An effects addition model based on bioaccumulation of metals from exposure to mixtures of metals can predict chronic mortality in the aquatic invertebrate Hyalella azteca.

Chronic toxicity tests of mixtures of 9 metals and 1 metalloid (As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Tl, and Zn) at equitoxic concentrations over an increasing concentration range were conducted with the epibenthic, freshwater amphipod Hyalella azteca. The authors conducted 28-d, water-only tests. The bioaccumulation trends changed for 8 of the elements in exposures to mixtures of the metals compared with individual metal exposures. The bioaccumulation of Co and Tl were affected the most. These changes may be due to interactions between all the metals as well as interactions with waterborne ligands. A metal effects addition model (MEAM) is proposed as a more accurate method to assess the impact of mixtures of metals and to predict chronic mortality. The MEAM uses background-corrected body concentration to predict toxicity. This is important because the chemical characteristics of different waters can greatly alter the bioavailability and bioaccumulation of metals, and interactions among metals for binding at the site of action within the organism can affect body concentration. The MEAM accurately predicted toxicity in exposures to mixtures of metals, and predicted results were within a factor of 1.1 of the observed data, using 24-h depurated body concentrations. The traditional concentration addition model overestimated toxicity by a factor of 2.7.

Cadmium bioavailability to Hyalella azteca from a periphyton diet compared to an artificial diet and application of a biokinetic model.

Differences between the bioavailability of cadmium in a periphyton diet and an artificial laboratory diet (TetraMin(®)) have important consequences for predicting bioaccumulation and toxicity in the freshwater amphipod Hyalella azteca. The assimilation efficiency (AE) of Cd was compared between periphyton and TetraMin(®) at low (1510 and 358 nmol/g ash-free dry mass respectively) and chronically lethal (31,200 and 2890 nmol/g ash-free dry mass respectively) Cd concentrations and in fresh and dry forms using a (109)Cd radiotracer pulse-chase feeding technique. Assimilation efficiency of Cd from periphyton (AE=3-14%) was lower than that for TetraMin(®) (AE=44-86%) regardless of Cd concentration or food form. Ingestion rate (IR) was lower for dry than fresh forms of periphyton (0.042 and 0.16 g AFDM/g H. azteca/day respectively) and TetraMin(®) (0.19 and 0.87 AFDM/g H. azteca/day respectively) and depuration rate (k(e)) did not differ statistically with food type, form or Cd concentration (0.032-0.094 d(-1)). Biokinetic models with parameters of AE, IR and k(e) were used to estimate bioaccumulation from the separate food types. These estimates were compared to those from an independent chronic Cd saturation bioaccumulation model. While the model estimates did not concur, a sensitivity analysis indicated that AE and IR were the most influential biokinetic model parameters for Cd in periphyton and TetraMin(®) respectively. It was hypothesized that AE was underestimated for Cd in periphyton due to a non-adapted gut enzyme system and IR was overestimated for Cd in TetraMin(®) due to an initial rapid ingestion phase in H. azteca's feeding habits. This research demonstrated the importance of using ecologically relevant food types in laboratory experiments and verifying acute biokinetic model predictions of dietary metal contribution with those derived from a chronic exposure which is more representative of a field exposure scenario.

Interactions of Pb and Cd mixtures in the presence or absence of natural organic matter with the fish gill.

Metal gill binding and toxicity can be modeled using the concentration addition model, in which the toxic unit (TU) concept is used to determine if constituent metals are acting in a strictly additive, less than, or greater than additive fashion. To test this hypothesis, rainbow trout (Oncorhynchus mykiss) were exposed to a matrix of Pb plus Cd mixtures (nominal concentrations=0.75, 1.5, 2.25, 3.0 µmol L(-1)), in the presence or absence of mainly terrigenous (allochthonous; 10 mg CL(-1)) natural organic matter (NOM), and metal-gill binding, and toxicity was quantified. Based on its greater affinity for metal-gill binding sites, Cd-gill binding was expected to exceed Pb-gill binding during metal mixture exposure, but this only occurred at the lowest metal concentrations (0.75 µmol L(-1)); at higher concentrations Pb-gill binding was greater than Cd-gill binding. These unexpected observations were because Pb and Cd likely bind to different populations of high affinity, low capacity binding sites on the gill, which was borne out in subsequent attempts to mathematically model metal-gill interactions during metal-mixture exposure. The presence of an additional low affinity, high capacity population of Pb-gill binding sites also contributed to higher Pb-gill accumulation. Metal-gill interactions were complicated by NOM, which exacerbated toxicity during Cd-only exposure despite lowering Cd-gill accumulation. NOM also promoted Cd-gill binding in the presence of low-moderate concentrations of Pb (0.75 and 1.50 µmol L(-1)). We suggest that direct interactions of Cd-NOM complexes with the gill, and increases in Cd bioavailability due to Pb outcompeting Cd for NOM-metal binding sites due to its greater affinity for such ligands, accounted for greater Cd-gill binding and toxicity. We conclude that interactions of Pb and Cd with the gill cannot be predicted using the concentration addition model, and that NOM is not universally protective against metal-gill binding and toxicity when fish are exposed to metal mixtures.

