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

Abstract

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.

Development of cardiovascular and neurodevelopmental metrics as sublethal endpoints for the Fish embryo toxicity test

“…Survival among control embryos exceeded 90% in each of the 3 FET test replicates. The mean LC50 value (± standard deviation) was estimated at 8.8 ± 2.7 mg DCA/L, a value consistent with previously reported DCA LC50 values, which range from 4.4 to 9.6 mg DCA/L (Braunbeck et al ; Böhler ; Jeffries et al ). None of the embryos in the 16 mg DCA/L group survived the duration of the FET test, and thus, sublethal endpoints were not measured in embryos from this group.…”

“…Methods for a fathead minnow FET test have already been developed (Braunbeck et al ), and studies have shown that the sensitivity of the fathead minnow FET test is comparable to that of other test methods, including the larval growth and survival test, for some chemical classes (Braunbeck et al ; Jeffries et al , 2015; Roush et al ), suggesting that the fathead minnow can be used as a FET test species. In addition, previous studies have provided evidence that metrics of growth (i.e., growth‐related gene expression, wet wt, and length), neurological dysfunction (i.e., neurological‐related gene expression, spontaneous contraction frequency, and eye size), and cardiovascular dysfunction (i.e., cardiovascular system–related gene expression, heart rate, and pericardial edema) in embryos and eleutheroembryos may be sensitive to contaminant‐induced alterations (Cheng et al ; Antkiewicz et al ; Usenko et al ; Chen ; Incardona et al ; Jeffries et al ; Roush et al ).…”

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

“…Each test included a positive control group exposed to the reference toxicant, 3,4-dichloroanile (DCA). Stock solutions of 100 mg/L were prepared in dechlorinated water using DCA (Sigma-Aldrich), as described in Sellin Jeffries et al [8]. Tests with mock MWWTP effluents included an ammonia-exposed positive control group as well as an effluent-only group (exposed to MWWTP effluents that had not been spiked with ammonia), in addition to the DCA-exposed positive control group.…”

“…Fathead minnows and zebrafish were obtained according to the procedures outlined in Sellin Jeffries et al [8]. Briefly, early-life stage zebrafish and fathead minnows for use in the FET and LGS tests were obtained from sexually mature adult zebrafish purchased from Aquatica BioTech (Sun City Center) or fathead minnows from Miami University's in-house culture (originally stocked using fish obtained from Aquatic Biosystems and the USEPA Research Facility [Cincinnati, OH]).…”

“…Efforts to develop the fathead minnow FET test have already begun [2,8], and it has been shown that with some minor modifications the zebrafish FET test can be adapted for use with the fathead minnow. The sensitivity of the fathead minnow FET test has previously been compared with that of other standardized testing methods, including the zebrafish FET and fathead minnow larval growth and survival (LGS; US Environmental Protection Agency [USEPA] method 1000.0 [9]) tests [8]. However, no clear-cut relationships between the sensitivities of these testing strategies have become apparent, likely because of the relatively limited data set available for the fathead minnow FET test.…”