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Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish-based tests using 3,4-dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;43:1285-1299. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

The fathead minnow fish embryo toxicity (FET) test has been proposed as a more humane alternative to current toxicity testing methods as younger organisms are thought to experience less distress during toxicant exposure. However, the FET test protocol does not include endpoints that allow for the prediction of sublethal adverse outcomes, limiting its utility relative to other test types. Researchers have proposed the development of sublethal endpoints for the FET test to increase its utility. The present study 1) developed methods for previously unmeasured sublethal metrics in fathead minnows (i.e., spontaneous contraction frequency and heart rate) and 2) investigated the responsiveness of several sublethal endpoints related to growth (wet wt, length, and growth-related gene expression), neurodevelopment (spontaneous contraction frequency, eye size, and neurodevelopmental gene expression), and cardiovascular function and development (pericardial area, heart rate, and cardiovascular system-related gene expression) as additional FET test metrics using the model toxicant 3,4-dichloroaniline. Of the growth, neurological, and cardiovascular endpoints measured, length, eye size, and pericardial area were found to be more responsive than the other endpoints evaluated. Future studies linking alterations in these endpoints to longer-term adverse impacts are needed to fully evaluate the predictive power of these metrics in chemical and whole-effluent toxicity testing. Environ Toxicol Chem 2018;37:2530-2541. © 2018 SETAC., (© 2018 SETAC.)

The fathead minnow fish embryo toxicity (FET) test has been identified as a potential alternative to toxicity test methods that utilize older fish. However, several challenges have been identified with the fathead minnow FET test, including: 1) difficulties in obtaining appropriately-staged embryos for FET test initiation, 2) a paucity of data comparing fathead minnow FET test performance to the fathead minnow larval growth and survival (LGS) test and 3) a lack of sublethal endpoints that could be used to estimate chronic toxicity and/or predict adverse effects. These challenges were addressed through three study objectives. The first objective was to optimize embryo production by assessing the effect of breeding group composition (number of males and females) on egg production. Results showed that groups containing one male and four females produced the largest clutches, enhancing the likelihood of procuring sufficient numbers of embryos for FET test initiation. The second study objective was to compare the performance of the FET test to that of the fathead minnow LGS test using three reference toxicants. The FET and LGS tests were similar in their ability to predict the acute toxicity of sodium chloride and ethanol, but the FET test was found to be more sensitive than the LGS test for sodium dodecyl sulfate. The last objective of the study was to evaluate the utility and practicality of several sublethal metrics (i.e., growth, developmental abnormalities and growth- and stress-related gene expression) as FET test endpoints. Developmental abnormalities, including pericardial edema and hatch success, were found to offer the most promise as additional FET test endpoints, given their responsiveness, potential for predicting adverse effects, ease of assessment and low cost of measurement., (Copyright © 2018 Elsevier Inc. All rights reserved.)