Comparisons of PNEC derivation logic flows under example regulatory schemes and implications for ecoTTC.

Derivation of Predicted No Effect Concentrations (PNECs) for aquatic systems is the primary deterministic form of hazard extrapolation used in environmental risk assessment. Depending on the data availability, different regulatory jurisdictions apply application factors (AFs) to the most sensitive measured endpoint to derive the PNEC for a chemical. To assess differences in estimated PNEC values, two PNEC determination methodologies were applied to a curated public database using the EnviroTox Platform (www.EnviroToxdatabase.org). PNECs were derived for 3647 compounds using derivation procedures based on example US EPA and a modified European Union chemical registration procedure to allow for comparisons. Ranked probability distributions of PNEC values were developed and 5th percentile values were calculated for the entire dataset and scenarios where full acute or full chronic data sets were available. The lowest PNEC values indicated categorization based on chemical attributes and modes of action would lead to improved extrapolations. Full acute or chronic datasets gave measurably higher 5th percentile PNEC values. Algae were under-represented in available ecotoxicity data but drove PNECs disproportionately. Including algal inhibition studies will be important in understanding chemical hazards. The PNEC derivation logic flows are embedded in the EnviroTox Platform providing transparent and consistent PNEC derivations and PNEC distribution calculations.

SSDs Revisited: Part I-A Framework for Sample Size Guidance on Species Sensitivity Distribution Analysis.

We propose a framework on sample size for species sensitivity distribution (SSD) analyses, with perspectives on Bayesian, frequentist, and even nonparametric approaches to estimation. The intent of a statistical sample size analysis is to ensure that the implementation of a statistical model will satisfy a minimum performance standard when relevant conditions are met. It requires that a statistical model be fully specified and that the means of measuring its performance as a function of sample size be detailed. Defining the model conditions under which sample size is calculated is often the most difficult, and important, aspect of sample size analysis because if the model is not representative, then the sample size analysis will provide incorrect guidance. Definitive guidance on sample size requires general agreement on representative models and their performance from stakeholders in important domains such as chemical safety assessments involving government regulators and industry; the present study provides an initial framework that could be used to this end in the future. In addition, our analysis provides immediate value for understanding how well current SSD analyses perform under a few basic models, sample sizes, and quantitative performance criteria. The results confirm that many analyses are adequately sized to estimate hazardous concentration percentile values (typically the 5th percentile for chemical hazard assessments). However, on the low end of sizes seen in common practice, hazardous concentration estimates can be more than 1 order of magnitude greater than the model-defined value. Environ Toxicol Chem 2019;38:1514-1525. © 2019 SETAC.

SSDs revisited: part II-practical considerations in the development and use of application factors applied to species sensitivity distributions.

Application factors are routinely applied in the extrapolation of laboratory aquatic toxicity data to ensure protection from exposure to chemicals in the natural environment. The magnitude of the application factor is both a scientific and a policy decision, but in any case, it should be rooted in scientific knowledge so as to not be arbitrary. Information-rich chemicals are often subjected to species sensitivity distribution (SSD) analysis to transparently describe certain aspects of assessment uncertainty and are normally subjected to much smaller application factors than screening information data sets. We describe a new set of tools useful to assess the quality of SSDs. Twenty-two data sets and 19 chemicals representing agrochemicals, biocides, surfactants, metals, and common wastewater contaminants were compiled to demonstrate how the tools can be used. "Add-one-in" and "leave-one-out" simulations were used to investigate SSD robustness and develop quantitative evidence for the use of application factors. Theoretical new toxicity data were identified for add-one-in simulations based on the expected probabilities necessary to lower the hazardous concentration to 5% of a species (HC5) by a factor of 2, 3, 5, or 10. Simulations demonstrate the basis for application factors in the range of 1 to 5 for well-studied chemicals with high-quality SSDs. Leave-one-out simulations identify the fact that the most influential values in the SSD come from the extremes of the sensitive and tolerant toxicity values. Mesocosm and field data consistently demonstrate that HC5s are conservative, further justifying the use of small application factors for high-quality SSDs. Environ Toxicol Chem 2019;38:1526-1541. © 2019 SETAC.

Mode of Action (MOA) Assignment Classifications for Ecotoxicology: An Evaluation of Approaches.

