Javier Terriente - 23 April 2026

Animal Testing Alternatives for Ecotoxicity

Each year, tens of thousands of fish and other aquatic vertebrates are used in regulatory ecotoxicity studies across Europe alone. As scientific advances and regulatory pressure converge to reduce animal use in chemical safety testing, the industry is undergoing a directional shift to progressively expand the use of new approach methodologies (NAMs).

The Need for Alternatives to Animal Testing in Ecotoxicity Research

Since Russell and Burch formulated the 3Rs (Reduction, Refinement, and Replacement of animals in research) in 1959, the momentum behind animal testing alternatives has grown substantially. Under Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), the European Chemicals Agency (ECHA) requires that animal testing be used only as a last resort, and actively promotes non-animal alternatives for chemical safety assessment. 

The OECD is developing  Integrated Approaches to Testing and Assessment (IATA) frameworks that integrate in vitro, in silico, and non-mammalian in vivo data to inform regulatory decisions without relying on single animal studies. As an example, project 2.54 for the OECD Test Guidelines program integrates algae and daphnid testing, the zebrafish embryo acute toxicity test (zFET), the RTgill-W1 cell line assay, and computational approaches such as quantitative structure-activity relationship (QSAR) methods to replace Fish Acute Toxicity Testing (OECD TG 203). The project is currently on hold pending an ECHA analysis of new data from alternative methods, expected by Q1 2026. 

The regulatory focus on reducing animal use is strong for mammalian models, such as rodents and rabbits, used in repeated-dose toxicity, reproductive toxicity, and carcinogenicity studies, where the number of animals involved is highest and where validated non-animal alternatives are most actively being developed. As these endpoints progressively move toward in vitro batteries and computational approaches, the expectation within the field is that the need for whole-organism, in vivo data will shift toward non-mammalian models that offer biological relevance but a lower regulatory and ethical burden. Zebrafish are a natural candidate for this role, and the trajectory of current regulatory initiatives suggests their use is likely to expand well beyond the endpoints already covered by approved guidelines.

Modern Alternatives to Animal Testing in Ecotoxicology: In Vitro, In Silico, and Zebrafish Models

Several categories of alternative methodologies are now in different stages of development and regulatory validation for ecotoxicity assessment. In silico approaches, including QSAR models, can screen large chemical inventories and predict aquatic toxicity for fish, daphnids, and algae cost-effectively. Tools such as ECOSAR, the OECD QSAR Toolbox, are already used routinely for chemical prioritization. 

In vitro approaches are also advancing: the RTgill-W1 cell line assay (OECD TG 249), based on rainbow trout gill cells, is a validated example specifically developed for aquatic ecotoxicology. Both in silico and in vitro tools are increasingly integrated into IATA  frameworks that combine multiple data streams in a weight-of-evidence approach to inform regulatory decisions, with the aim of increasing the pace of toxicological evaluation while reducing reliance on animal studies.

Among non-mammalian whole-organism models, zebrafish hold a distinct position. Their small size, transparent embryos, short lifecycle, and high fecundity allow direct in vivo evaluation of chemical toxicity at cellular, organ, and organism levels in a high-throughput format. Zebrafish embryos under 5 days post-fertilization (dpf) are not currently protected under most animal experimentation legislation, reducing regulatory barriers to their use. The most established application is the zFET  (OECD TG 236) (Figure 1), validated through multi-laboratory ring studies demonstrating good reproducibility (CV below 30%) and formally proposed as an alternative to the acute fish toxicity test (OECD TG 203), which alone accounts for approximately 50% of all aquatic vertebrates used in regulatory ecotoxicity testing in Europe.

Figure 1. Lack of somite formation after incubation with tested compound: Although retarded in development by approx. 10 hours, the 24 hours post-fertilization (hpf) zebrafish embryo in (a) shows well-developed somites (→), whereas the embryo in (b) does not show any sign of somite formation (→). Although showing a pronounced yolk sac oedema (*), the 48 hpf zebrafish embryo in (c) shows distinct formation of somites (→), whereas the 96 hpf zebrafish embryo depicted in (d) does not show any sign of somite formation (→). Note also the spinal curvature (scoliosis) and the pericardial oedema (*) in the embryo shown in (d). Source: OECD Guidelines for the Testing of Chemicals. Test Guideline No. 236. Fish Embryo Acute Toxicity (FET) Test. 2025. 

Regulatory Frameworks Supporting Alternative Ecotoxicity Testing

The regulatory framework is moving rapidly, and recent developments signal that zebrafish are on track to cover substantially more ground than TG 236 alone.

In June 2025, the OECD released 56 updated Test Guidelines, including revisions to TG 203, TG 210, and TG 236 that now incorporate optional omics endpoints, enabling tissue collection for transcriptomic and mode-of-action analyses alongside standard toxicity readouts. This integrates mechanistic data into regulatory ecotoxicity testing and aligns these guidelines with next-generation risk assessment frameworks. 

