Weight of evidence tools in the prediction of acute fish toxicity.

Acute fish toxicity (AFT) is a key endpoint in nearly all regulatory implementations of environmental hazard assessments of chemicals globally. Although it is an early tier assay, the AFT assay is complex and uses many juvenile fish each year for the registration and assessment of chemicals. Thus, it is imperative to seek animal alternative approaches to replace or reduce animal use for environmental hazard assessments. A Bayesian Network (BN) model has been developed that brings together a suite of lines of evidence (LoEs) to produce a probabilistic estimate of AFT without the testing of additional juvenile fish. Lines of evidence include chemical descriptors, mode of action (MoA) assignment, knowledge of algal and daphnid acute toxicity, and animal alternative assays such as fish embryo tests and in vitro fish assays (e.g., gill cytotoxicity). The effort also includes retrieval, assessment, and curation of quality acute fish toxicity data because these act as the baseline of comparison with model outputs. An ideal outcome of this effort would be to have global applicability, acceptance and uptake, relevance to predominant fish species used in chemical assessments, be expandable to allow incorporation of future knowledge, and data to be publicly available. The BN model can be conceived as having incorporated principles of tiered assessment and whose outcomes will be directed by the available evidence in combination with prior information. We demonstrate that, as additional evidence is included in the prediction of a given chemical's ecotoxicity profile, both the accuracy and the precision of the predicted AFT can increase. Ultimately an improved environmental hazard assessment will be achieved. Integr Environ Assess Manag 2023;19:1220-1234. © 2022 SETAC.

Interlaboratory Validation of Toxicity Testing Using the Duckweed Lemna minor Root-Regrowth Test.

The common duckweed (Lemna minor), a freshwater monocot that floats on the surfaces of slow-moving streams and ponds, is commonly used in toxicity testing. The novel Lemna root- regrowth test is a toxicity test performed in replicate test vessels (24-well plates), each containing 3 mL test solution and a 2-3 frond colony. Prior to exposure, roots are excised from the plant, and newly developed roots are measured after 3 days of regrowth. Compared to the three internationally standardized methods, this bioassay is faster (72 h), simpler, more convenient (requiring only a 3-mL) and cheaper. The sensitivity of root regrowth to 3,5-dichlorophenol was statistically the same as using the conventional ISO test method. The results of interlaboratory comparison tests conducted by 10 international institutes showed 21.3% repeatability and 27.2% reproducibility for CuSO4 and 21.28% repeatability and 18.6% reproducibility for wastewater. These validity criteria are well within the generally accepted levels of <30% to 40%, confirming that this test method is acceptable as a standardized biological test and can be used as a regulatory tool. The Lemna root regrowth test complements the lengthier conventional protocols and is suitable for rapid screening of wastewater and priority substances spikes in natural waters.

An ecotoxicological assessment of mine tailings from three Norwegian mines.

The study assessed the environmental toxicity of three Norwegian mine tailings from Omya Hustadmarmor, Sydvaranger, and Sibelco, which are all released into a seawater recipient. Ecotoxicity assessments were performed on the overlying water extracted from the mine tailings, the transformation/dissolution waters obtained from the mine tailings, and whole sediment assessment using a suite of marine organisms including algae, Crustacea, and Mollusca. Overall, based on the toxicity evaluation of the transformation/dissolution data, Sibelco tailings resulted in the highest toxicity albeit at relatively high concentrations, followed by Sydvaranger and Hustadmarmor. Sibelco was the only mine where process chemicals were not used. In contrast, the Corophium sediment contact assay revealed a significantly higher toxicity exerted by Hustadmarmor tailings, which may indicate a physical impact of the fine tailings. The effects observed were discussed with respect to both the measured chemical concentrations of the tailings and the potential physical impact of the tailing particles on organism health.

Defining the chronic impacts of atenolol on embryo-larval development and reproduction in the fathead minnow (Pimephales promelas).

