Influence of Test Method Variables on Sensitivity of Neocloeon Triangulifer to a Reference Toxicant in Short-Term, Effluent Style Evaluations.

Recent literature has demonstrated the sensitivity of mayflies to environmental contaminants. However, to date, there are no methods approved by the US Environmental Protection Agency for using sensitive insects like mayflies in whole-effluent toxicity or receiving water toxicity tests. The parthenogenetic mayfly Neocloeon triangulifer has been shown to be amenable to continuous culture in the laboratory, and methods have been described for its use in both acute and chronic toxicity studies. The goal of the present study was to investigate aspects of N. triangulifer testing and culturing methods that might require adjustment so that they are applicable for testing effluents and receiving waters in a short-term exposure. To this end, the influence of organism age, test duration, and test temperature on sensitivity to NaCl as a reference toxicant were tested (concentrations ranging from 182 to 2489 mg/L). Further studies were conducted to assess the utility of commercially available diets and the influence of nutrient amendment of water on organism growth and sensitivity. Seven-day NaCl tests started with less than 24-h-old larvae were similar in sensitivity to 14-day and full life chronic tests, and were much more sensitive than those started with 7-day-old organisms. Reducing test temperature from 25 °C to 22 °C had a minor influence on culture timing, and little impact on sensitivity to NaCl. In other experiments, reconstituted test water supplemented with nutrients to potentially improve in-test food quality had minimal effect on growth at 7 days and did not significantly alter acute sensitivity to NaCl relative to unamended reconstituted water. A suitable commercially available, ready-to-feed diet substitute for cultured diatoms was not found. Testing N. triangulifer in effluents or receiving waters with the methods recommended will complement similar methods for Ceriodaphnia dubia. Environ Toxicol Chem 2022;41:2758-2768. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Acute Toxicity of Major Geochemical Ions to Fathead Minnows (Pimephales promelas): Part B-Modeling Ion Toxicity.

Mathematical models are presented for the acute median lethal concentrations of major geochemical ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO4 2- , HCO3 - /CO3 2- ) to fathead minnows (Pimephales promelas), based on an extensive series of experiments presented in a companion article. Toxicity relationships across different dilution waters, individual salts, and salt mixtures suggest six independent mechanisms of toxicity to consider in modeling efforts, including Mg/Ca-specific toxicity, osmolarity-related toxicity, SO4 -specific toxicity, K-specific toxicity, effects of high pH/alkalinity, and a multiple ion-related toxicity at low Ca distinct from the other mechanisms. Models are evaluated using chemical activity-based exposure metrics pertinent to each mechanism, but concentration-based alternative models that are simpler to apply are also addressed. These models are compared to those previously provided for Ceriodaphnia dubia, and various issues regarding their application to risk assessments are discussed. Environ Toxicol Chem 2022;41:2095-2106. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Current ecotoxicity testing needs among selected U.S. federal agencies.

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies' regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations.

Acute Toxicity of Major Geochemical Ions to Fathead Minnows (Pimephales Promelas): Part A-Observed Relationships for Individual Salts and Salt Mixtures.

The results of a series of experiments on the acute toxicity of major geochemical ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO4 2- , HCO3 - /CO3 2- ) to fathead minnows (Pimephales promelas) are reported. Tests of individual major ion salts in various dilution waters demonstrated that the toxicities of Na, Mg, and K salts decrease as the overall ion content of the dilution water increases. For Na and Mg salts, this is attributable to Ca content as previously reported for Ceriodaphnia dubia. For K salts, the cause is unclear, but it is not due to Na as reported for C. dubia. In an unregulated test at high pH (9.3), NaHCO3 was also found to be twice as toxic compared to when the pH was reduced to 8.4. Experiments with binary salt mixtures indicated the existence of multiple independent mechanisms of action. These include K-specific toxicity and Ca/Mg-specific toxicity previously reported for C. dubia, but also apparent toxicities related to SO4 and to high pH/alkalinity in CO3 /HCO3 -dominated exposures. Previous work with C. dubia also suggested a general ion toxicity involving all ions that was correlated with osmolarity. For fathead minnow, similar correlations were observed, but multiple mechanisms were indicated. At higher Ca, this general toxicity could be attributable to osmotic effects, but at lower Ca, osmolarity may be more a covariate than a cause, with this toxicity being related to a combined effect of ions other than via osmolarity. Environ Toxicol Chem 2022;41:2078-2094. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Daphnia magna and Ceriodaphnia dubia Have Similar Sensitivity in Standard Acute and Chronic Toxicity Tests.