The mode of toxic action (MOA) is recognized as a key determinant of chemical toxicity and as an alternative to chemical class-based predictive toxicity modeling. However, MOA classification has never been standardized in ecotoxicology, and a comprehensive comparison of classification tools and approaches has never been reported. Here we critically evaluate three MOA classification methodologies using an aquatic toxicity data set of 3448 chemicals, compare the approaches, and assess utility and limitations in screening and early tier assessments. The comparisons focused on three commonly used tools: Verhaar prediction of toxicity MOA, the U.S. Environmental Protection Agency (EPA) ASsessment Tool for Evaluating Risk (ASTER) QSAR (quantitative structure activity relationship) application, and the EPA Mode of Action and Toxicity (MOAtox) database. Of the 3448 MOAs predicted using the Verhaar scheme, 1165 were classified by ASTER, and 802 were available in MOAtox. Of the subset of 432 chemicals with MOA assignments for each of the three schemes, 42% had complete concordance in MOA classification, and there was no agreement for 7% of the chemicals. The research shows the potential for large differences in MOA classification between the five broad groups of the Verhaar scheme and the more mechanism-based assignments of ASTER and MOAtox. Harmonization of classification schemes is needed to use MOA classification in chemical hazard and risk assessment more broadly.

Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians--screening for estrogen, androgen and thyroid hormone disruption.

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

Environmental properties of long-chain alcohols. Part 2: Structure-activity relationship for chronic aquatic toxicity of long-chain alcohols.

Daphnia magna reproduction tests were performed with C(10), C(12), C(14) and C(15) alcohols to establish a structure-activity relationship of chronic effects of long-chain alcohols. The data generation involved substantial methodological efforts due to the exceptionally rapid biodegradability of the test substances and the need to test as close as possible to their water solubility limits. Test concentrations were determined by GC-MS before and after test solution renewal. Whereas apparent toxicity based on survival and reproduction increased with increasing C-chain lengths up to C(14), observations of toxicity to C(15) alcohol were not in line with lower chain lengths due to the lack of toxicity below the level of water solubility. When omitting C(15), the slope of most (Q)SARs approach -1, being consistent with the expectation of a non-polar narcotic mode of action. Further testing at higher chain lengths is not sensible due to progressively lower solubility, at remaining biodegradability. Effects on mortality and reproduction are not expected below the level of water solubility.

Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test?

The fish acute toxicity test is a mandatory component in the base set of data requirements for ecotoxicity testing. The fish acute toxicity test is not compatible with most current animal welfare legislation because mortality is the primary endpoint and it is often hypothesized that fish suffer distress and perhaps pain. Animal alternative considerations have also been incorporated into new European REACH regulations through strong advocacy for the reduction of testing with live animals. One of the most promising alternative approaches to classical acute fish toxicity testing with live fish is the fish embryo toxicity (FET) test. The FET has been a mandatory component in routine whole effluent testing in Germany since 2005 and has already been standardized at the international level. In order to analyze the applicability of the FET also in chemical testing, a comparative re-evaluation of both fish and fish embryo toxicity data was carried out for a total of 143 substances, and statistical approaches were developed to evaluate the correlation between fish and fish embryo toxicity data. Results confirm that fish embryo tests are neither better nor worse than acute fish toxicity tests and provide strong scientific support for the FET as a surrogate for the acute fish toxicity test.

Assessment of the environmental risk of long-chain aliphatic alcohols.

An environmental assessment of long-chain alcohols (LCOH) has recently been conducted under the OECD SIDS High Production Volume (HPV) Program via the Global International Council of Chemical Associations (ICCA) Aliphatic Alcohols Consortium. LCOH are used primarily as intermediates, as a precursor to alcohol-based surfactants and as alcohol per se in a wide variety of consumer product applications. Global production volume is approximately 1.58 million metric tonnes. The OECD HPV assessment covers linear to slightly branched LCOH ranging from 6 to 22 alkyl carbons (C). LCOH biodegrade exceptionally rapidly in the environment (half-lives on the order of minutes); however, due to continuous use and distribution to wastewater treatment systems, partitioning properties, biodegradation of alcohol-based surfactants, and natural alcohol sources, LCOH are universally detected in wastewater effluents. An environmental risk assessment of LCOH is presented here by focusing on the most prevalent and toxic members of the linear alcohols, specifically, from C(12-15). The assessment includes environmental monitoring data for these chain lengths in final effluents of representative wastewater treatment plants and covers all uses of alcohol (i.e., the use of alcohol as a substance and as an intermediate for the manufacturing of alcohol-based surfactants). The 90th percentile effluent discharge concentration of 1.979microg/L (C(12)-C(15)) was determined for wastewater treatment plants in 7 countries. Chronic aquatic toxicity studies with Daphnia magna demonstrated that between C(13) and C(15) LCOH solubility became a factor and that the structure-activity relationship was characterized by a toxicity maximum between C(13) and C(14). Above C(14) the LCOH was less toxic and become un-testable due to insolubility. Risk quotients based on a toxic units (TU) approach were determined for various scenarios of exposure and effects extrapolation. The global average TU ranged from 0.048 to 0.467 depending on the scenario employed suggesting a low risk to the environment. The fact that environmental exposure calculations include large fractions of naturally derived alcohol from animal, plant, and microbially mediated biotransformations further supports a conclusion of low risk.

