Environmental hazard of cationic polymers relevant in personal and consumer care products: A critical review.

Historically, polymers have been excluded from registration and evaluation under the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) program, the European chemical management program. Recently, interest has increased to include polymers. A tiered registration system has been envisioned and would begin with classes of polymers of greater interest based on certain properties. Cationic polymers are one such class. There is a pressing need to understand the quality and limitations of historical cationic polymer studies and to identify key sources of uncertainty in environmental hazard assessments so we can move toward scientifically robust analyses. To that end, we performed a critical review of the existing cationic polymer environmental effects literature to evaluate polymer characterization and test methodologies to understand how these parameters may affect test interpretation. The relationship between physicochemical parameters, acute and chronic toxicity, and relative trophic level sensitivity were explored. To advance our understanding of the environmental hazard and subsequent risk characterization of cationic polymers, there is a clear need for a consistent testing approach as many polymers are characterized as difficult-to-test substances. Experimental parameters such as dissolved organic carbon and solution renewal approaches can alter cationic polymer bioavailability and toxicity. It is recommended that OECD TG 23 "Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Substances" testing considerations be applied when conducting environmental toxicity assays with cationic polymers. Integr Environ Assess Manag 2023;19:312-325. © 2021 SETAC.

Understanding Ecotoxicological Responses of Fish Embryos and Gill Cells to Cationic Polymers.

Cationic polymers are considered by the scientific and regulatory communities as a group of greater interest amongst the polymers in commerce. As a category, relatively little hazard information is available in the public literature. Very few examples exist of published, high-quality polymer characterization and quantification of exposure. In the present study we describe a series of fish embryo toxicity (FET) and fish gill cytotoxicity assays used to establish a baseline understanding of several representative polyquaternium categories (PQ-6, PQ-10, PQ-16) in animal alternative models, accompanied by high-quality analytical characterization. Materials were chosen to encompass a range of molecular weights and charge densities to determine the influence of test material characteristics on toxicity. Both chorionated and dechorionated FET assays were generally similar to published acute fish toxicity data. Toxicity was correlated with cationic polymer charge density, and not with molecular weight, and was a combination of physical effects and likely toxicity at the site of action. Toxicity could be ameliorated by humic acid in a dose-dependent manner. Fish gill cytotoxicity results were orders of magnitude less sensitive than FET test responses. Environ Toxicol Chem 2022;41:2259-2272. © 2022 SETAC.

Evaluation of a Bayesian Network for Strengthening the Weight of Evidence to Predict Acute Fish Toxicity from Fish Embryo Toxicity Data.

The use of fish embryo toxicity (FET) data for hazard assessments of chemicals, in place of acute fish toxicity (AFT) data, has long been the goal for many environmental scientists. The FET test was first proposed as a replacement to the standardized AFT test nearly 15 y ago, but as of now, it has still not been accepted as a standalone replacement by regulatory authorities such as the European Chemicals Agency (ECHA). However, the ECHA has indicated that FET data can be used in a weight of evidence (WoE) approach, if enough information is available to support the conclusions related to the hazard assessment. To determine how such a WoE approach could be applied in practice has been challenging. To provide a conclusive WoE for FET data, we have developed a Bayesian network (BN) to incorporate multiple lines of evidence to predict AFT. There are 4 different lines of evidence in this BN model: 1) physicochemical properties, 2) AFT data from chemicals in a similar class or category, 3) ecotoxicity data from other trophic levels of organisms (e.g., daphnids and algae), and 4) measured FET data. The BN model was constructed from data obtained from a curated database and conditional probabilities assigned for the outcomes of each line of evidence. To evaluate the model, 20 data-rich chemicals, containing a minimum of 3 AFT and FET test data points, were selected to ensure a suitable comparison could be performed. The results of the AFT predictions indicated that the BN model could accurately predict the toxicity interval for 80% of the chemicals evaluated. For the remaining chemicals (20%), either daphnids or algae were the most sensitive test species, and for those chemicals, the daphnid or algal hazard data would have driven the environmental classification. Integr Environ Assess Manag 2020;16:452-460. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Fish embryo tests and acute fish toxicity tests are interchangeable in the application of the threshold approach.

