Subchronic Toxicity Testing with Zebrafish: Meeting REACH Requirements with Early-Life Stage Fish Studies

Data requirements for chemical registrants working under REACH increase as tonnage grows. From 100 tonnes per year onwards, acute fish toxicity data is no longer sufficient, long-term toxicity data becomes a standard requirement, and the Fish Early-Life Stage Toxicity Test (OECD TG 210) is the accepted method to generate it.

Zebrafish (Danio rerio) are one of the accepted species for TG 210 studies under REACH, and in many cases, the most practical choice. Their high fecundity, small size, and short developmental timeline make them well-suited for subchronic testing in a laboratory setting.

What Is Subchronic Toxicity Testing and When Does REACH Require It?

In the context of aquatic ecotoxicology, subchronic toxicity testing evaluates the effects of sustained exposure on aquatic organisms over a developmentally significant window. In fish, this window is defined from the fertilized egg through the onset of active juvenile feeding. 

Under REACH, the required evaluations depend on the annual tonnage of the chemical, increasing with higher tonnage. Long-term fish toxicity testing is required for chemicals with quantities over 100 tonnes/year, as specified in Annex IX. REACH also requires the long-term fish toxicity evaluation for chemicals of 10-100 tons/year if they are poorly soluble. It is important to remember that REACH mandates animal testing only as a last resort. Information requirements can be met with available results from fish tests, read-across studies, or quantitative structure-activity relationship (QSAR) models. If no data exists, then the chemical undergoes a new experimental test with fish. 

^a OECD test guidelines (TG) are internationally agreed upon test methods for assessing chemical safety: https://www.oecd.org/chemicalsafety/ testing/oecdguidelinesforthetestingofchemicals.htm 

^b For 10-100 ton/year chemicals, the standard information requirement depends on the solubility of the chemical. If the chemical is poorly soluble, the registrant must submit a proposal for a long-term fish toxicity test (TG 210) if no existing long-term data is available. Otherwise, only information on short-term fish toxicity is required. 

^c REACH has allowed OECD TG 212 (until deleted in the 2022 REACH revision (EC, 2022)) and OECD TG 215, but our review of compliance check and test proposal decisions indicates that, in practice, ECHA has rejected proposals for TG 212 and TG 215 and required TG 210 instead. 

^d Usually as part of a substance evaluation (Section 1.3).

Source: Knight J et al.  Fish count, too - The animal toll of REACH aquatic toxicity tests. ALTEX. 2026;43(1):142-157. 

Currently, REACH (Annex IX) accepts OECD TG 210 “Fish, Early-life Stage Toxicity Test” for subchronic toxicity testing. Originally, OECD TG 212 and 215 were also accepted, but TG 212 was deleted in 2022, and, in practice, ECHA has only considered TG 210 sufficient to examine all sensitive points in the fish life-cycle.  For endocrine-active substances, additional testing may be required. The commonly required test is TG 234 “Fish Sexual Development Test”, where the duration is extended until fish reach sexual maturity. Although the endocrine disruption test is not a standard REACH requirement, concerns about endocrine activity can trigger it, independent of tonnage band. 

Subchronic Zebrafish Study Endpoints: Growth, Reproduction, and Organ-Level Readouts

The OECD TG 210 study runs from fertilized eggs to the point when control fish reach the free-feeding juvenile stage, exposing them throughout to a constant concentration of the chemical in the water. This window covers embryogenesis, hatching, yolk sac absorption, and early juvenile development: the phases where chemical exposure is most likely to produce measurable sublethal effects. 

The test ends when the fish in a parallel control group of fish, which are not exposed to the chemical, reach a juvenile life stage. This is about 28 or 30 days after hatching, depending on the species. For freshwater studies: rainbow trout, fathead minnow, zebrafish, and medaka. For estuarine and marine studies: sheepshead minnow and silverside. 

The mandatory endpoints are survival, hatching rate, and growth (body length and weight). These generate the NOEC, LOEC, or ECx values by using a regression model to estimate the concentration that would cause a x % change in the effect measured. Any morphological abnormalities or behavioral effects observed during the study should also be recorded. Moreover, the June 2025 revision of OECD TG 210 introduced optional parameters for histopathology and omics, including tissue collection for transcriptomic and metabolomic analyses. 