The cladocerans Daphnia magna and Ceriodaphnia dubia have been used for decades to assess the hazards of chemicals and effluents, but toxicity data for these species have traditionally been treated separately. Numerous standard acute and chronic test guidelines have been developed for both species. In the present study, data were compiled and curated for acute survival (48 h) and growth and reproduction tests with D. magna (21 days chronic) and C. dubia (7 days chronic) toxicity assays. Orthogonal regressions were developed to statistically compare the acute and chronic sensitivity of D. magna and C. dubia across a diversity of chemicals and modes of action. Acute orthogonal regressions between D. magna and D. pulex, a widely accepted surrogate species, were used to set a data-driven benchmark for what would constitute a suitable D. magna surrogate. The results indicate that there is insufficient evidence to suggest a difference in acute or chronic sensitivity of D. magna and C. dubia in standard toxicity tests. Further, the variability in the acute D. magna and C. dubia regressions were of the same magnitude as that in D. magna and D. pulex regressions. Slope and y-intercept values were also comparable. The absence of significant differences in toxicity values suggests similar species sensitivity in standard tests across a range of chemical classes and modes of action. Environ Toxicol Chem 2022;41:134-147. © 2021 SETAC.

Method Development for a Short-Term 7-Day Toxicity Test with Unionid Mussels.

The US Environmental Protection Agency's short-term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short-term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field-collected effluent in 7-day exposures. Both mussel species were equally or more sensitive (more than two-fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7-day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7-day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%-52% length increase) when the 1- or 2-week-old mussels were fed 2 ml twice daily and the 3-week-old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314-520 mg Cl/L) among the three age groups, indicating that an age range of 1- to 3-week-old mussels can be used for a 7-day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7-day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392-3409. © 2021 SETAC.

Bioaccumulation of Bis-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate and Mono-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate by Lumbriculus variegatus.

The brominated flame retardant bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBPH) is used widely in consumer items including polyurethane foam used in furniture. Information on its bioaccumulation in aquatic species is limited. In the current study, sediment bioaccumulation tests with the oligochaete Lumbriculus variegatus were performed on a spiked natural sediment equilibrated for 14.5 months. Analysis showed the TBPH used to spike the sediment contained a small amount (0.046% by mass) of mono-(2-ethylhexyl)-3,4,5,6-tetrabromophthalate (TBMEHP), a potential biotransformation product of the parent chemical. Steady-state biota-sediment accumulation factors (BSAFs) of 0.254 and 1.50 (kg organic carbon/kg lipid) were derived for TBPH and TBMEHP, respectively. TBPH had biphasic elimination behavior where 94% of the body burden was depleted within the first 12 h of elimination (i.e., half-life of 1.2 h or less) and the remaining 6% eliminated very slowly thereafter (half-life of 15 days). There was little evidence for biotransformation of either chemical by L. variegatus. This investigation confirms the extremely hydrophobic behavior of TBPH and its impact on its bioavailability.

Mode of Action Classifications in the EnviroTox Database: Development and Implementation of a Consensus MOA Classification.