Validation of a chronic dietary cadmium bioaccumulation and toxicity model for Hyalella azteca exposed to field-contaminated periphyton and lake water.

A model previously developed in the laboratory to predict chronic bioaccumulation and toxicity of cadmium to Hyalella azteca from a diet of periphyton was validated by comparing predictions with measurements of Cd in two exposure scenarios: laboratory-cultured H. azteca exposed for 28 d to field-contaminated water and periphyton, and Cd measured in field-collected H. azteca. In both exposure scenarios, model predictions of bioaccumulation were shown to be robust; however, effects on Cd bioaccumulation from complexation with dissolved organic carbon (DOC) and inhibition of Cd bioaccumulation by Ca²âº must be incorporated into the model to permit its wider application. The model predicted that 80 to 84% of Cd in H. azteca came from periphyton when H. azteca were chronically exposed to dissolved Cd in lake water at 2.63 to 3.01 nmol/L and periphyton at 1,880 to 2,630 nmol/g ash-free dry mass. Dietary Cd contributed markedly to the model-predicted decrease in 28-d survival to 74% at environmental Cd concentrations in food and water. In reality, survival decreased to 10%. The lower than predicted survival likely was due to the higher nutritional quality of periphyton used to develop the model in the laboratory compared with the field-collected periphyton. Overall, this research demonstrated that Cd in a periphyton diet at environmental concentrations can contribute to chronic toxicity in H. azteca.

Modeling chronic dietary cadmium bioaccumulation and toxicity from periphyton to Hyalella azteca.

A chronic (28-d) Cd saturation bioaccumulation model was developed to quantify the Cd contribution from a natural periphyton diet to Cd in the freshwater amphipod Hyalella azteca. Bioaccumulation was then linked to chronic toxic effects. Juvenile H. azteca were exposed to treatments of Cd in water (3.13-100 nmol/L nominal) and food (389-26,300 nmol/g ash-free dry mass). Cadmium bioaccumulation, survival, and growth were recorded. Dietary Cd was estimated to contribute 21 to 31, 59 to 94, and 40 to 55% to bioaccumulated Cd in H. azteca exposed to treatments of Cd primarily in water, food, and food + water, respectively. Survival as a function of Cd lethal body concentration (679 nmol/g; 95% confidence limits, 617-747) was the most robust endpoint. Body concentration integrated all exposure routes. Based on the lethal body concentration, dietary Cd was predicted to contribute markedly (26-90%) to Cd in H. azteca. Cadmium concentration and food nutritional quality (biomass, chlorophyll a, total lipid, fatty acids, total protein) had no effect on H. azteca nutritional quality (total lipid, fatty acids, total protein) but did influence H. azteca dry weight. This research highlighted the importance of including a dietary component when modeling chronic effects of Cd and when refining endpoints for use in ecological risk assessment and water quality guidelines.

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.

Bioaccumulation of the synthetic hormone 17alpha-ethinylestradiol in the benthic invertebrates Chironomus tentans and Hyalella azteca.

The present study investigated the bioaccumulation of the synthetic hormone 17alpha-ethinylestradiol (EE2) in the benthic invertebrates Chironomus tentans and Hyalella azteca, in water-only and spiked sediment assays. Water and sediment residue analysis was performed by LC/MS-MS, while biota extracts were analyzed using both LC/MS-MS and a recombinant yeast estrogen receptor assay. At the lowest exposure concentration, C. tentans accumulated less EE2 than H. azteca in the water-only assays (p=0.0004), but due to different slopes, this difference subsided with increasing concentrations; at the exposure concentration of 1mg/L, C. tentans had a greater body burden than H. azteca (p=0.02). In spiked sediments, C. tentans had the greatest EE2 accumulation (1.2+/-0.14 vs. 0.5+/-0.05 microg/gdw, n=4). Measurements in H. azteca indicated a negligible contribution from the sediments to the uptake of EE2 in this species. These differences were likely due to differences in the behavior and life history of the two species (epibenthic vs. endobenthic). Water-only bioaccumulation factors (BAFs) calculated at the lowest exposure concentration were significantly smaller in C. tentans than in H. azteca (31 vs. 142, respectively; p<0.0001). In contrast, the sediment bioaccumulation factor (BSAF) of C. tentans was larger than that of H. azteca (0.8 vs. 0.3; p<0.0001). Extracts of the exposed animals caused a response in a recombinant yeast estrogen receptor assay, thus confirming the estrogenic activity of the samples, presumably from EE2 and its estrogenic metabolites. The results of the present study suggest that consumption of invertebrate food items could provide an additional source of exposure to estrogenic substances in vertebrate predators.

