Kevin V. Brix

Utility of Tissue Residues for Predicting Effects of Metals on Aquatic Organisms

As part of a SETAC Pellston Workshop, we evaluated the potential use of metal tissue residues for predicting effects in aquatic organisms. This evaluation included consideration of different conceptual models and then development of several case studies on how tissue residues might be applied for metals, assessing the strengths and weaknesses of these different approaches. We further developed a new conceptual model in which metal tissue concentrations from metal-accumulating organisms (principally invertebrates) that are relatively insensitive to metal toxicity could be used as predictors of effects in metal-sensitive taxa that typically do not accumulate metals to a significant degree. Overall, we conclude that the use of tissue residue assessment for metals other than organometals has not led to the development of a generalized approach as in the case of organic substances. Species-specific and site-specific approaches have been developed for one or more metals (e.g., Ni). The use of gill tissue residues within the biotic ligand model is another successful application. Aquatic organisms contain a diverse array of homeostatic mechanisms that are both metal- and species-specific. As a result, use of whole-body measurements (and often specific organs) for metals does not lead to a defensible position regarding risk to the organism. Rather, we suggest that in the short term, with sufficient validation, species- and site-specific approaches for metals can be developed. In the longer term it may be possible to use metal-accumulating species to predict toxicity to metal-sensitive species with appropriate field validation.

Acute and chronic toxicity of nickel to a cladoceran (Ceriodaphnia dubia) and an amphipod (Hyalella azteca)

This study evaluated acute and chronic nickel (Ni) toxicity to Ceriodaphnia dubia and Hyalella azteca with the objective of generating information for the development of a biotic ligand model for Ni. Testing with C. dubia was used to evaluate the effect of ambient hardness on Ni toxicity, whereas the larger H. azteca was used to derive lethal body burden information for Ni toxicity. As was expected, acute C. dubia median lethal concentrations (LC50s) for Ni increased with increasing water hardness. The 48-h LC50s were 81, 148, 261, and 400 microg/L at hardnesses of 50, 113, 161, and 253 mg/L (as CaCO3), respectively. Ceriodaphnia dubia was found to be significantly more sensitive in chronic exposures than other species tested (including other daphnids such as Daphnia magna); chronic toxicity was less dependent on hardness than was acute toxicity. Chronic 20% effective concentrations (EC20s) were estimated at <3.8, 4.7, 4.0, and 6.9 microg/L at hardnesses of 50, 113, 161, and 253 mg/L, respectively. Testing with H. azteca resulted in a 96-h LC50 of 3,045 microg/L and a 14-d EC20 of 61 microg/L at a hardness of 98 mg/L (as CaCO3). Survival was more sensitive than was growth in the chronic study with H. azteca. The 20% lethal accumulation effect level based on measured Ni body burdens was 247 nmol/g wet weight.

Chronic toxicity of lead to three freshwater invertebrates-Brachionus calyciflorus, Chironomus tentans, and Lymnaea stagnalis

Chronic lead (Pb) toxicity tests with Brachionus calyciflorus, Chironomus tentans, and Lymnaea stagnalis were performed in artificial freshwaters. The no-observable-effect concentration (NOEC), lowest-observable-effect concentration (LOEC), and calculated 20% effect concentration (EC20) for the rotifer B. calyciflorus were 194, 284, and 125 microg dissolved Pb/L, respectively. The midge C. tentans was less sensitive, with NOEC and LOEC of 109 and 497 microg dissolved Pb/L, respectively, and the snail L. stagnalis exhibited extreme sensitivity, evident by NOEC, LOEC, and EC20 of 12, 16, and < 4 microg dissolved Pb/L, respectively. Our findings are presented in the context of other reports on chronic Pb toxicity in freshwater organisms. The L. stagnalis results are in agreement with a previous report on pulmonate snails and should be viewed in the context of current U.S. Environmental Protection Agency (U.S. EPA) hardness adjusted water quality criteria of 8 microg Pb/L. The present findings and earlier reports indicate that freshwater pulmonate snails may not be protected by current regulatory standards. Measurements of whole-snail Na+ and Ca2+ concentrations following chronic Pb exposure revealed that Na+ homeostasis is disturbed by Pb exposure in juvenile snails in a complicated pattern, suggesting two physiological modes of action depending on the Pb exposure concentration. Substantially reduced growth in the snails that exhibit very high Ca2+ requirements may be related to reduced Ca2+ uptake and thereby reduced shell formation.