Aquatic risk assessment of alcohol ethoxylates in North America and Europe.

An environmental risk assessment for alcohol ethoxylates (AE) is presented that integrates wastewater treatment plant monitoring, fate, and ecotoxicity research with a new application of mixture toxicity theory based on simple similar concentration addition of AE homologs in a species-sensitivity distribution (SSD) context. AEs are nonionic surfactants composed of a homologous series of molecules that range in alkyl chain length from 12 to 18 carbons and ethoxylates from 0 to 18 units. Chronic ecotoxicity of AE is summarized for 17 species in 60 tests and then normalized to monitoring data for AE mixtures. To do so, chronic aquatic toxicity was first expressed as EC10 per species (the concentration predicted to cause a 10% reduction in an important ecological endpoint). Normalization integrated several new quantitative structure-activity relationships for algae, daphnids, fish, and mesocosms and provided an interpretation of toxicity test data as a function of individual homologs in an AE mixture. SSDs were constructed for each homolog and the HC5 (hazardous concentration protective of 95% of species based on a small biological effect [the chronic EC10]) was predicted. Total mass of AE in monitored effluents from 29 sites in Europe, Canada, and the United States averaged 6.8, 2.8, and 3.55 microg/L, respectively. For risk assessment purposes, correction of exposure to account for fatty alcohol derived from sources other than AE and for sorbed components based on experimental evidence was used to determine AE concentrations in undiluted (100%) effluents from North America and Europe. Exposure and effect findings were integrated in a toxic unit (TU)-based model that considers the measured distribution of individual AE homologs in effluent with their corresponding SSDs. Use of environmentally relevant exposure corrections (bioavailability and accounting for AE-derived alcohol) resulted in TUs ranging from 0.015 to 0.212. Low levels of risk are concluded for AE in the aquatic environments of Europe and North America.

Ecotoxicity quantitative structure-activity relationships for alcohol ethoxylate mixtures based on substance-specific toxicity predictions.

Traditionally, ecotoxicity quantitative structure-activity relationships (QSARs) for alcohol ethoxylate (AE) surfactants have been developed by assigning the measured ecotoxicity for commercial products to the average structures (alkyl chain length and ethoxylate chain length) of these materials. Acute Daphnia magna toxicity tests for binary mixtures indicate that mixtures are more toxic than the individual AE substances corresponding with their average structures (due to the nonlinear relation of toxicity with structure). Consequently, the ecotoxicity value (expressed as effects concentration) attributed to the average structures that are used to develop the existing QSARs is expected to be too low. A new QSAR technique for complex substances, which interprets the mixture toxicity with regard to the "ethoxymers" distribution (i.e., the individual AE components) rather than the average structure, was developed. This new technique was then applied to develop new AE ecotoxicity QSARs for invertebrates, fish, and mesocosms. Despite the higher complexity, the fit and accuracy of the new QSARs are at least as good as those for the existing QSARs based on the same data set. As expected from typical ethoxymer distributions of commercial AEs, the new QSAR generally predicts less toxicity than the QSARs based on average structure.

Predicting the sorption of fatty alcohols and alcohol ethoxylates to effluent and receiving water solids.

Alcohol ethoxylates (AEs) are an important group of nonionic surfactants. Commercial AEs consist of a mixture of several homologues of varying carbon chain length (Cx) and degree of ethoxylation (EOy). The major disposal route of AE is down the drain to municipal wastewater treatment plants that discharge into receiving surface waters. Sorption of AE homologues onto activated sludge and river water solids is an important factor in assessing exposure of AE in the environment. This study presents the experimental determination of sorption coefficients for a wide array of AE homologues including five alcohols under environmentally relevant conditions and combines these data with literature data to generate a predictive model for the sorption of AEs in the environment. These results demonstrate that sorption can be effectively modeled using a log Kd vs. Cx and EOy predictive equation having the form log Kd = 0.331C - 0.00897EO - 1.126(R2 = 0.64).