Multiple mode of action (MOA) frameworks have been developed in aquatic ecotoxicology, mainly based on fish toxicity. These frameworks provide information on a key determinant of chemical toxicity, but the MOA categories and level of specificity remain unique to each of the classification schemes. The present study aimed to develop a consensus MOA assignment within EnviroTox, a curated in vivo aquatic toxicity database, based on the following MOA classification schemes: Verhaar (modified) framework, Assessment Tool for Evaluating Risk, Toxicity Estimation Software Tool, and OASIS. The MOA classifications from each scheme were first collapsed into one of 3 categories: non-specifically acting (i.e., narcosis), specifically acting, or nonclassifiable. Consensus rules were developed based on the degree of concordance among the 4 individual MOA classifications to attribute a consensus MOA to each chemical. A confidence rank was also assigned to the consensus MOA classification based on the degree of consensus. Overall, 40% of the chemicals were classified as narcotics, 17% as specifically acting, and 43% as unclassified. Sixty percent of chemicals had a medium to high consensus MOA assignment. When compared to empirical acute toxicity data, the general trend of specifically acting chemicals being more toxic is clearly observed for both fish and invertebrates but not for algae. EnviroTox is the first approach to establishing a high-level consensus across 4 computationally and structurally distinct MOA classification schemes. This consensus MOA classification provides both a transparent understanding of the variation between MOA classification schemes and an added certainty of the MOA assignment. In terms of regulatory relevance, a reliable understanding of MOA can provide information that can be useful for the prioritization (ranking) and risk assessment of chemicals. Environ Toxicol Chem 2019;38:2294-2304. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Bioaccumulation of Highly Hydrophobic Chemicals by Lumbriculus variegatus.

Bioaccumulation of highly hydrophobic chemicals (log KOW > 8) from contaminated sediments by Lumbriculus variegatus has been studied for relatively few chemicals, and the measured and model predicted biota-sediment accumulation factors (BSAFs) can differ by orders of magnitude. In the current study, sediment bioaccumulation tests with L. variegatus were performed on sediments dosed with chemicals having a wide range of predicted n-octanol/water partition coefficients (KOW; 106-1018), including some higher than most highly hydrophobic chemicals studied to date. The highly hydrophobic chemicals had biphasic elimination kinetics with compartments A and B having fast and slow elimination kinetics, respectively, and for compartment B, elimination followed first-order kinetics. For compartment A with fast elimination kinetics, the mechanism and its kinetic-order could not be determined. Steady-state BSAFs (kg organic carbon/kg lipid) of 0.015, 0.024, and 0.022 were derived for tetradecachloro-p-terphenyl, tetradecachloro-m-terphenyl, and octadecachloro-p-quaterphenyl, respectively. The high uncertainty in predicted KOWs for highly hydrophobic chemicals limited the comparison and evaluation of predicted BSAFs from the Arnot-Gobas food web model and BSAFs measured in this study. The results of this study point to the need to perform dietary assimilation efficiency studies with highly hydrophobic compounds to resolve uncertainties surrounding the estimation of their KOW and the need to understand mechanism and models for the biphasic elimination kinetics.

Chronic toxicity of major ion salts and their mixtures to Ceriodaphnia dubia.

In previous work we intensively studied the acute responses of the cladoceran Ceriodaphnia dubia to major geochemical ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO42- , and HCO3- /CO32- ), culminating in the development of models to predict acute toxicity from ionic composition. To quantitatively evaluate whether the toxicological behavior of major ions observed for C. dubia extends to chronic toxicity, we conducted 58 chronic toxicity tests with individual major salts and binary mixtures thereof. Chronic responses paralleled those demonstrated previously for acute exposure, specifically 1) similar relative toxicity of individual salts; 2) different Na salts showing similar potency when exposure is expressed as osmolarity; 3) toxicity of Mg, Ca, and K salts related to cation activity; 4) decreased toxicity of Na and Mg salts when Ca activity is increased at less than toxic concentrations; 5) additive behavior for salt mixtures sharing a common cation; and 6) independent behavior for salt mixtures with dissimilar cations, except Mg/Ca mixtures which appeared additive. Acute-to-chronic ratios were fairly consistent among salts, with values of approximately 1.8 for acute 50% lethal concentration (LC50) to chronic 50% effect concentration (EC50) and 2.8 for LC50/EC20 when expressed on an activity basis. Adjusting the previous acute toxicity model for acute-to-chronic ratios yielded chronic models that predict chronic toxicity within the range of intertest variability. Because these models are informed by a wide range of ion mixtures, they should provide robust assessment tools for natural waters enriched with major ions. Environ Toxicol Chem 2019;38:769-783. © Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

An International Perspective on the Tools and Concepts for Effluent Toxicity Assessments in the Context of Animal Alternatives: Reduction in Vertebrate Use.

