Physical-Chemical Recovery of a Montane Stream After Remediation of Acid Mine Drainage: Timing and Extent After Turning off the Tap.

We monitored physical-chemical conditions in the North Fork of Clear Creek in Colorado (USA) before, during, and after the start of remediation (lime treatment) to remove metals from two major inputs of acid mine drainage (AMD) water. In addition, we analyzed historical monitoring data that extended back more than two decades. Concentration-discharge (C-D) and load-discharge (L-D) plots accounted for discharge dependence in concentrations and loads of metals, major ions, and other water chemistry parameters. Total and dissolved concentrations, and loads of the metals decreased after remediation began, with the largest decreases usually during low stream flow. However, postremediation concentrations and loads remained slightly to considerably higher than reference, probably because of unidentified groundwater seeps and/or small surface flows. Dissolved Cu concentrations decreased much less than total Cu concentrations, because the percentage of total Cu in the dissolved phase increased considerably as particulate Fe (PFe) concentration decreased. We conclude that 1) water chemistry can change to a new steady state or pseudo-steady state relatively quickly after major AMD inputs to a stream are remediated; 2) elevated flows during snowmelt and rainfall periods can mobilize additional amounts of major ions and metals, resulting in in-stream concentrations that are manifestations of both dilution and mobilization; 3) although lime treatment of AMD-related waters can decrease metal concentrations, it does not decrease elevated concentrations of major ions that might impair sensitive stream invertebrates; 4) although Fe is toxic to aquatic organisms, PFe adsorbs other metals and thereby provides protection against their toxicity; and 5) use of C-D and L-D plots and element ratios can indicate the presence of unidentified AMD inputs to a stream. Environ Toxicol Chem 2023;42:495-511. © 2022 SETAC.

Influence of Geochemical Fractionation of Fulvic Acid on its Spectral Characteristics and its Protection Against Copper Toxicity to Daphnia magna.

Dissolved copper (Cu) can contribute to toxicity in aquatic systems impacted by acid mine drainage (AMD), and its bioavailability is influenced by aqueous complexation with organic ligands that predominantly include fulvic acids (FAs). Because the geochemical fractionation of FAs that accompanies sorption to hydrous aluminum oxides (HAOs) and hydrous iron oxides (HFOs) can alter Cu complexation with FA, we investigated FAs isolated from three categories of water (pristine, AMD, and in situ-fractionated mixtures of pristine and AMD collected at stream confluences) in three mining-impacted alpine watersheds in central Colorado, USA. We also conducted geochemical fractionation of field-collected FAs and Suwannee River FAs by precipitating HAOs and HFOs in the laboratory. Spectral properties of the FAs (e.g., UV-VIS absorbance) were altered by geochemical fractionation, and in acute toxicity tests with an aquatic invertebrate (Daphnia magna) Cu was more toxic in the presence of in situ- and laboratory-fractionated FAs (median effect concentration [EC50] 19-50 µg Cu L-1 ) than in the presence of nonfractionated FAs (EC50 48-146 µg Cu L-1 ). After adjusting for the strain-specific sensitivity of our D. magna, we improved the accuracy of Biotic Ligand Model predictions of Cu EC50 values for AMD-related FAs by using an "effective dissolved organic carbon" based on spectral properties that account for among-FA differences in protectiveness against Cu toxicity. However, some differences remained between predicted and measured EC50 values, especially for FAs from AMD-related waters that might contain important metal-binding moieties not accounted for by our measured spectral indices. Environ Toxicol Chem 2023;42:449-462. © 2022 SETAC.

Integrated Assessment of Chemical and Biological Recovery After Diversion and Treatment of Acid Mine Drainage in a Rocky Mountain Stream.