Critical Review of Proposed Residue-Based Selenium Toxicity Thresholds for Freshwater Fish

Proposed fish toxicity thresholds for interpreting the biological significance of selenium concentrations measured in environmental media include 2 to 5 µg/L in water, 4 mg/kg dw in fish whole body tissue, 10 mg/kg dw in fish ovaries, and 3 mg/kg dw in fish diets. Use of these thresholds would likely identify fish populations as being at risk at numerous sites across the U.S. However, selenium effects on fish populations in the field have only been conclusively demonstrated at a few locations. Based on our critical review, these threshold values are not consistent with USEPA methodology for deriving criteria, in many cases are not supported by the scientific literature, and, as a result, are generally overly conservative. Based on currently available information, we believe the scientific literature is not supportive of generic sediment or water thresholds, but is supportive of alternative separate whole body thresholds of 9 mg/kg dw for warmwater fish and 6 mg/kg dw for larval coldwater anadromous fish, ...

The sensitivity of aquatic insects to divalent metals: a comparative analysis of laboratory and field data.

Laboratory studies have traditionally indicated that aquatic insects are relatively insensitive to metals while field studies have suggested them to be among the most sensitive aquatic invertebrate taxa. We reviewed and synthesized available studies in the literature to critically assess why this discrepancy exists. Despite the intense effort to study the effects of metals on aquatic biota over the past several decades, we found studies specific to insects to still be relatively limited. In general, the discrepancy between laboratory and field studies continues with few efforts having been made to elucidate the ecological and physiological mechanisms that underlie the relative sensitivity (or insensitivity) of aquatic insects to metals. However, given the limited data available, it appears that aquatic insects are indeed relatively insensitive to acute metal exposures. In contrast, we suggest that some aquatic insect taxa may be quite sensitive to chronic metal exposure and in some cases may not be protected by existing water quality criteria for metals. The discrepancy between laboratory and field studies with respect to chronic sensitivity appears to largely be driven by the relatively short exposure periods in laboratory studies as compared to field studies. It also appears that, in some cases, the sensitivity of aquatic insects in field studies may be the result of direct effects on primary producers, which lead to indirect effects via the food chain on aquatic insects. Finally, available evidence suggests that diet is an important source of metal accumulation in insects, but to date there have been no conclusive studies evaluating whether dietary metal accumulation causes toxicity. There is a clear need for developing a more mechanistic understanding of aquatic insect sensitivity to metals in long-term laboratory and field studies.

Acute and chronic toxicity of nickel to rainbow trout (Oncorhynchus mykiss)

Of the fish species tested in chronic Ni exposures, rainbow trout (Oncorhynchus mykiss) is the most sensitive. To develop additional Ni toxicity data and to investigate the toxic mode of action for Ni, we conducted acute (96-h) and chronic (85-d early life-stage) flow-through studies using rainbow trout. In addition to standard toxicological endpoints, we investigated the effects of Ni on ionoregulatory physiology (Na, Ca, and Mg). The acute median lethal concentration for Ni was 20.8 mg/L, and the 24-h gill median lethal accumulation was 666 nmol/g wet weight. No effects on plasma Ca, Mg, or Na were observed during acute exposure. In the chronic study, no significant effects on embryo survival, swim-up, hatching, or fingerling survival or growth were observed at dissolved Ni concentrations up to 466 microg/L, the highest concentration tested. This concentration is considerably higher than the only other reported chronic no-observed-effect concentration (<33 microg/L) for rainbow trout. Accumulation of Ni in trout eggs indicates the chorion is only a partial barrier with 36%, 63%, and 1% of total accumulated Ni associated with the chorion, yolk, and embryo, respectively. Whole-egg ion concentrations were reduced by Ni exposure. However, most of this reduction occurred in the chorion rather than in the embryos, and no effects on hatching success or larval survival were observed as a result. Plasma ion concentrations measured in swim-up fingerlings at the end of the chronic-exposure period were not significantly reduced by exposure to Ni. Nickel accumulated on the gill in an exponential manner but plateaued in trout plasma at waterborne Ni concentrations of 118 microg/L or greater. Consistent with previous studies, Ni did not appear to disrupt ionoregulation in acute exposures of rainbow trout. Our results also suggest that Ni is not an ionoregulatory toxicant in long-term exposures, but the lack of effects in the highest Ni treatment precludes a definitive conclusion.