Toxicity of a cadmium-contaminated diet to Hyalella azteca.

Four- and 10-week chronic toxicity tests were conducted using the freshwater amphipod Hyalella azteca and Cd-contaminated Chlorella sp. as a food source. Chlorella sp. was cultured in various Cd concentrations, filtered from solution, rinsed, dried, and ground into food flakes for the H. azteca. Unlike Cd toxicity from water sources, growth was found to be a more sensitive toxicological endpoint than survival, with calculated 50 and 25% effect concentrations (EC50s and EC25s, respectively) of 5.43 and 2.82 nmol/g, respectively, for Cd measured in food. Based on the regression of Cd in Chlorella sp. against Cd in filtered culture medium, the EC50 and EC25 corresponded to dissolved Cd concentrations of 11.30 and 5.09 nmol/L, respectively. Little or no bioaccumulation of Cd was found in the tissues of H. azteca that were fed contaminated food. These results demonstrate an apparent toxicological effect (either direct or indirect) of Cd-contaminated Chlorella sp. to H. azteca that is not associated with Cd accumulation. Toxicity of Cd-contaminated Chlorella sp. differs from waterborne Cd toxicity both in terms of the most sensitive endpoint (growth vs survival) and the relationship between toxicity and bioaccumulation. Unlike Cd toxicity through water exposure, Cd bioaccumulation by H. azteca cannot, therefore, be used to infer toxicity of Cd in a diet of Chlorella sp. Although the concentration of Cd in the algal culture medium that ultimately reduced growth of H. azteca in the present study was higher than Cd in water, which caused mortality to H. azteca in water-only tests during previous studies, further research regarding the contribution of dietary Cd to overall Cd toxicity is needed to verify that water-quality guidelines and risk assessments based on water-only exposures are fully protective.

Assessment of the toxicity of mixtures of copper, 9,10-phenanthrenequinone, and phenanthrene to Daphnia magna: evidence for a reactive oxygen mechanism.

Polycyclic aromatic hydrocarbons and their derivatives are ubiquitous environmental contaminants. They are commonly present in complex mixtures with other contaminants, such as metals. The toxicities of phenanthrene (PHE) and 9,10-phenanthrenequinone (PHQ) with or without Cu were determined using Daphnia magna. Copper was the most toxic among the three chemicals tested, followed by PHQ and then PHE, with 48-h median effective concentrations (EC50s) of 0.96, 1.72, and 5.33 microM, respectively. Copper at 0.31 microM, or approximately the 5% effective concentration, decreased the EC50 of PHQ from 1.72 to 0.28 microM. Likewise, PHQ at 1.2 microM, or approximately the 10% effective concentration, significantly lowered the EC50 of Cu from 0.96 to 0.30 microM. This synergistic effect was not observed, however, in mixtures of Cu and PHE based on the response addition model. Assimilation of Cu wasfound to be similar with or without PHQ at increasing external concentrations of Cu, indicating that the increased toxicity of their mixtures is physiologically based. The ability of Cu plus PHQ to generate reactive oxygen species (ROS) was measured as well. Copper alone caused elevated ROS levels at a low concentration (0.63 microM). With PHQ present, however, this elevation in ROS occurred at an even lower Cu level (0.31 microM). Possible attenuation effects of ascorbic acid (vitamin C) on toxicity and ROS production induced by Cu, PHQ, and their mixtures were then examined. Ascorbic acid protected against Cu and Cu-plus-PHQ mixture-mediated toxicity but did not affect PHQ toxicity. Ascorbic acid also lowered ROS levels in the presence of Cu and Cu plus PHQ. We conclude that there exist potential toxic interactions between metals and modified PAHs and that these interactions can involve ROS formation.

Lac Dufault sediment core trace metal distribution, bioavailability and toxicity to Hyalella azteca.