Responses of stream ecosystems to gradual reductions in metal loading following remediation or restoration activities have been well documented in the literature. However, much less is known about how these systems respond to the immediate or more rapid elimination of metal inputs. Construction of a water treatment plant on the North Fork of Clear Creek (NFCC; CO, USA), a US Environmental Protection Agency Superfund site, captured, diverted, and treated the two major point-source inputs of acid mine drainage (AMD) and provided an opportunity to investigate immediate improvements in water quality. We conducted a 9-year study that included intensive within- and among-year monitoring of receiving-stream chemistry and benthic communities before and after construction of the treatment plant. Results showed a 64%-86% decrease in metal concentrations within months at the most contaminated sites. Benthic communities responded with increased abundance and diversity, but downstream stations remained impaired relative to reference conditions, with significantly lower taxonomic richness represented by a few dominant taxa (i.e., Baetis sp., Hydropsyche sp., Simulium sp., Orthocladiinae). Elevated metal concentrations from apparent residual sources, and relatively high conductivity from contributing major ions not removed during the treatment process, are likely limiting downstream recovery. Our study demonstrates that direct AMD treatment can rapidly improve water quality and benefit aquatic life, but effectiveness is limited, in part, to the extent that inputs of metals are captured and treated. Consideration should also be given to the effects of elevated major ion concentrations from the treated effluent not removed during the lime treatment process. Continued chemical and biological monitoring will be needed to quantify the NFCC recovery trajectory and to inform future remediation strategies. Environ Toxicol Chem 2023;42:512-524. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Method to assess the potential magnitude of terrestrial European avian population reductions from ingestion of lead ammunition.

Current estimates of terrestrial bird losses across Europe from ingestion of lead ammunition are based on uncertain or generic assumptions. A method is needed to develop defensible European-specific estimates compatible with available data that does not require long-term field studies. We propose a 2-step method using carcass data and population models. The method estimates percentage of deaths diagnosed as directly caused by lead poisoning as a lower bound and, as an upper bound, the percentage of possible deaths from sublethal lead poisoning that weakens birds, making them susceptible to death by other causes. We use these estimates to modify known population-level annual mortality. Our method also allows for potential reductions in reproduction from lead shot ingestion because reductions in survival and reproduction are entered into population models of species with life histories representative of the most groups of susceptible species. The models estimate the sustainability and potential population decreases from lead poisoning in Europe. Using the best available data, we demonstrate the method on two taxonomic groups of birds: gallinaceous birds and diurnal raptors. The direction of the population trends affects the estimate, and we incorporated such trends into the method. Our midpoint estimates of the reduction in population size of the European gallinaceous bird (< 2%) group and raptor group (2.9-7.7%) depend on the species life history, maximum growth rate, population trend, and if reproduction is assumed to be reduced. Our estimates can be refined as more information becomes available in countries with data gaps. We advocate use of this method to improve upon or supplement approaches currently being used. As we demonstrate, the method also can be applied to individual species of concern if enough data across countries are available.

Denitrification and Nitrogen Burial in Swiss Lakes.

Earth's nitrogen (N) cycle is imbalanced because of excessive anthropogenic inputs. Freshwater lakes efficiently remove N from surface waters by transformation of NO3- to atmospheric N2 and/or N2O (denitrification; DN) and by burial of organic N in sediments (net sedimentation; NS). However, relatively little is known about the controlling environmental conditions, and few long-term measurements on individual lakes are available to quantify conversion rates. We report N-elimination rates in 21 Swiss lakes estimated from whole-lake N budgets covering up to ∼20 years of monitoring. The NO3- concentration in the bottom water was the main predictor of DN. Additionally, DN rates were positively correlated with external N load and the area-specific hydraulic loading rate (mean depth/water residence time; Qs). NS of N was strongly related to total phosphorus (P) concentration. Nitrogen removal efficiency (NRE), the fraction of the load of dissolved N to a lake removed by DN and NS, was strongly negatively related to Qs. This previously unconsidered variable improves the predictability of NRE and does not require knowledge of N and P loading rates or concentrations. We conclude that P management alone intended to oligotrophy lakes only slightly increases N export unless it is accompanied by N management.

Toxicity of Nanoparticulate Nickel to Aquatic Organisms: Review and Recommendations for Improvement of Toxicity Tests.

We reviewed the literature on toxicity of nanoparticulate nickel (nano-Ni) to aquatic organisms, from the perspective of relevance and reliability in a regulatory framework. Our main findings were 1) much of the published nano-Ni toxicity data is of low or medium quality in terms of reporting key physical-chemical properties, methodologies, and results, compared with published dissolved nickel studies; and 2) based on the available information, some common findings about nanoparticle (NP) toxicity are not supported for nano-Ni. First, we concluded that nanoparticulate elemental nickel and nickel oxide, which differ in chemical composition, generally did not differ in their toxicity. Second, there is no evidence that the toxicity of nano-Ni increases as the size of the NPs decreases. Third, for most organisms tested, nano-Ni was not more toxic on a mass-concentration basis than dissolved Ni. Fourth, there is conflicting evidence about whether the toxicity is directly caused by the NPs or by the dissolved fraction released from the NPs. However, no evidence suggests that any of the molecular, physiological, and structural mechanisms of nano-Ni toxicity differ from the general pattern for many metal-based nanomaterials, wherein oxidative stress underlies the observed effects. Physical-chemical factors in the design and conduct of nano-Ni toxicity tests are important, but often they are not adequately reported (e.g., characteristics of dry nano-Ni particles and of wetted particles in exposure waters; exposure-water chemistry). Environ Toxicol Chem 2020;39:1861-1883 © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Validation of Bioavailability-Based Toxicity Models for Metals.