Do Cd, Cu, Ni, Pb, and Zn biomagnify in aquatic ecosystems?

In this review, we sought to assess from a study of the literature whether five in organic metals (viz., cadmium, copper, lead, nickel, and zinc) bio magnify in aquatic food webs. We also examined whether accumulated metals were toxic to consumers/predators and whether the essential metals (Cu and Zn and possibly Ni) behaved differently from non-essential ones (Cd and Pb). Biomagnification potential was indexed by the magnitude of single and multiple trophic transfers in food chains. In this analysis, we used three lines of evidence-laboratory empirical, biokinetic modeling, and field studies-to make assessments. Trophic transfer factors, calculatedfrom lab studies, field studies, and biokinetic modeling, were generally congruent.Results indicated that Cd, Cu, Pb, and Zn generally do not biomagnify in food chains consisting of primary producers, macro invertebrate consumers, and fish occupying TL 3 and higher. However, bio magnification of Zn (TTFs of 1-2) is possible for circumstances in which dietary Zn concentrations are below those required for metabolism. Cd, Cu, Ni, and Zn may biomagnify in specific marine food chains consisting of bivalves, herbivorous gastropods, and barnacles at TL2 and carnivorous gastropods at TL3. There was an inverse relationship between TTF and exposure concentration for Cd, Cu, Pb, and Zn, a finding that is consistent with previous reviews of bioconcentration factors and bioaccumulation factors for metals. Our analysis also failed to demonstrate a relationship between the magnitude of TTFsand dietary toxicity to consumer organisms. Consequently, we conclude that TTFs for the metals examined are not an inherently useful predictor of potential hazard(i.e., toxic potential) to aquatic organisms. This review identified several uncertainties or data gaps, such as the relatively limited data available for nickel, reliance upon highly structured food chains in laboratory studies compared to the unstructured food webs found in nature, and variability in TTFs between the organisms found in different habitats, and years sampled.

Toxicogenomics of water chemistry influence on chronic lead exposure to the fathead minnow (Pimephales promelas)

Establishment of water quality criteria (WQC), intended to protect aquatic life, continues to rely principally on water hardness (i.e. Ca(2+)) for lead (Pb) despite growing evidence that other chemical parameters also strongly influence toxicity. To more clearly define the water chemistry parameters mediating Pb toxicity, we evaluated the effects of hardness as CaSO(4) and dissolved organic carbon (DOC) as humic acid during chronic (150 days) exposures to the fathead minnow. Measured Pb concentrations ranged from 157+/-5 nM (33+/-1 microg/L) Pb in base water to 177+/-7 (37+/-1 microg/L) and 187+/-7 nM (39+/-1 microg/L) Pb in CaSO(4)- or HA-supplemented water, respectively. Fish were collected at 2, 4, 10, 30, 63, 90 and 150 days of exposure. Traditional toxicological endpoints were examined alongside gene transcription analyses to help clarify the underlying mechanisms of Pb toxicity and to identify candidate molecular markers that might ultimately serve as robust indicators of exposure and effect. Addition of CaSO(4) did not prevent whole body Pb accumulation whereas DOC afforded strong protection (about half the amount accumulated by fish in base water) suggesting that current, hardness-based WQC are likely inaccurate for predicting chronic Pb effects in aquatic systems. Custom-made microarrays were co-hybridized with base water samples+/-Pb up to the 30 days time point. Quantitative PCR was employed to verify gene transcription responses and to extend analysis to the CaSO(4) and HA treatments and the 150 days time point. Identification of four genes by microarray analysis revealed clear Pb-induced responses over time: glucose-6-phosphate dehydrogenase, glutathione-S-transferase, ferritin and beta-globin. Results obtained by qPCR were in strong agreement with microarray data by regression analysis (r(2)=0.82, slope=1.28). The associated pathways implicated herein for these genes provide further evidence supporting roles for anemia and neurological disorders in chronic Pb toxicity. Effects of water chemistry on Pb accumulation and gene transcription responses were in close parallel, though alterations in ionoregulatory and morphological endpoints were not observed. Whereas DOC was protective against Pb accumulation and mRNA expression changes, Ca(2+) was not. Additionally, several hypothesis-driven genes (ECaC, DMT-1, and ALA-D) were examined by qPCR but revealed either no change or small Pb-induced responses lacking any clear influence attributable to water chemistry. These findings should help pave the way toward development of a new chronic Pb BLM and a Pb-responsive gene transcript profile for fathead minnows, both of which would greatly aid future environmental monitoring and regulatory strategies for Pb.