To determine changes in metal distribution, bioavailability and toxicity with sediment depth, two 20-cm-long replicate cores were collected from a lake historically subjected to the influence of metal mining and smelting activity. The vertical distribution of Pb, Cd and Cu in sediment was similar for all three metals, with the surface layers showing enrichment and the deeper (pre-industrial) layers showing lower concentrations. Toxicity of each sediment core section was determined in laboratory tests with the freshwater amphipod Hyalella azteca. Bioavailable metal in each sediment slice was estimated from metal concentrations in overlying water in these toxicity tests and, for Cd, also from metal bioaccumulation. The profile for Cd in tissue was comparable to Cd in sediment and overlying water, but relative Cd bioavailability from sediment increased with sediment depth. Survival increased with increasing sediment depth, suggesting that surface sediments were probably less or non-toxic before industrialization.

Effect of major ions on the toxicity of copper to Hyalella azteca and implications for the biotic ligand model.

The effect of major ions (Ca, Mg, Na, and K) and pH on Cu toxicity (LC50) to Hyalella azteca was determined in 1 week exposures. The simplest equation for describing Cu toxicity is a linear relationship between the total dissolved Cu LC50 and Ca and Na in water, ignoring pH. This equation would be useful in tier one of a two-tiered approach; if the measured dissolved Cu exceeds the value predicted from the equation, the sample should either be tested for toxicity, or a more detailed chemical speciation analysis can be conducted. The data were not consistent with a single-binding-site biotic ligand model, assuming that toxicity was due to the free Cu ion alone. However, toxicity could be predicted using a two-binding-site model. This requires separate coefficients to account for the effects of Ca and Na at low and high pH values (6.5-8.4), corresponding to the different binding sites (Mg and K did not affect toxicity). The single-binding-site BLM does not allow for this. Toxicity of Cu hydroxide or carbonate complexes does not need to be invoked, but cannot be excluded, and several models invoking the toxicity of these complexes can also explain the data. The free ion LC50 is strongly dependent on pH, but the LC50 for total dissolved Cu is almost pH independent. The effects of Ca and Na on the free ion LC50 are very different at high and low pH (contrary to single-site biotic ligand model predictions), but similar for total dissolved Cu. Published data suggest that the same model, with different coefficients, can also be applied to Daphnia and fish. A more critical evaluation of the effects of cations at both low and high pH for organisms other than Hyalella is needed to determine if the BLM needs to be adjusted to incorporate more than one binding site for other species as well. Hydrogen ions reduce the toxicity of free Cu ions to Hyalella, but Cu also reduces the toxicity of hydrogen ions. A mixture model accounting for the joint toxicity of Cu and pH, as well as their mutual antagonistic effects, is presented.

Toxicity of sixty-three metals and metalloids to Hyalella azteca at two levels of water hardness.

The toxicity of all atomically stable metals in the periodic table, excluding Na, Mg, K, and Ca, was measured in one-week exposures using the freshwater amphipod Hyalella azteca in both Lake Ontario, Canada, and soft water (10% Lake Ontario). Metals were added as atomic absorption standards (63 metals), and also as anion salts for 10 metals. Lethal concentrations resulting in 50% mortality (LC50s) were obtained for 48 of the metals tested; the rest were not toxic at 1,000 microg/L. The most toxic metals on a molar basis were Cd, Ag, Pb, Hg, Cr (anion), and Tl, with nominal LC50s ranging from 5 to 58 nmol/L (1 to 58 nmol/L measured). These metals were followed by U, Co, Os, Se (anion), Pt, Lu, Cu, Ce, Zn, Pr, Ni, and Yb with nominal LC50s ranging from 225 to 1,500 nmol/L (88-1,300 nmol/L measured). Most metals were similarly or slightly more toxic in soft water, but Al, Cr, Ge, Pb, and U were >17-fold more toxic in soft water; Pd was less toxic in soft water. Atomic absorption (AA) standards of As and Se in acid had similar toxicity as anions, Sb was more toxic as the AA standard, and Cr and Mn were more toxic as anions. One-week LC50s for H. azteca correlate strongly with three-week LC50s and three-week effect concentrations resulting in 50% reduction in reproduction (EC50s) in Daphnia magna.

Uptake and depuration of cadmium, nickel, and lead in laboratory-exposed Tubifex tubifex and corresponding changes in the concentration of a metallothionein-like protein.