Acute and chronic aquatic toxicity structure-activity relationships for alcohol ethoxylates.

Acute and chronic toxicity tests using the cladoceran Daphnia magna were conducted on several alcohol ethoxylate surfactants. Exposure and homologue distributions were confirmed using specific analytical methods. These data were used to test currently available acute structure-activity relationships (SARs) and to develop a new chronic SAR to extrapolate test data to effluent or receiving water mixtures. Existing acute SARs adequately predicted the toxicity of these materials with an r(2) of 0.96 based on alkyl chain length and number of ethoxylates and 0.98 based on logK(ow). The additional chronic toxicity data allowed for development of a chronic SAR based on logK(ow) and produced an r(2) of 0.93. Slopes of new and published acute, chronic, and mesocosm SARs based on logK(ow) ranged from -0.61 to -0.87. These new data and refined SARs will assist in the toxicity prediction of mixtures in the environment.

Integration of aquatic fate and ecological responses to linear alkyl benzene sulfonate (LAS) in model stream ecosystems.

An integrated model stream ecosystem fate and effect study of dodecyl linear alkylbenzene sulfonate (C(12)LAS) was performed in the summer and fall of 1996. The study addressed responses of periphytic microbes, immature benthic fauna including abundance, drift, and emergence of adult insects in a 56-day exposure. Exposures ranged from 126 to 2978 microg/L and were continuously presented in a single-pass, flow-through test system. Microbial heterotrophs acclimated to C(12)LAS exposure quickly (14 days) and biodegraded C(12)LAS at all concentrations. Blue-green algae responded by increasing in abundance with increasing C(12)LAS concentration. Invertebrates responded by increased drift and reduced benthic abundances at concentrations exceeding 293 microg/L. Emergence at 927 microg/L also declined relative to the control. Adverse responses for mayflies and chironomids were indicated using univariate statistical techniques. Multivariate techniques indicated these taxa plus mollusks, aquatic worms, caddisflies, and stoneflies were impaired at some concentrations. Bioavailability of C(12)LAS was investigated in streams as a function of the total suspended solid load in the water column driven by local weather and watershed patterns. A continuous bioavailability model indicated exposure was reduced by an average of 8.5+/-8.9%. A model ecosystem no-observed-effect concentration (NOEC) was concluded to be 293 microg/L based on measured water column exposure and adjusted to 268 microg/L by the bioavailability model. A literature review of 13 available model ecosystem studies was conducted and NOEC conclusions were adjusted by a structure-activity relationship to a dodecyl chain length (sulfophenyl position and distribution being ignored due to lack of information in the reviewed studies). Lentic studies (n=7) were found to have higher NOECs than lotic studies (n=6) and were more variable. Mean NOECs+/-SD for all studies, lentic studies only, and lotic studies only were 3320+/-6040, 5720+/-7640, and 530+/-430 microg/L, respectively. Interpretation of results for anomalies from specific studies suggests the importance of experimental design, use of laboratory versus natural surface water, biological complexity of the test system, and physical test system design as relevant factors for consideration. The specific results of the new model ecosystem study presented in this article can be well defended on the basis of a robust experimental and physical design and because the system contained a diverse and sensitive aquatic community. A low or no uncertainty factor could be applied to the result.

The effect of the oil dispersant Omni-Clean on the toxicity of fuel oil no. 2 in two bioassays with the sheepshead minnow Cyprinodon variegatus.

Bioassays (7-day early life stage and 96 h acute bioassays) were conducted with the sheepshead minnow, Cyprinodon variegatus, to determine the toxicity of the dispersant Omni-Clean by itself and in combination with fuel oil no. 2. Performance characteristics of both bioassay types were also compared. Bioassays used oil by itself, dispersant by itself, and oil and dispersant in various ratios. Omni-Clean was less toxic than many other dispersants, and had a relatively small effect on individual biomass. Toxicities of the oil/dispersant combinations were generally higher than expected from the toxicities of the oil and dispersant by themselves, indicating a more-than-additive effect on toxicity. The comparison of performance characteristics between the 7-day and the 96-hour bioassays showed that the early life stage test is generally more sensitive, and has the added advantage of an additional and sensitive endpoint (fish biomass).