Regulatory jurisdictions worldwide are increasingly incorporating bioavailability-based toxicity models into development of protective values (PVALs) for freshwater and saltwater aquatic life (e.g., water quality criteria, standards, and/or guidelines) for metals. Use of such models for regulatory purposes should be contingent on their ability to meet performance criteria as specified through a model-validation process. Model validation generally involves an assessment of a model's appropriateness, relevance, and accuracy. We review existing guidance for validation of bioavailability-based toxicity models, recommend questions that should be addressed in model-validation studies, discuss model study type and design considerations, present several new ways to evaluate model performance in validation studies, and suggest a framework for use of model validation in PVAL development. We conclude that model validation should be rigorous but flexible enough to fit the user's purpose. Although a model can never be fully validated to a level of zero uncertainty, it can be sufficiently validated to fit a specific purpose. Therefore, support (or lack of support) for a model should be presented in such a way that users can choose their own level of acceptability. We recommend that models be validated using experimental designs and endpoints consistent with the data sets that were used to parameterize and calibrate the model and validated across a broad range of geographically and ecologically relevant water types. Environ Toxicol Chem 2019;39:101-117. © 2019 SETAC.

Effects of copper on olfactory, behavioral, and other sublethal responses of saltwater organisms: Are estimated chronic limits using the biotic ligand model protective?

There is concern over whether regulatory criteria for copper (Cu) are protective against chemosensory and behavioral impairment in aquatic organisms. We compiled Cu toxicity data for these and other sublethal endpoints in 35 tests with saltwater organisms and compared the Cu toxicity thresholds with biotic ligand model (BLM)-based estimated chronic limits (ECL values, which are 20% effect concentrations [EC20s] for the embryo-larval life stage of the blue mussel [Mytilus edulis], a saltwater species sensitive to Cu that has historically been used to derive saltwater Cu criteria). Only 8 of the 35 tests had sufficient toxicity and chemistry data to support unequivocal conclusions (i.e., a Cu EC20 or no-observed-effect concentration could be derived, and Cu and dissolved organic carbon [DOC] concentrations were measured [or DOC concentrations could be inferred from the test-water source]). The BLM-based ECL values would have been protective (i.e., the ECL was lower than the toxicity threshold) in 7 of those 8 tests. In the remaining 27 tests, this meta-analysis was limited by several factors, including 1) the Cu toxicity threshold was a "less than" value in 19 tests because only a lowest-observed-effect concentration could be calculated and 2) Cu and/or DOC concentrations often were not measured. In 2 of those 27 tests, the ECL would not have been protective if based only on a conservatively high upper-bound DOC estimate. To facilitate future evaluations of the protectiveness of aquatic life criteria for metals, we urge researchers to measure and report exposure-water chemistry and test-metal concentrations that bracket regulatory criteria. Environ Toxicol Chem 2018;37:1515-1522. © 2018 SETAC.

Protectiveness of Cu water quality criteria against impairment of behavior and chemo/mechanosensory responses: An update.

A meta-analysis was conducted of studies that reported behavior and chemo/mechanosensory responses by fish, amphibians, and aquatic invertebrates in Cu-containing waters and also reported sufficient water chemistry for calculation of hardness-based and biotic ligand model (BLM)-based water quality criteria (WQC) for Cu. The calculated WQC concentrations were then compared with the corresponding 20% impairment concentrations (IC20) of Cu for those behavior and chemo/mechanosensory responses. The hardness-based acute and chronic WQC for Cu would not have been protective (i.e., the IC20 would have been lower than the WQC) in 33.6 and 26.2%, respectively, of the 107 combined behavior- and chemo/mechanosensory-response cases that also had adequate water chemistry data for BLM-based WQC calculations (32.7% inconclusive). In comparison, the BLM-based acute and chronic WQC for Cu would not have been protective in only 10.3 and 4.7%, respectively, of the same 107 cases (29.9% inconclusive). To improve evaluations of regulatory effectiveness, researchers conducting aquatic Cu toxicity tests should measure and report complete BLM-input water chemistry and bracket the hardness-based and BLM-based WQC concentrations for Cu that would be applicable in their exposure waters. This meta-analysis demonstrates that, overall, the BLM-based WQC for Cu were considerably more protective than the hardness-based WQC for Cu against impairment of behavior and chemo/mechanosensory responses. Environ Toxicol Chem 2018;37:1260-1279. © 2018 SETAC.