A database was compiled for algal Organisation for Economic Co-operation and Development (OECD) test guideline 201, for Daphnia magna OECD test guideline 202, for the acute fish toxicity (AFT) OECD test guideline 203, and for the fish embryo toxicity (FET) OECD test guideline 236 to assess the suitability and applicability of the FET test in a threshold approach context. In the threshold approach, algal and Daphnia toxicity are assessed first, after which a limit test is conducted at the lower of the 2 toxicity values using fish. If potential fish toxicity is indicated, a full median lethal concentration assay is performed. This tiered testing strategy can significantly reduce the number of fish used in toxicity testing because algae or Daphnia are typically more sensitive than fish. A total of 165 compounds had AFT and FET data available, and of these, 82 had algal and Daphnia acute toxicity data available. Algae and Daphnia were more sensitive 75 to 80% of the time. Fish or FET tests were most sensitive 20 and 16% of the time, respectively, when considered as the sole fish toxicity indicator and 27% of the time when both were considered simultaneously. When fish were the most sensitive trophic level, different compounds were identified as the most toxic in FET and to AFT tests; however, the differences were not so large that they resulted in substantially different outcomes when potencies were binned using the United Nations categories of aquatic toxicity under the Globally Harmonized System for classification and labeling. It is recommended that the FET test could be used to directly replace the AFT test in the threshold approach or could be used as the definitive test if an AFT limit test indicated toxicity potential for a chemical. Environ Toxicol Chem 2019;38:671-681. © 2019 SETAC.

Advances in understanding the response of fish to linear alcohols in the environment.

Short to long chain alcohols have a range of ecotoxicity to aquatic life driven by hydrophobic interactions with biological membranes. Carbon chain length and octanol:water partitioning coefficients are surrogates for hydrophobicity and strongly relate to aquatic toxicity. In these investigations, the toxicity of ethanol to 1-n-dodecanol to juvenile fish in standard acute toxicity tests is reviewed. Toxicity tests employing fish embryos (zebrafish Danio rerio and fathead minnow Pimephales promelas) in the Fish Embryo Test (OECD 236) format were conducted from C2 to C10 to compare against standard juvenile fish toxicity. Quantitative structure activity relationships for FET and fish individually and combined demonstrate that embryos are not different in sensitivity to juvenile fish. A combined QSAR was developed of the form Log 96 h LC50 (mM/L) = -0.925*log Kow + 2.060 (R2 10 = 0.954). Alcohols of 11-12 carbons show a deflection in the QSAR as toxicity approaches the solubility limit. Alcohols with longer chain lengths may only be tested at lower exposures relevant for chronic toxicity. Decanol was evaluated in a 33-d fish early life stage test (OECD 210) and survival was the most sensitive endpoint (EC10 = 0.43 mg/L, 0.0027 mM/L). This study suggests a reasonable acute to chronic ratio of 6.5 in line with historical literature for non-polar narcotic compounds. Fish are not uniquely more sensitive than Daphnia magna which suggests estimations of environmental hazard can be confidently made with either taxon. The overall environmental risk assessments for the longer chain alcohols included in this research remain largely unchanged primarily due to previous research demonstrating a very minimal environmental exposure even for highly toxic members of the category.

On the impact of sample size on median lethal concentration estimation in acute fish toxicity testing: Is n = 7/group enough?

The fish acute toxicity test method is foundational to aquatic toxicity testing strategies, yet the literature lacks a concise sample size assessment. Although various sources address sample size, historical precedent seems to play a larger role than objective measures. We present a novel and comprehensive quantification of the effect of sample size on estimation of the median lethal concentration (LC50), covering a wide range of scenarios. The results put into perspective the practical differences across a range of sample sizes, from n = 5/concentration up to n = 23/concentration. We also provide a framework for setting sample size guidance illustrating ways to quantify the performance of LC50 estimation, which can be used to set sample size guidance given reasonably difficult (or worst-case) scenarios. There is a clear benefit to larger sample size studies: they reduce error in the determination of LC50s, and lead to more robust safe environmental concentration determinations, particularly in cases likely to be called worst-case (shallow slope and true LC50 near the edges of the concentration range). Given that the use of well-justified sample sizes is crucial to reducing uncertainty in toxicity estimates, these results lead us to recommend a reconsideration of the current de minimis 7/concentration sample size for critical studies (e.g., studies needed for a chemical registration, which are being tested for the first time, or involving difficult test substances). Environ Toxicol Chem 2018;37:1565-1578. © 2018 SETAC.

Aquatic toxicity structure-activity relationships for the zwitterionic surfactant alkyl dimethyl amine oxide to several aquatic species and a resulting species sensitivity distribution.