Seasonal temperature declines do not decrease periphytic surfactant biodegradation or increase algal species sensitivity.

The effects of seasonally decreasing river water temperature on surfactant biodegradation and algal sensitivity are reviewed from four stream mesocosm studies conducted over a 5-year period. Seasonal temperatures ranged from 28 to 0 degree C over all studies and temperature declines were approximately 9 to 14 degrees C over the course of each individual study. Mesocosm periphyton were naturally colonized on tile substrata with in-flowing river water for a period of 3 to 8 weeks prior to the initiation of sampling. Streams were dosed for 8 to 11 weeks with microgram/L (ppb) quantities of the surfactants C12-alkyl sulfate (C12-AS), C45E2.17S-alkyl ethoxysulfate (AES), C25E6-alkyl ethoxylate (AE) or 0 to 13% final effluent during the sampling period. Mineralization of C12-AS and AE by periphyton in the dosed streams generally increased over the dosing period while mineralization remained approximately constant in the control streams. The results from the AE study occurred with an increase in periphyton heterotrophic respiration. Mineralization of AES increased over the dosing period in streams receiving the highest dose of AES and remained constant in streams receiving lower doses. All studies involving surfactant exposure demonstrated a positive correlation between surfactant concentration and mineralization during periods of seasonal temperature decline. Mineralization of AE by periphyton dosed with final effluent increased slightly over the testing period. Periphytic algal taxonomy and biovolume were evaluated during the AES study. Overall, these tests showed no increases in species sensitivity over the testing period. Taken collectively, these results indicate that there is no correlation between naturally decreasing seasonal temperatures and lower rates of surfactant mineralization or increased species sensitivity by naturally acclimated periphyton.

Stream periphytic biodegradation of the anionic surfactant C12-alkyl sulfate at environmentally relevant concentrations.

The effects of continuous exposure to C12-alkyl sulfate on a periphytic microbial community were determined in an 8-week stream mesocosm study. C12-alkyl sulfate concentrations ranged from environmentally relevant (< 10-20 micrograms/liter) to unrealistically high concentrations (> 1500 micrograms/liter). Endpoints evaluated included turnover rates, bacterial cell density, heterotrophic mixed amino acid uptake, and fatty acid profile evaluations. Predosed periphyton demonstrated a mean turnover rate for C12-alkyl sulfate of 0.08/hr. During the 8-week dosing period, a significant increase in mean turnover rates was observed in streams dosed with > or = 61 micrograms C12-alkyl sulfate/liter, despite a 10 degrees C drop in stream temperature. A significant correlation between turnover rate and C12-alkyl sulfate concentration was also observed. While bacterial cell density increased during the study, it was determined that the biodegradation acclimation to C12-alkyl sulfate was not biomass-specific. Likewise, bacterial activity generally increased over the study, but it did not correlate with either biodegradation or bacterial cell density. Lastly, phospholipid fatty acid profiles indicate that a shift in the microbial community occurred in the high-dose stream as opposed to the control stream. This study demonstrates that C12-alkyl sulfate is rapidly degraded and induces a biodegradative acclimation response at environmentally relevant concentrations.

Literature review and analysis of biological complexity in model stream ecosystems: influence of size and experimental design.

A meta-analysis of more than 150 model stream ecosystem studies employed in hazard assessment was conducted to assess the effect of model ecosystem size on biological complexity and experimental design. Test system length was highly correlated to duration of colonization and chemical exposure period; however, size was generally unrelated to taxa richness, diversity, abundance, or biomass for algal, protozoan, and invertebrate assemblages. Fish were usually caged and not free-roaming, even in the relatively large (> 10 m) systems. Experimental designs, especially in recent years, have been less concerned with pseudoreplication, resulting in studies emphasizing numerous treatments with few or no replicates. Test systems have evolved that emphasize flow through exposures, use of natural source waters, and regression-style experimental designs. These factors collectively reduce problems historically associated with pseudoreplication. Larger model ecosystems (> 6 m) are advantageous for increasing the ability to sample more intensively through time and space without imposing sampling artifacts. Fish (caged and free-roaming) are also more often used in larger systems. Several model ecosystem studies that were compared with natural field-based communities demonstrated a high degree of correspondence for biological complexity attributes. Well-designed model stream ecosystems have a high degree of biological complexity and reasonable levels of variability for ecologically relevant endpoints. The use of small application factors (approximately equal to 1) is applicable to ecotoxicological results from these studies for risk assessment.