From Acute to Subchronic Toxicity: How Zebrafish Early-Life Stage Studies Reduce Testing Costs

A standard TG 210 protocol requires at least 80 fertilized eggs per concentration level across four replicate chambers. In zebrafish, a single spawning event produces hundreds of eggs, making this straightforward to achieve. Their small size means studies can be run in compact, temperature-controlled aquatic systems that require significantly less space and infrastructure than equivalent setups for rainbow trout or fathead minnow.

The study duration is also shorter. In zebrafish, the test runs for approximately 30 days post-hatch. In rainbow trout, the recommended duration of the test is 2 weeks after controls are free-feeding  (or 60 days post-hatch). For registrants working under regulatory timelines, that difference matters. A shorter study also means less test substance consumed. Rainbow trout are larger at every developmental stage, require higher water volumes, and therefore need greater quantities of the test compound to maintain the target concentrations throughout the exposure period. 

Finally, there is a scientific argument against relying on acute-to-chronic extrapolation as an alternative to running a TG 210 study. Acute-to-chronic ratios (ACRs) are widely used to estimate chronic NOECs from acute LC50 data. Compiled datasets of fish and invertebrate ACRs report a median value around 8–12, but a range spanning more than 16,000-fold (from 1.1 to 18,550) (Raimondo, Montague, and Barron, 2007). For 1 in 10 substances, the ACR exceeds 70. This means that for a significant proportion of chemicals, extrapolation from acute data will substantially underestimate chronic toxicity. Measured ELS data removes that uncertainty. 

At ZeClinics, we have the expertise and the capabilities to help you generate that data efficiently and within regulatory timelines. As a zebrafish CRO, we offer zebrafish assays for chemical safety assessment. Besides long-term toxicity testing, our services include acute toxicity screening and reproductive toxicity to go deeper into the safety profile of your compound: 

• Fish Embryo Acute Test (FET) - OECD TG 236
• Fish early-life stages toxicity test – OECD TG 210
• Fish Short-term Toxicity Test on Embryo and Sac-Fry Stages – OECD TG 212
• 21-day Fish Assay – OECD TG 230
• Fish Juvenile Growth Test – OECD TG 215
• Reproductive toxicity – OECD TG 229
• Fish Sexual Development Test (FSDT) – OECD TG 234
• Zebrafish extended one generation reproductive toxicity study (ZEOGRT) – OECD ZEOGRT

If your registration is approaching the 100 t/year threshold or you are dealing with a substance with an uncertain chronic risk profile, we can help you. 

Contact us!

Sources

European Parliament and Council. Regulation (EC) No 1907/2006 (REACH). 2006. Available from: https://eur-lex.europa.eu/eli/reg/2006/1907/oj/eng

Knight J, Rovida C, Willett K, Ingram J. Fish count, too - The animal toll of REACH aquatic toxicity tests. ALTEX. 2026;43(1):142-157. doi: 10.14573/altex.2506011. 

OECD (2025). Test No. 210: Fish, Early-Life Stage Toxicity Test. OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, Paris. https://doi.org/10.1787/9789264203785-en

Raimondo S, Montague BJ, Barron MG. Determinants of variability in acute to chronic toxicity ratios for aquatic invertebrates and fish. Environ Toxicol Chem. 2007 Sep;26(9):2019-23. doi: 10.1897/07-069R.1.

By Javier Terriente

Javier is the co-founder of ZeClinics and ZeCardio Therapeutics, two biotech firms specializing in zebrafish-based preclinical drug discovery for cardiovascular, neural, and toxicology applications. He combines scientific leadership with business acumen, having successfully driven fundraising efforts and strategic partnerships.

Currently leading scientific efforts at ZeCardioTx (and formerly CSO at ZeClinics), Javier also serves on the Board of Directors of AseBio, where he advocates for industry collaboration. His academic background includes a PhD in Molecular Biology and a Marie Curie Fellowship. Recognized as an expert in zebrafish models, he has published extensively and has supervised five industrial PhD theses.