Is the Factor-of-2 Rule Broadly Applicable for Evaluating the Prediction Accuracy of Metal-Toxicity Models?

In aquatic toxicology, a toxicity-prediction model is generally deemed acceptable if its predicted median lethal concentrations (LC50 values) or median effect concentrations (EC50 values) are within a factor of 2 of their paired, observed LC50 or EC50 values. However, that rule of thumb is based on results from only two studies: multiple LC50 values for the fathead minnow (Pimephales promelas) exposed to Cu in one type of exposure water, and multiple EC50 values for Daphnia magna exposed to Zn in another type of exposure water. We tested whether the factor-of-2 rule of thumb also is supported in a different dataset in which D. magna were exposed separately to Cd, Cu, Ni, or Zn. Overall, the factor-of-2 rule of thumb appeared to be a good guide to evaluating the acceptability of a toxicity model's underprediction or overprediction of observed LC50 or EC50 values in these acute toxicity tests.

Acute Toxicity of Ternary Cd-Cu-Ni and Cd-Ni-Zn Mixtures to Daphnia magna: Dominant Metal Pairs Change along a Concentration Gradient.

Multiple metals are usually present in surface waters, sometimes leading to toxicity that currently is difficult to predict due to potentially non-additive mixture toxicity. Previous toxicity tests with Daphnia magna exposed to binary mixtures of Ni combined with Cd, Cu, or Zn demonstrated that Ni and Zn strongly protect against Cd toxicity, but Cu-Ni toxicity is more than additive, and Ni-Zn toxicity is slightly less than additive. To consider multiple metal-metal interactions, we exposed D. magna neonates to Cd, Cu, Ni, or Zn alone and in ternary Cd-Cu-Ni and Cd-Ni-Zn combinations in standard 48 h lethality tests. In these ternary mixtures, two metals were held constant, while the third metal was varied through a series that ranged from nonlethal to lethal concentrations. In Cd-Cu-Ni mixtures, the toxicity was less than additive, additive, or more than additive, depending on the concentration (or ion activity) of the varied metal and the additivity model (concentration-addition or independent-action) used to predict toxicity. In Cd-Ni-Zn mixtures, the toxicity was less than additive or approximately additive, depending on the concentration (or ion activity) of the varied metal but independent of the additivity model. These results demonstrate that complex interactions of potentially competing toxicity-controlling mechanisms can occur in ternary-metal mixtures but might be predicted by mechanistic bioavailability-based toxicity models.

Prevalence of feral swine disturbance at important archaeological sites over a large landscape in Florida.

Feral swine are globally known as one of the most destructive invasive vertebrates, damaging native habitats, native plants and animals, agriculture, infrastructure, spreading diseases. There has been little quantification on their disturbance to archaeological sites across a broad landscape. Over 6 years we inspected 293 significant archaeological sites for swine disturbance across a vast area. We found a 42% prevalence of swine disturbance among all sites, with prevalence not distinguishable among prehistoric sites, historic sites, and sites with both components. The areas of disturbance mapped within three historic homestead sites showed 5-26% of total site surface area rooted. Disturbance was not evident upon re-inspection of one of these sites after 18 months, indicating how evidence of disturbance can be obscured in this environment. Thus, our observed 42% prevalence of disturbance should be considered a minimum for disturbance occurring through time. Artifacts depths were <10 cm of the surface at 85% of the sites and <20 cm of the surface for 90% of the sites. Feral swine rooting commonly exceeds 20 cm in depth, especially in soft sandy substrates typical of Florida, making the great majority of the studied sites highly vulnerable to artifact damage or displacement.

Effect of age on acute toxicity of cadmium, copper, nickel, and zinc in individual-metal exposures to Daphnia magna neonates.