High net calcium uptake explains the hypersensitivity of the freshwater pulmonate snail, Lymnaea stagnalis, to chronic lead exposure

Previous studies have shown that freshwater pulmonate snails of the genus Lymnaea are exceedingly sensitive to chronic Pb exposure. An EC20 of <4microgl(-1)Pb for juvenile snail growth has recently been determined for Lymnaea stagnalis, which is at or below the current USEPA water quality criterion for Pb. We characterized ionoregulation and acid-base balance in Pb-exposed L. stagnalis (young adults approximately 1g) to investigate the mechanisms underlying this hypersensitivity. After 21-day exposure to 18.9microgl(-1)Pb, Ca(2+) influx was significantly inhibited (39%) and corresponding net Ca(2+) flux was significantly reduced from 224 to -23nmolg(-1)h(-1). An 85% increase in Cl(-) influx was also observed, while Na(+) ion transport appeared unaffected. Finally, a marked alkalosis of extracellular fluid was observed with pH increasing from 8.35 in the control to 8.65 in the 18.9microgl(-1) Pb-exposed group. Results based on direct measurement of Ca(2+) influx in 1g snails gave an influx nearly an order of magnitude higher (750nmolg(-1)h(-1)) than in comparably sized fish in similar water chemistry. Under control conditions, specific growth rate in newly hatched snails was estimated at 16.7% per day over the first 38-day post-hatch and whole body Ca(2+) concentrations were relatively constant at approximately 1100nmolg(-1) over this period. Based on these data, it is estimated that newly hatched snails have net Ca(2+) uptake rates on the order of 7600nmolg(-1)h(-1). A model was developed integrating these data and measured inhibition of Ca(2+) influx rates of 13.4% and 38.7% in snails exposed to 2.7 and 18.9microgl(-1)Pb, respectively. The model estimates 45% and 83% reductions in newly hatched snail growth after 30-day exposure in these two Pb-exposed groups. These results compare well with previous direct measurements of 47% and 90% reductions in growth at similar Pb concentrations, indicating the high net Ca(2+) uptake is the controlling factor in observed Pb hypersensitivity.

Egg Selenium Concentrations as Predictors of Avian Toxicity

Aquatic birds are exposed to selenium through their diet by ingesting aquatic invertebrates that have accumulated selenium from water and the food chain. However, dietary composition is highly variable among species, over time, and across sites, making it difficult to provide accurate estimations of dietary exposure for particular species at specific locations. Selenium accumulates in the egg, resulting in embryo malformation, embryonic death, and decreased survival of juveniles. If the relationship between egg concentration and these reproductive parameters can be defined with sufficient certainty, then risk assessments can be performed through analysis of egg selenium concentrations. Other researchers have proposed egg toxicity thresholds that lead to conclusions of widespread selenium toxicoses in waterbirds. However, we believe these values are overly conservative and that it is unlikely that selenium is posing a significant risk to wild birds in areas where the current water quality criterion is bein...