Amine oxide (AO) is a cationically charged surfactant at environmental pH and has previously been assessed in the OECD (Organization for Economic Cooperation and Development) High Production Volume (HPV) chemicals program. Typical of cationic chemicals, AO is highly aquatically toxic. In this study we vastly improve the knowledge of AO toxicity by developing acute Quantitative Structure Activity Relationships (QSARs) for an alga (Desmodesmus subspicatus), an invertebrate (Daphnia magna) and a fish (Danio rerio) using the appropriate array of OECD Test Guidelines. A chronic toxicity QSAR was also determined for the most sensitive taxon, Desmodesmus. Pure AO spanning the chain lengths of C8 to C16 were tested individually with trace analytical confirmation of exposures in all tests. The QSARs were all of high quality (R2 0.92-0.98) with slopes ranging from -0.338 to -0.484. QSARs were then used to normalize toxicity outcomes for a larger, previously published data set used in HPV, European REACH (Registration, Evaluation, and Authorization of Chemicals), and peer reviewed publications. Two additional species, Lemna gibba (macrophyte) and Ankistrodesmus falcatus (alga) were studied in exposures to dodecyl (C12) AO to provide sufficient taxonomic diversity to conduct a Species Sensitivity Distribution (SSD) analysis. The SSD 5th percentile hazardous concentration (HC5) to C12 AO was found to be 0.052mg/L which is similar to an existing AO 28-d, 3-community periphyton community bioassay normalized to C12 AO (No-observed-effect-concentration or NOEC=0.152mg/L). The statistical properties of the SSD was probed suggesting that new studies of additional taxa would be required that were at least 10-fold more sensitive than the most sensitive taxon to move the HC5 lower by a factor of 3. The overall AO hazard assessment suggests a large margin of safety relative to published environmental exposure data.

Development of acute toxicity quantitative structure activity relationships (QSAR) and their use in linear alkylbenzene sulfonate species sensitivity distributions.

Linear Alkylbenzene Sulfonate (LAS) is high tonnage and widely dispersed anionic surfactant used by the consumer products sector. A range of homologous structures are used in laundry applications that differ primarily on the length of the hydrophobic alkyl chain. This research summarizes the development of a set of acute toxicity QSARs (Quantitative Structure Activity Relationships) for fathead minnows (Pimephales promelas) and daphnids (Daphnia magna, Ceriodaphnia dubia) using accepted test guideline approaches. A series of studies on pure chain length LAS from C10 to C14 were used to develop the QSARs and the robustness of the QSARs was tested by evaluation of two technical mixtures of differing compositions. All QSARs were high quality (R(2) were 0.965-0.997, p < 0.0001). Toxicity normalization employing QSARs is used to interpret a broader array of tests on LAS chain length materials to a diverse group of test organisms with the objective of developing Species Sensitivity Distributions (SSDs) for various chain lengths of interest. Mixtures include environmental distributions measured from exposure monitoring surveys of wastewater effluents, various commercial mixtures, or specific chain lengths. SSD 5th percentile hazardous concentrations (HC5s) ranged from 0.129 to 0.254 mg/L for wastewater effluents containing an average of 11.26-12 alkyl carbons. The SSDs are considered highly robust given the breadth of species (n = 19), use of most sensitive endpoints from true chronic studies and the quality of the underlying statistical properties of the SSD itself. The data continue to indicate a low hazard to the environment relative to expected environmental concentrations.

The fish embryo toxicity test as a replacement for the larval growth and survival test: A comparison of test sensitivity and identification of alternative endpoints in zebrafish and fathead minnows.

The fish embryo toxicity (FET) test has been proposed as an alternative to the larval growth and survival (LGS) test. The objectives of the present study were to evaluate the sensitivity of the FET and LGS tests in fathead minnows (Pimephales promelas) and zebrafish (Danio rerio) and to determine if the inclusion of sublethal metrics as test endpoints could enhance test utility. In both species, LGS and FET tests were conducted using 2 simulated effluents. A comparison of median lethal concentrations determined via each test revealed significant differences between test types; however, it could not be determined which test was the least and/or most sensitive. At the conclusion of each test, developmental abnormalities and the expression of genes related to growth and toxicity were evaluated. Fathead minnows and zebrafish exposed to mock municipal wastewater-treatment plant effluent in a FET test experienced an increased incidence of pericardial edema and significant alterations in the expression of genes including insulin-like growth factors 1 and 2, heat shock protein 70, and cytochrome P4501A, suggesting that the inclusion of these endpoints could enhance test utility. The results not only show the utility of the fathead minnow FET test as a replacement for the LGS test but also provide evidence that inclusion of additional endpoints could improve the predictive power of the FET test.

