variability in acute toxicity tests with glochidia and newly transformed juvenile mussels using the standard methods outlined in American Society for Testing and Materials (ASTM). Multiple 48-h toxicity tests with glochidia and 96-h tests with juvenile mussels were conducted within a single laboratory and among five laboratories. All tests met the test acceptability requirements (e.g., >or=90% control survival). Intralaboratory tests were conducted over two consecutive mussel-spawning seasons with mucket (Actinonaias ligamentina) or fatmucket (Lampsilis siliquoidea) using copper, ammonia, or chlorine as a toxicant. For the glochidia of both species, the variability of intralaboratory median effective concentrations (EC50s) for the three toxicants, expressed as the coefficient of variation (CV), ranged from 14 to 27% in 24-h exposures and from 13 to 36% in 48-h exposures. The intralaboratory CV of copper EC50s for juvenile fatmucket was 24% in 48-h exposures and 13% in 96-h exposures. Interlaboratory tests were conducted with fatmucket glochidia and juveniles by five laboratories using copper as a toxicant. The interlaboratory CV of copper EC50s for glochidia was 13% in 24-h exposures and 24% in 48-h exposures, and the interlaboratory CV for juveniles was 22% in 48-h exposures and 42% in 96-h exposures. The high completion success and the overall low variability in test results indicate that the test methods have acceptable precision and can be performed routinely.

Toxicity of sediments potentially contaminated by coal mining and natural gas extraction to unionid mussels and commonly tested benthic invertebrates

Sediment toxicity tests were conducted to assess potential effects of contaminants associated with coal mining or natural gas extraction activities in the upper Tennessee River basin and eastern Cumberland River basin in the United States. Test species included two unionid mussels (rainbow mussel, Villosa iris, and wavy-rayed lampmussel, Lampsilis fasciola, 28-d exposures), and the commonly tested amphipod, Hyalella azteca (28-d exposure) and midge, Chironomus dilutus (10-d exposure). Sediments were collected from seven test sites with mussel communities classified as impacted and in proximity to coal mining or gas extraction activities, and from five reference sites with mussel communities classified as not impacted and no or limited coal mining or gas extraction activities. Additional samples were collected from six test sites potentially with high concentrations of polycyclic aromatic hydrocarbons (PAHs) and from a test site contaminated by a coal ash spill. Mean survival, length, or biomass of one or more test species was reduced in 10 of 14 test samples (71%) from impacted areas relative to the response of organisms in the five reference samples. A higher proportion of samples was classified as toxic to mussels (63% for rainbow mussels, 50% for wavy-rayed lampmussels) compared with amphipods (38%) or midge (38%). Concentrations of total recoverable metals and total PAHs in sediments did not exceed effects-based probable effect concentrations (PECs). However, the survival, length, or biomasses of the mussels were reduced significantly with increasing PEC quotients for metals and for total PAHs, or with increasing sum equilibrium-partitioning sediment benchmark toxic units for PAHs. The growth of the rainbow mussel also significantly decreased with increasing concentrations of a major anion (chloride) and major cations (calcium and magnesium) in sediment pore water. Results of the present study indicated that (1) the findings from laboratory tests were generally consistent with the field observations of impacts on mussel populations; (2) total recoverable metals, PAHs, or major ions, or all three in sediments might have contributed to the sediment toxicity; (3) the mussels were more sensitive to the contaminants in sediments than the commonly tested amphipod and midge; and (4) a sediment toxicity benchmark of 1.0 based on PECs may not be protective of mussels.

Influence of dissolved organic carbon on toxicity of copper to a unionid mussel (Villosa iris) and a cladoceran (Ceriodaphnia dubia) in acute and chronic water exposures

Acute and chronic toxicity of copper (Cu) to a unionid mussel (Villosa iris) and a cladoceran (Ceriodaphnia dubia) were determined in water exposures at four concentrations of dissolved organic carbon (DOC; nominally 0.5, 2.5, 5, and 10 mg/L as carbon [C]). Test waters with DOC concentrations of 2.5 to 10 mg C/L were prepared by mixing a concentrate of natural organic matter (Suwannee River, GA, USA) in diluted well water (hardness 100 mg/L as CaCO(3) , pH 8.3, DOC 0.5 mg C/L). Acute median effect concentrations (EC50s) for dissolved Cu increased approximately fivefold (15-72 µg Cu/L) for mussel survival in 4-d exposures and increased about 11-fold (25-267 µg Cu/L) for cladoceran survival in 2-d exposures across DOC concentrations from 0.5 to 10 mg C/L. Similarly, chronic 20% effect concentrations (EC20s) for the mussel in 28-d exposures increased about fivefold (13-61 µg Cu/L for survival; 8.8-38 µg Cu/L for biomass), and the EC20s for the cladoceran in 7-d exposures increased approximately 17-fold (13-215 µg Cu/L) for survival or approximately fourfold (12-42 µg Cu/L) for reproduction across DOC concentrations from 0.5 to 10 mg C/L. The acute and chronic values for the mussel were less than or approximately equal to the values for the cladoceran. Predictions from the biotic ligand model (BLM) used to derive the U.S. Environmental Protection Agencys ambient water quality criteria (AWQC) for Cu explained more than 90% of the variation in the acute and chronic endpoints for the two species, with the exception of the EC20 for cladoceran reproduction (only 46% of variation explained). The BLM-normalized acute EC50s and chronic EC20s for the mussel and BLM-normalized chronic EC20s for the cladoceran in waters with DOC concentrations of 2.5 to 10 mg C/L were equal to or less than the final acute value and final chronic value in the BLM-based AWQC for Cu, respectively, indicating that the Cu AWQC might not adequately protect the mussel from acute and chronic exposure, and the cladoceran from chronic exposure.

Evaluation of influence of sediment on the sensitivity of a unionid mussel (Lampsilis siliquoidea) to ammonia in 28-day water exposures.

A draft update of the U.S. Environmental Protection Agency ambient water quality criteria (AWQC) for ammonia substantially lowers the ammonia AWQC, primarily due to the inclusion of toxicity data for freshwater mussels. However, most of the mussel data used in the updated AWQC were generated from water-only exposures and limited information is available on the potential influence of the presence of a substrate on the response of mussels in laboratory toxicity tests. Our recent study demonstrated that the acute sensitivity of mussels to ammonia was not influenced by the presence of substrate in 4-d laboratory toxicity tests. The objective of the current study was to determine the sensitivity of mussels to ammonia in chronic 28-d water exposures with the sediment present (sediment treatment) or absent (water-only treatment). The chronic toxicity test was conducted starting with two-month-old juvenile mussels (fatmucket, Lampsilis siliquoidea) in a flow-through diluter system, which maintained consistent pH (≈8.3) and six concentrations of total ammonia nitrogen (N) in overlying water and in sediment pore water. The chronic value (ChV, geometric mean of the no-observed-effect concentration and the lowest-observed-effect concentration) was 0.36 mg N/L for survival or biomass in the water-only treatment, and was 0.66 mg N/L for survival and 0.20 mg N/L for biomass in the sediment treatment. The 20% effect concentration (EC20) for survival was 0.63 mg N/L in the water-only treatment and was 0.86 mg N/L in the sediment treatment (with overlapping 95% confidence intervals; no EC20 for biomass was estimated because the data did not meet the conditions for any logistic regression analysis). The similar ChVs or EC20s between the water-only treatment and the sediment treatment indicate that the presence of sediment did not substantially influence the sensitivity of juvenile mussels to ammonia in the 28-d chronic laboratory water exposures.