Since the 1940s, effluent toxicity testing has been used to assess potential ecological impacts of effluents and help determine necessary treatment options for environmental protection prior to release. Strategic combinations of toxicity tests, analytical tools, and biological monitoring have been developed. Because the number of vertebrates utilized in effluent testing is thought to be much greater than that used for individual chemical testing, there is a new need to develop strategies to reduce the numbers of vertebrates (i.e., fish) used. This need will become more critical as developing nations begin to use vertebrates in toxicity tests to assess effluent quality. A workshop was held to 1) assess the state of science in effluent toxicity testing globally; 2) determine current practices of regulators, industry, private laboratories, and academia; and 3) explore alternatives to vertebrate (fish) testing options and the inclusion of modified/new methods and approaches in the regulatory environment. No single approach was identified, because of a range of factors including regulatory concerns, validity criteria, and wider acceptability of alternatives. However, a suite of strategies in a weight-of-evidence approach would provide the flexibility to meet the needs of the environment, regulators, and the regulated community; and this "toolbox" approach would also support reduced reliance on in vivo fish tests. The present Focus article provides a brief overview of wastewater regulation and effluent testing approaches. Alternative methodologies under development and some of the limitations and barriers to regulatory approaches that can be selected to suit individual country and regional requirements are described and discussed. Environ Toxicol Chem 2018;37:2745-2757. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The acute toxicity of major ion salts to Ceriodaphnia dubia. III. Mathematical models for mixture toxicity.

Based on previous research on the acute toxicity of major ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO42- , and HCO3- /CO32- ) to Ceriodaphnia dubia, a mathematical model was developed for predicting the median lethal concentration (LC50) for any ion mixture, excepting those dominated by K-specific toxicity. One component of the model describes a mechanism of general ion toxicity to which all ions contribute and predicts LC50s as a function of osmolarity and Ca activity. The other component describes Mg/Ca-specific toxicity to apply when such toxicity exceeds the general ion toxicity and predicts LC50s as a function of Mg and Ca activities. This model not only tracks well the observed LC50s from past research used for model development but also successfully predicts LC50s from new toxicity tests on synthetic mixtures of ions emulating chemistries of various ion-enriched effluents and receiving waters. It also performs better than a previously published model for major ion toxicity. Because of the complexities of estimating chemical activities and osmolarity, a simplified model based directly on ion concentrations was also developed and found to provide useful predictions. Environ Toxicol Chem 2018;37:247-259. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

The acute toxicity of major ion salts to Ceriodaphnia dubia. II. Empirical relationships in binary salt mixtures.

Many human activities increase concentrations of major geochemical ions (Na+1 , K+1 , Ca+2 , Mg+2 , Cl-1 , SO4-2 , and HCO3-1 /CO3-2 ) in freshwater systems, and can thereby adversely affect aquatic life. Such effects involve several toxicants, multiple toxicity mechanisms, various ion interactions, and widely varying ion compositions across different water bodies. Previous studies of individual salt toxicities have defined some useful relationships; however, adding single salts to waters results in atypical compositions and does not fully address mixture toxicity. To better understand mechanisms and interactions for major ion toxicity, 29 binary mixture experiments, each consisting of 7 to 8 toxicity tests, were conducted on the acute toxicity of major ion salts and mannitol to Ceriodaphnia dubia. These tests showed multiple mechanisms of toxicity, including: 1) nonspecific ion toxicity, correlated with osmolarity and to which all ions contribute; and 2) cation-dependent toxicities for potassium (K), magnesium (Mg), and calcium (Ca) best related to their chemical activities. These mechanisms primarily operate independently, except for additive toxicity of Mg-dependent and Ca-dependent toxicities. These mixture studies confirmed ameliorative effects of Ca on sodium (Na) and Mg salt toxicities and of Na on K salt toxicity, and further indicated lesser ameliorative effects of Ca on K salt toxicity and Mg on Na salt toxicity. These results provide a stronger basis for assessing risks from the complex mixtures of ions found in surface waters. Environ Toxicol Chem 2017;36:1525-1537. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Alternative approaches to vertebrate ecotoxicity tests in the 21st century: A review of developments over the last 2 decades and current status.