​​At the EU level, Commission Regulation (EU) 2025/2573 was adopted in 2025 and entered into force in early 2026. It introduces three new ecotoxicity test methods: OECD TG 252 (REACTIV), which screens substances for estrogenic activity in vivo; OECD TG 253 (JHASA), which evaluates juvenile hormone-related effects using Daphnia magna; and OECD TG 321 (HYBIT), which assesses bioconcentration potential in aquatic invertebrates using Hyalella azteca. Notably, two of the three new methods target endocrine activity. 

Separately, the European Commission is preparing a formal Roadmap towards Phasing Out Animal Testing for Chemical Safety Assessments, expected to be published by the first quarter of 2026. Stakeholder consultations carried out in 2024-2025 consistently identified endocrine disruption, reproductive toxicity, and developmental effects as the priority areas where validated non-animal alternatives are most needed.

How ZeClinics Is Contributing to the Future of Ecotoxicity Testing

Developmental neurotoxicity (DNT) is one of the clearest examples of where regulation is heading. Current OECD guidelines for DNT assessment (TG 426 and 443) rely on mammalian models and are lengthy, expensive, and of uncertain human relevance. The OECD has prioritized the development of a DNT in vitro battery (DNT-IVB) as a faster and more effective alternative. The battery combines human cell-based assays evaluating key events in neurodevelopment, including proliferation, migration, differentiation, and network formation, with whole-organism zebrafish assays that capture functional responses to chemical exposure that cell models cannot fully replicate. The zebrafish light/dark transition behavioral assay has been identified as the gold standard whole-organism component of this battery.

At ZeClinics, we are active participants in this process. As members of the OECD Expert Group on Developmental Neurotoxicity, we have contributed to the international inter-laboratory effort to establish a harmonized zebrafish DNT protocol, including the assessment of approximately 28 chemicals with available rodent data across four laboratories. Our DNT assay uses the well-validated light/dark transition test: early-life zebrafish embryos are exposed to multiple concentrations of the compound of interest based on a previously determined Benchmark Dose (BMD), and locomotion parameters are assessed at 120 hpf using the DanioVision device (Figure 2). Behavioral alterations are associated with specific defects in CNS development, providing a rapid, cost-effective, and biologically relevant in vivo readout.

Figure 2. Representative plot of locomotion response to light/dark stimulus of 96 larvae at 120 hpf treated with 7 concentrations of a compound of interest. The higher concentration (0.5uM) of compound X induces hypermobility.

Beyond DNT, ZeClinics’ ecotoxicity portfolio covers the full range of endpoints where zebrafish are either already accepted or under active regulatory development: the Fish Embryo Acute Test (TG 236), developmental toxicity, reproductive toxicity, and endocrine disruption. As regulatory frameworks expand the acceptance of zebrafish data, we can help organizations generate robust, regulatorily relevant ecotoxicity data that reduces reliance on mammalian testing while maintaining the whole-organism complexity that in vitro systems cannot replicate.

Discover how zebrafish can fit into your ecotoxicity testing strategy

Sources

Busquet F, Strecker R, Rawlings JM, Belanger SE, Braunbeck T, Carr GJ, Cenijn P, Fochtman P, Gourmelon A, Hübler N, Kleensang A, Knöbel M, Kussatz C, Legler J, Lillicrap A, Martínez-Jerónimo F, Polleichtner C, Rzodeczko H, Salinas E, Schneider KE, Scholz S, van den Brandhof EJ, van der Ven LT, Walter-Rohde S, Weigt S, Witters H, Halder M. OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing. Regul Toxicol Pharmacol. 2014 Aug;69(3):496-511. doi: 10.1016/j.yrtph.2014.05.018.

Commission Regulation (EU) 2025/2573 amending Regulation (EC) No 440/2008 as regards test methods. Official Journal of the European Union; 2025. Available from: https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=OJ:L_202502573

ECHA. Alternatives to animal testing under REACH [Internet]. Available from: https://echa.europa.eu/animal-testing-under-reach

European Commission. Roadmap towards phasing out animal testing [Internet]. Available from: https://single-market-economy.ec.europa.eu/sectors/chemicals/reach/roadmap-towards-phasing-out-animal-testing_en

OECD. In vitro assays for developmental neurotoxicity (DNT-IVB) [Internet]. Available from: https://www.oecd.org/en/topics/sub-issues/testing-of-chemicals/in-vitro-assays-for-developmental-neurotoxicity.html

OECD. Publications on Testing and Assessment of Chemicals [Internet]. Available from: https://www.oecd.org/en/topics/sub-issues/testing-of-chemicals/publications-on-testing-and-assessment-of-chemicals.html

OECD. Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals. OECD Publishing, Paris; updated June 2025.

Schaupp CM, Maloney EM, Mattingly KZ, Olker JH, Villeneuve DL. Comparison of in silico, in vitro, and in vivo toxicity benchmarks suggests a role for ToxCast data in ecological hazard assessment. Toxicol Sci. 2023 Sep 28;195(2):145-154. doi: 10.1093/toxsci/kfad072.

Schür C, Paparella M, Faßbender C, Stoddart G, Baity Jesi M, Schirmer K. Daphnids can safeguard the use of alternative bioassays to the acute fish toxicity test: a focus on neurotoxicity. Environ Toxicol Chem. 2025 Sep 1;44(9):2635-2647. doi: 10.1093/etojnl/vgaf014.