Alternative methods for toxicity assessments in fish: comparison of the fish embryo toxicity and the larval growth and survival tests in zebrafish and fathead minnows.

An increased demand for chemical toxicity evaluations has resulted in the need for alternative testing strategies that address animal welfare concerns. The fish embryo toxicity (FET) test developed for zebrafish (Danio rerio) is one such alternative, and the application of the FET test to other species such as the fathead minnow (Pimephales promelas) has been proposed. In the present study, the performances of the FET test and the larval growth and survival (LGS; a standard toxicity testing method) test in zebrafish and fathead minnows were evaluated. This required that testing methods for the fathead minnow FET and zebrafish LGS tests be harmonized with existing test methods and that the performance of these testing strategies be evaluated by comparing the median lethal concentrations of 2 reference toxicants, 3,4-dicholoraniline and ammonia, obtained via each of the test types. The results showed that procedures for the zebrafish FET test can be adapted and applied to the fathead minnow. Differences in test sensitivity were observed for 3,4-dicholoraniline but not ammonia; therefore, conclusions regarding which test types offer the least or most sensitivity could not be made. Overall, these results show that the fathead minnow FET test has potential as an alternative toxicity testing strategy and that further analysis with other toxicants is warranted in an effort to better characterize the sensitivity and feasibility of this testing strategy.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals.

The fish embryo test (FET) is a potential animal alternative for the acute fish toxicity (AFT) test. A comprehensive validation program assessed 20 different chemicals to understand intra- and interlaboratory variability for the FET. The FET had sufficient reproducibility across a range of potencies and modes of action. In the present study, the suitability of the FET as an alternative model is reviewed by relating FET and AFT. In total, 985 FET studies and 1531 AFT studies were summarized. The authors performed FET-AFT regressions to understand potential relationships based on physical-chemical properties, species choices, duration of exposure, chemical classes, chemical functional uses, and modes of action. The FET-AFT relationships are very robust (slopes near 1.0, intercepts near 0) across 9 orders of magnitude in potency. A recommendation for the predictive regression relationship is based on 96-h FET and AFT data: log FET median lethal concentration (LC50) = (0.989 × log fish LC50) - 0.195; n = 72 chemicals, r = 0.95, p < 0.001, LC50 in mg/L. A similar, not statistically different regression was developed for the entire data set (n = 144 chemicals, unreliable studies deleted). The FET-AFT regressions were robust for major chemical classes with suitably large data sets. Furthermore, regressions were similar to those for large groups of functional chemical categories such as pesticides, surfactants, and industrial organics. Pharmaceutical regressions (n = 8 studies only) were directionally correct. The FET-AFT relationships were not quantitatively different from acute fish-acute fish toxicity relationships with the following species: fathead minnow, rainbow trout, bluegill sunfish, Japanese medaka, and zebrafish. The FET is scientifically supportable as a rational animal alternative model for ecotoxicological testing of acute toxicity of chemicals to fish.

Saltatory ontogeny of fishes and sensitive early life stages for ecotoxicology tests.

Fish display a wide range of developmental ontogenies. These distinctions have taxonomic, evolutionary, and ecological importance in addition to practical implications on the use of fish in aquatic toxicity tests. With respect to animal welfare, vertebrates are afforded protected or non-protected status in the European Union based upon whether they feed endogenously off the yolk or exogenously by procurement and ingestion of food. The concept of saltatory ontogeny suggests development is not gradual but proceeds in leaps separated by a series of stable developmental states. In this context, endogenous/exogenous feeding also distinguishes the developmental phases of embryo (egg), eleutheroembryo (feeding off the yolk sac) and larvae (exogenous feeding) in fish. The recent proposal for the Fish Embryo Test (FET) as an animal alternative to the standard fish acute toxicity test (OECD 203 and equivalent tests) puts a clear focus on the need to identify the non-protected and protected life intervals in test species as well as their sensitivities which coincides with the developmental phases identified in saltatory ontogeny. In this paper we described a method to quantify embryo, eleutheroembryo, and larva phases in Danio rerio, the zebrafish. Danio eleutheroembryos preyed upon 5 different protozoan species (Euglena, Euplotes, Paramecium aurelia, Paramecium bursaria and Paramecium multimicronucleatum) between 24 and 48hr following hatching (85-95% of fish, n=20 per species, 25 degrees C). Based upon these data it is recommended that testing of developing zebrafish embryos should be terminated between 24 and 48hr after hatching in order to be compliant with existing animal welfare legislation within Europe.