The need for alternative approaches to the use of vertebrate animals for hazard assessment of chemicals and pollutants has become of increasing importance. It is now the first consideration when initiating a vertebrate ecotoxicity test, to ensure that unnecessary use of vertebrate organisms is minimized wherever possible. For some regulatory purposes, the use of vertebrate organisms for environmental risk assessments has been banned; in other situations, the number of organisms tested has been dramatically reduced or the severity of the procedure refined. However, there is still a long way to go to achieve a complete replacement of vertebrate organisms to generate environmental hazard data. The development of animal alternatives is based not just on ethical considerations but also on reducing the cost of performing vertebrate ecotoxicity tests and in some cases on providing better information aimed at improving environmental risk assessments. The present Focus article provides an overview of the considerable advances that have been made toward alternative approaches for ecotoxicity assessments over the last few decades. Environ Toxicol Chem 2016;35:2637-2646. © 2016 SETAC.

The acute toxicity of major ion salts to Ceriodaphnia dubia: I. influence of background water chemistry.

The ions Na+ , K+ , Ca2+ , Mg2+ , Cl- , SO42- , and HCO3- /CO32- (referred to in the present study as "major ions") are present in all freshwaters and physiologically required by aquatic organisms but can increase to harmful levels from a variety of anthropogenic activities. It is also known that the toxicities of major ion salts can vary depending on the concentrations of other ions, and understanding these relationships is key to establishing appropriate environmental limits. The authors present a series of experiments with Ceriodaphnia dubia to evaluate the acute toxicity of 12 major ion salts and to determine how toxicity of these salts varies as a function of background water chemistry. All salts except CaSO4 and CaCO3 were acutely toxic below saturation, with the lowest median lethal concentrations found for K salts. All 10 salts that showed toxicity also showed some degree of reduced toxicity as the ionic content of the background water increased. Experiments that independently varied Ca:Mg ratio, Na:K ratio, Cl:SO4 ratio, and alkalinity/pH demonstrated that Ca concentration was the primary factor influencing the toxicities of Na and Mg salts, whereas the toxicities of K salts were primarily influenced by the concentration of Na. These experiments also indicated multiple mechanisms of toxicity and suggested important aspects of dosimetry; the toxicities of K, Mg, and Ca salts were best related to the chemical activity of the cation, whereas the toxicities of Na salts also reflected an influence of the anions and were well correlated with osmolarity. Understanding these relationships between major ion toxicity and background water chemistry should aid in the development of sensible risk-assessments and regulatory standards. Environ Toxicol Chem 2016;35:3039-3057. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Sediment Bioaccumulation Test with Lumbriculus variegatus: Effects of Organism Loading.