In previous studies, variability was high among replicate acute cadmium (Cd) Daphnia magna lethality tests (e.g., >10-fold range of median effect concentrations [EC50s]), less among zinc (Zn) tests, and relatively low for copper (Cu) and nickel (Ni) tests. Although the US Environmental Protection Agency's (USEPA's) protocol includes starting toxicity tests with neonates less than 24 h old, the authors hypothesized that age-related differences in sensitivity to metals might occur even within that relatively narrow age range. Daphnia magna neonates were collected during 3 age windows (0-4 h, 10-14 h, and 20-24 h old) and immediately exposed to each of the 4 metals for 48 h using the standard USEPA protocol. In repeated sets of tests during different weeks, the Cd EC50 of the youngest neonates was approximately 10-fold greater than the EC50 of the oldest neonates (i.e., Cd was less toxic to the youngest neonates) and the EC50 of neonates aged 10 h to 14 h was intermediate. Age-related differences were negligible in Cu, Ni, and Zn tests. Therefore, variability in toxicity of Cd may partly be caused by temporal variability in neonate age at the start of toxicity tests. Decreasing the age range of D. magna used in toxicity tests could help to improve the accuracy and precision of toxicity models, particularly for metal mixtures. Environ Toxicol Chem 2017;36:113-119. © 2016 SETAC.

The Use of Field and Mesocosm Experiments to Quantify Effects of Physical and Chemical Stressors in Mining-Contaminated Streams.

Identifying causal relationships between acid mine drainage (AMD) and ecological responses in the field is challenging. In addition to the direct toxicological effects of elevated metals and reduced pH, mining activities influence aquatic organisms indirectly through physical alterations of habitat. The primary goal of this research was to quantify the relative importance of physical (metal-oxide deposition) and chemical (elevated metal concentrations) stressors on benthic macroinvertebrate communities. Mesocosm experiments conducted with natural assemblages of benthic macroinvertebrates established concentration-response relationships between metals and community structure. Field experiments quantified effects of metal-oxide contaminated substrate and showed significant differences in sensitivity among taxa. To predict the recovery of dominant taxa in the field, we integrated our measures of metal tolerance and substrate tolerance with estimates of drift propensity obtained from the literature. Our estimates of recovery were consistent with patterns observed at downstream recovery sites in the NFCC, which were dominated by caddisflies and baetid mayflies. We conclude that mesocosm and small-scale field experiments, particularly those conducted with natural communities, provide an ecologically realistic complement to laboratory toxicity tests. These experiments also control for the confounding variables associated with field-based approaches, thereby supporting causal relationships between AMD stressors and responses.

Can Ingestion of Lead Shot and Poisons Change Population Trends of Three European Birds: Grey Partridge, Common Buzzard, and Red Kite?

Little is known about the magnitude of the effects of lead shot ingestion alone or combined with poisons (e.g., in bait or seeds/granules containing pesticides) on population size, growth, and extinction of non-waterbird avian species that ingest these substances. We used population models to create example scenarios demonstrating how changes in these parameters might affect three susceptible species: grey partridge (Perdix perdix), common buzzard (Buteo buteo), and red kite (Milvus milvus). We added or subtracted estimates of mortality due to lead shot ingestion (4-16% of mortality, depending on species) and poisons (4-46% of mortality) reported in the UK or France to observed mortality of studied populations after models were calibrated to observed population trends. Observed trends were decreasing for partridge (in continental Europe), stable for buzzard (in Germany), and increasing for red kite (in Wales). Although lead shot ingestion and poison at modeled levels did not change the trend direction for the three species, they reduced population size and slowed population growth. Lead shot ingestion at modeled rates reduced population size of partridges by 10%, and when combined with bait and pesticide poisons, by 18%. For buzzards, decrease in mean population size by lead shot and poisons combined was much smaller (≤ 1%). The red kite population has been recovering; however, modeled lead shot ingestion reduced its annual growth rate from 6.5% to 4%, slowing recovery. If mortality from poisoned baits could be removed, the kite population could potentially increase at a rapid annual rate of 12%. The effects are somewhat higher if ingestion of these substances additionally causes sublethal reproductive impairment. These results have uncertainty but suggest that declining or recovering populations are most sensitive to lead shot or poison ingestion, and removal of poisoned baits can have a positive impact on recovering raptor populations that frequently feed on carrion.