Based on weight loss in water, 24 h is recommended for Tubifex tubifex gut clearance. Biota-to-sediment accumulation factors (BSAFs) in gut-cleared T. tubifex following six weeks of exposure to Cd-, Ni-, and Pb-spiked sediment were 12.4, 3.0, and 19.0, respectively. Tissue Ni concentrations peaked after 12 h, whereas Cd and Pb were accumulated for the duration of the exposure. Tubifex tubifex were transferred to either water (24 h) or sediment (10 weeks) to monitor changes in internal metal concentrations. After 24 h in water, only Ni concentration had declined significantly (p < 0.05), suggesting that the majority of Ni was associated with the gut content, while Cd and Pb were accumulated in the tissues. Metal depuration in sediment was described with two-compartment, first-order kinetic models (r2 = 0.7-0.8; p < 0.001), indicating that T. tubifex has both a quickly depurated and a more tightly bound pool of accumulated metal. Tubifex tubifex were also exposed to sediment spiked with just Cd (3.66 micromol/g). Cadmium uptake and induction of metallothionein-like protein (MTLP) were rapid; both parameters were significantly elevated within 24 h of exposure. Metallothionein-like protein (8.7 +/- 1.8 nmol/g) and Cd (60.8 +/- 11.0 micromol/g) reached maximum concentrations after 96 h and four weeks, respectively.

Tributyltin uptake and depuration in Hyalella azteca: implications for experimental design.

The purpose of this study was to address four aspects of the kinetics of tributyltin (TBT) in the freshwater amphipod Hyalella azteca: time to steady state, route of uptake, depuration rates, and effect of gut clearance. The amphipods accumulated TBT rapidly, reaching steady state within 14 d. Body concentrations were similar between caged and sediment-exposed animals, indicating that the primary route of uptake is via dissolved TBT. However, the rate of uptake was significantly higher in sediment-exposed amphipods. During depuration, body concentrations of TBT exhibited a biphasic decline, with a stronger decrease over the first 24 h that is attributed primarily to gut clearance, followed by a more gradual decrease most likely due to excretion from the body. Gut contents contributed significantly to body concentrations of TBT, accounting for 30% of the initial total body burden in sediment-exposed amphipods. Half-lives of TBT in gut-cleared H. azteca were 8 d and 14 d for amphipods exposed to spiked water and spiked sediment, respectively. The results of this study have significant implications in the experimental design and interpretation of studies involving the effects of TBT in H. azteca.

Accumulation of tributyltin in Hyalella azteca as an indicator of chronic toxicity: survival, growth, and reproduction.

The chronic toxicity of tributyltin (TBT) was examined by exposing two successive generations of the freshwater amphipod, Hyalella azteca, to sediments spiked with TBT. Survival was the most sensitive measure of effect, with lethal concentration resulting in 50% mortality (LC50) values on a water and body concentration basis ranging from 76 to 145 ng Sn/L and 2,790 to 4,300 ng Sn/g, respectively. Individual growth of amphipods was not negatively affected by TBT, and although reproduction might be more sensitive than survival, the data were too variable to use on a routine basis. There were no detectable TBT-induced differences in the response between first- and second-generation animals. The relationship between toxicity and bioaccumulation of TBT in H. azteca was determined and can be used as a tool to predict the toxicity of TBT in environmental samples. Body concentrations exceeding 2,000 ng Sn/g in H. azteca exposed to field-collected samples would indicate that chronic toxicity due to TBT is likely occurring in amphipod populations at those sites.

The relative sensitivity of four benthic invertebrates to metals in spiked-sediment exposures and application to contaminated field sediment.

The relative sensitivity of four benthic invertebrates (Hyalella azteca, Chironomus riparius, Hexagenia spp., and Tubifex tubifex) was determined for Cd, Cu, and Ni in water-only and in spiked-sediment exposures. Survival (median lethal concentrations [LC50s] and the concentrations estimated to be lethal to 25% of test organisms [LC25s]), and endpoints for growth and reproduction (mean inhibitory concentrations [IC25s]) were compared. The sensitivities differed depending on the species and metal, although some trends emerged. In water-only exposures, H. azteca is the most sensitive species to cadmium and nickel, with mean LC50s of 0.013 and 3.6 mg/L, respectively; C. riparius is the most sensitive species to copper, with a mean LC50 of 0.043 mg/L. In the spiked-sediment exposures, the order in decreasing sensitivity to copper is Hyalella = Hexagenia < Chironomus < Tubifex for survival and growth/reproduction. For cadmium, the order in decreasing sensitivity is Hyalella = Chironomus < Hexagenia < Tubifex, and for nickel is Hyalella << Hexagenia < Chironomus < Tubifex. Chironomus riparius and Hexagenia spp. survival can be used to distinguish between toxicity caused by different metals. Species test responses in field-collected sediment(Collingwood Harbour, ON, Canada) were examined in an attempt to determine the causative agent of toxicity throughout, using the established species sensitivities. Sediment toxicity was categorized first by comparing species responses to those established for a reference database. Test responses in the field-collected sediment do not support causality by Cu, a suspected toxicant based on comparison of sediment chemistry with sediment quality guidelines.