At contaminated sediment sites, the bioavailability of contaminants in sediments is assessed using sediment-bioaccumulation tests with Lumbriculus variegates (Lv). The testing protocols recommend that ratio of total organic carbon (TOC) in sediment to L. variegatus (dry weight) (TOC/Lv) should be no less than 50:1. Occasionally, this recommendation is not followed, especially with sediments having low TOC, e.g., <1 %. This study evaluated the impacts and resulting biases in the testing results when the recommendation of "no less than 50:1" is not followed. In the study, seven sediments were tested with a series of TOC/Lv ratios that spanned the recommendation. With increasing loading of organisms, growth of the organisms decreased in six of the seven sediments tested. Residues of polychlorinated biphenyls (PCBs) in the L. variegatus were measured in six of the seven sediments tested, and differences in PCB residues among loading ratios across all sediments were small, i.e., ±50 %, from those measured at the minimum recommended ratio of 50:1 TOC/Lv. In all sediment, PCB residues increased with increasing loading of the organisms for the mono-, di-, and tri-chloro-PCBs. For tetra-chloro and heavier PCBs, residues increased with increasing loading of organisms for only two of the six sediments. PCB residues were not significantly different between TOC/Lv loadings of 50:1 and mid-20:1 ratios indicating that equivalent results can be obtained with TOC/Lv ratios into the mid-20:1 ratios. Overall, the testing results suggest that when testing recommendation of 50:1 TOC/Lv is not followed, potential biases in the biota-sediment accumulations factors from the sediment-bioaccumulation test will be small.

Using an interlaboratory study to revise methods for conducting 10-d to 42-d water or sediment toxicity tests with Hyalella azteca.

Studies have been conducted to refine US Environmental Protection Agency, ASTM International, and Environment Canada standard methods for conducting 42-d reproduction tests with Hyalella azteca in water or in sediment. Modifications to the H. azteca method include better-defined ionic composition requirements for exposure water (i.e., >15 mg/L of chloride and >0.02 mg/L of bromide) and improved survival, growth, and reproduction with alternate diets provided as increased rations over time in water-only or whole-sediment toxicity tests. A total of 24 laboratories volunteered to participate in the present interlaboratory study evaluating the performance of H. azteca in 42-d studies in control sand or control sediment using the refined methods. Improved growth and reproduction of H. azteca was observed with 2 alternate diets of 1) ramped diatoms (Thalassiosira weissflogii) + ramped Tetramin or 2) yeast-cerophyll-trout chow (YCT) + ramped Tetramin, especially when compared with results from the traditional diet of 1.8 mg YCT/d. Laboratories were able to meet proposed test acceptability criteria and in most cases had lower variation in growth or reproduction compared with previous interlaboratory studies using the traditional YCT diet. Laboratory success in conducting 42-d H. azteca exposures benefited from adherence to several key requirements of the detailed testing, culturing, and handling methods. Results from the present interlaboratory study are being used to help revise standard methods for conducting 10-d to 42-d water or sediment toxicity exposures with H. azteca. Environ Toxicol Chem 2016;35:2439-2447. © 2016 SETAC.

Sediment bioaccumulation test with Lumbriculus variegatus: effects of feeding.

Standard sediment-bioaccumulation test methods specify that Lumbriculus variegatus should not be fed during the 28-day exposure. This lack of feeding can lead to decreases in L. variegatus weight and lipid content during the 28-day exposure period. Differences in intrinsic nutritional content of sediments could lead to additional variability in organism performance and/or contaminant uptake. To evaluate the potential benefits of feeding, sediment-bioaccumulation tests were performed comparing treatments with and without supplemental feeding with tropical fish food and also comparing performance food introduced as blended slurry versus fine flakes. The ration of food provided had to be limited to 6 mg/300-mL beaker with 250 mg of L. variegatus (ww) receiving three feedings per week to maintain acceptable dissolved oxygen (DO) in the test chambers. Relative weight change during exposure varied across sediments in the absence of food from very little change to as much as a 40 % decrease from starting weight. Feeding slurry and flake foods increased the total weight of recovered organisms by 32 and 48 %, respectively, but they did not decrease variability in weight changes across sediments. Lipid contents of the organisms decreased similarly across all feeding treatments during the test. At test termination, lipid contents of L. variegatus across unfed, slurry-fed, and flake-fed treatments were not significantly different per Tukey's honest significant difference test with 95 % family-wise confidence. Feeding resulted in polychlorinated biphenyl residues in L. variegatus being generally slightly less (median 78 %) and slightly greater (median 135 %) than the unfed treatments with slurry and flake formulated foods, respectively.

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.