A test of the additivity of acute toxicity of binary-metal mixtures of ni with Cd, Cu, and Zn to Daphnia magna, using the inflection point of the concentration-response curves.

Mixtures of metals are often present in surface waters, leading to toxicity that is difficult to predict. To provide data for development of multimetal toxicity models, Daphnia magna neonates were exposed to individual metals (Cd, Cu, Ni, Zn) and to binary combinations of those metals in standard 48-h lethality tests conducted in US Environmental Protection Agency moderately hard reconstituted water with 3 mg dissolved organic carbon (DOC)/L added as Suwannee River fulvic acid. Toxicity tests were performed with mixtures of Ni and 1) Cd, which is considerably more toxic than Ni; 2) Cu, which is less toxic than Cd but more toxic than Ni; and 3) Zn, which has a toxicity threshold similar to Ni. For each combination of metals in the binary mixtures, the concentration of 1 metal was held constant while the second metal was varied through a series that ranged from nonlethal to lethal concentrations; then the roles of the metals were reversed. Inflection points of the concentration-response curves were compared to test for additivity of toxicity. Sublethal concentrations of Ni caused less-than-additive toxicity with Cd, slightly less-than-additive toxicity with Zn, and greater-than-additive toxicity with Cu. One explanation of these results might be competition among the metals for binding to biological ligands and/or dissolved organic matter. Therefore, models might have to incorporate sometimes competing chemical interactions to accurately predict metal-mixture toxicity. Environ Toxicol Chem 2016;35:1843-1851. © 2015 SETAC.

Metal mixture modeling evaluation project: 2. Comparison of four modeling approaches.

As part of the Metal Mixture Modeling Evaluation (MMME) project, models were developed by the National Institute of Advanced Industrial Science and Technology (Japan), the US Geological Survey (USA), HDR|HydroQual (USA), and the Centre for Ecology and Hydrology (United Kingdom) to address the effects of metal mixtures on biological responses of aquatic organisms. A comparison of the 4 models, as they were presented at the MMME workshop in Brussels, Belgium (May 2012), is provided in the present study. Overall, the models were found to be similar in structure (free ion activities computed by the Windermere humic aqueous model [WHAM]; specific or nonspecific binding of metals/cations in or on the organism; specification of metal potency factors or toxicity response functions to relate metal accumulation to biological response). Major differences in modeling approaches are attributed to various modeling assumptions (e.g., single vs multiple types of binding sites on the organism) and specific calibration strategies that affected the selection of model parameters. The models provided a reasonable description of additive (or nearly additive) toxicity for a number of individual toxicity test results. Less-than-additive toxicity was more difficult to describe with the available models. Because of limitations in the available datasets and the strong interrelationships among the model parameters (binding constants, potency factors, toxicity response parameters), further evaluation of specific model assumptions and calibration strategies is needed.

Acute toxicity of binary and ternary mixtures of Cd, Cu, and Zn to Daphnia magna.

Standard static-exposure acute lethality tests were conducted with Daphnia magna neonates exposed to binary or ternary mixtures of Cd, Cu, and Zn in moderately hard reconstituted water that contained 3 mg dissolved organic carbon/L added as Suwannee River fulvic acid. These experiments were conducted to test for additive toxicity (i.e., the response to the mixture can be predicted by combining the responses obtained in single-metal toxicity tests) or nonadditive toxicity (i.e., the response is less than or greater than additive). Based on total metal concentrations (>90% dissolved) the toxicity of the tested metal mixtures could be categorized into all 3 possible additivity categories: less-than-additive toxicity (e.g., Cd-Zn and Cd-Cu-Zn mixtures and Cd-Cu mixtures when Cu was titrated into Cd-containing waters), additive toxicity (e.g., some Cu-Zn mixtures), or more-than-additive toxicity (some Cu-Zn mixtures and Cd-Cu mixtures when Cd was titrated into Cu-containing waters). Exposing the organisms to a range of sublethal to supralethal concentrations of the titrated metal was especially helpful in identifying nonadditive interactions. Geochemical processes (e.g., metal-metal competition for binding to dissolved organic matter and/or the biotic ligand, and possibly supersaturation of exposure waters with the metals in some high-concentration exposures) can explain much of the observed metal-metal interactions. Therefore, bioavailability models that incorporate those geochemical (and possibly some physiological) processes might be able to predict metal mixture toxicity accurately.