Atenolol is a beta-adrenergic receptor antagonist ('beta-blocker') widely used for the treatment of angina, glaucoma, high blood pressure and other related conditions. Since atenolol is not appreciably metabolized in humans, the parent compound is the predominant excretory product, and has been detected in sewage effluent discharges and surface waters. Consequently, atenolol has been chosen as a reference pharmaceutical for a European Union-funded research consortium, known as ERAPharm (http://www.erapharm.org), which focused on the fate and effects of pharmaceuticals in the environment. Here, we present data generated within this project from studies assessing population-relevant effects in a freshwater fish species. Using fathead minnows (Pimephales promelas) as a standard OECD test species, embryo-larval development (early life stage or ELS) and short-term (21 d) adult reproduction studies were undertaken. In the ELS study, the 4d embryo NOEC(hatching) and LOEC(hatching) values were 10 and >10mg/L, respectively, and after 28 d, NOEC(growth) and LOEC(growth) values were 3.2 and 10mg/L, respectively (arithmetic mean measured atenolol concentrations were >90% of these nominal values). In the short-term reproduction study, NOEC(reproduction) and LOEC(reproduction) values were 10 and >10mg/L, respectively (mean measured concentrations were 77-96% of nominal values), while the most sensitive endpoint was an increase in male fish condition index, giving NOEC(condition index) and LOEC(condition index) values of 1.0 and 3.2mg/L, respectively. The corresponding measured plasma concentration of atenolol in these fish was 0.0518 mg/L. These data collectively suggest that atenolol has low chronic toxicity to fish under the conditions described, particularly considering the low environmental concentrations reported. These data also allowed the assessment of two theoretical approaches proposed as predictors of the environmental impact of human pharmaceuticals: the Huggett 'mammalian-fish leverage model'; and the acute:chronic ratio (ACR). The Huggett model gave a measured human: fish effect ratio (ER) of 19.3 for atenolol, which compared well with the predicted ER of 40.98. Moreover, for an ER of 19.3, the model suggests that chronic testing may be warranted, and from our resultant effects data, atenolol does not cause significant chronic effects in fathead minnow at environmentally realistic concentrations. The calculated ACR for atenolol is >31.25, which is far lower than that of 17 alpha-ethinylestradiol and other potent steroidal oestrogens, thus further supporting the observed low toxicity. The data produced for atenolol here fit well with both approaches, but also highlight the importance of generating 'real' experimental data with which to calibrate and validate such models.

Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows (Pimephales promelas).

Laboratory studies were conducted to investigate potential adverse effects on development, growth, reproduction and biomarker responses (vitellogenin [VTG] and gonad histology) in fathead minnows (Pimephales promelas) exposed to tamoxifen citrate. Based on the results of a partial life cycle study (nominal [mean measured] concentrations ranged from 0.18 [0.11] to 18 [15.74] microg/L), a 284-d fish full life-cycle (FFLC) flow-through study was conducted using newly fertilized embryos (<24 h postfertilization) exposed to nominal (mean measured) concentrations of 14C-tamoxifen citrate that ranged from 0.01 (0.007) to 5.12 (4.08) microg/L. Triethylene glycol (2.0 microl/L) was used as a solvent carrier, with 17beta-estradiol (E2) as a positive control (nominal 0.1 microg/L). Among the biomarkers measured, significant effects on VTG and gonad histology were observed, although these results required care in their interpretation. Among important population-relevant endpoints, no effects on reproduction were observed at nominal concentrations < or = 5.12 microg/L. Effects on growth (length and weight) were observed in some treatments; however, some of these showed irregular concentration-response relationships, which made interpretation uncertain, or were deemed transient in nature (e.g., reduction in growth of F1 28-d posthatch larval fish at nominal concentrations of 0.08, 0.64, and 5.12 microg/L) and judged not to be biologically significant. Interpretation of results from fish chronic studies is challenging and frequently calls for scientific judgement about statistical and biological significance and what constitutes an adverse effect. Using the principles used in mammalian toxicology studies, data from partial and FFLC studies were evaluated from both statistical and biological perspectives in order to determine no-observed-adverse effect concentrations (expressed as (adverse)NOEC) for use in environmental risk assessment. Careful consideration of both biological and statistical outcomes from these studies suggested overall (adverse)NOEC concentration and lowest-observed-effect concentration ((adverse)LOEC) values for tamoxifen citrate of 5.12 microg/L and 5.6 microg/L, respectively.

A strategy to reduce the numbers of fish used in acute ecotoxicity testing of pharmaceuticals.

The pharmaceutical industry gives high priority to animal welfare in the process of drug discovery and safety assessment. In the context of environmental assessments of active pharmaceutical ingredients (APIs), existing U.S. Food and Drug Administration and draft European regulations may require testing of APIs for acute ecotoxicity to algae, daphnids, and fish (base-set ecotoxicity data used to derive the predicted no-effect concentration [PNECwater] from the most sensitive of three species). Subject to regulatory approval, it is proposed that testing can be moved from fish median lethal concentration (LC50) testing (typically using > or = 42 fish/API) to acute threshold tests using fewer fish (typically 10 fish/API). To support this strategy, we have collated base-set ecotoxicity data from regulatory studies of 91 APIs (names coded for commercial reasons). For 73 of the 91 APIs, the algal median effect concentration (EC50) and daphnid EC50 values were lower than or equal to the fish LC50 data. Thus, for approximately 80% of these APIs, algal and daphnid acute EC50 data could have been used in the absence of fish LC50 data to derive PNECwater values. For the other 18 APIs, use of an acute threshold test with a step-down factor of 3.2 is predicted to give comparable PNECwater outcomes. Based on this preliminary scenario of 91 APIs, this approach is predicted to reduce the total number of fish used from 3,822 to 1,025 (approximately 73%). The present study, although preliminary, suggests that the current regulatory requirement for fish LC50 data regarding APIs should be succeeded by fish acute threshold (step-down) test data, thereby achieving significant animal welfare benefits with no loss of data for PNECwater estimates.