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Ecotoxicological information

Short-term toxicity to fish

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Description of key information

LC50 (96 h) = 1.614 mg/l (measured)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
1.614 mg/L

Additional information

The toxic potential of the substance to fish is evaluated considering data on the substance itself and also experimental results obtained by using Similar Substance 01. Justification for Read Across is given in Section 13 of IUCLID.

The toxic potential of Similar Substance 01 to fish (Brachydanio rerio) was evaluated in the Key Study according to the ISO7346 -1. The concentrations to be tested in the main study were selected based on a range-finding test in which three fish were exposed to five concentrations of the substance (200 -100 -50 -25 -10 mg/l). In the main test seven fish were exposed to 50 -30 -25 -20 mg/l of the substance for 96 hours during which fish were observed for mortality. Negative and positive control run in parallel. Mortality, pH, temperature and oxygen concentration were measured every 24 hours. Concentrations of the substance were measured at the beginning and at the end of the test. LC50 (96 h) = 1.614 mg/l (measured)

The potential of the substance to induce toxic effects to fish is investigated in the two literature studies in which the fish embryo toxicity (FET) test and two in vitro bioassays (NR and EROD assays) were performed.

The test substance was investigated for its acute embryo toxicity and teratogenicity using the FET test according to the OECD Guideline 236 and DIN 15088 . The study was performed using the zebrafish Danio rerio, a model organism useful for the prediction of human teratogenicity. The embryos were exposed to five different concentrations of the substance ranging from 41 mg/l to 152 mg/l (nominal) for 48 hours. Positive and negative controls run in parallel. After exposure, teratogenic effects as well as lethal endpoints (coagulation of the embryo, non-detachment of the tail and non-detection of the heartbeat) were recorded. The embryos were assessed in the chorion. Microscopic examination revealed elongated heads for fish embryos treated with the substance compared to untreated embryos. Embryos' head lengths were measured after exposure to the substance. The test substance caused a statistically significant effect on head development resulting in elevated head lengths in zebrafish embryos. LC50 (48 h) = 83 mg/l (nominal)

A further evaluation of the toxic potential of the substance was investigated by means of the NR assay and the EROD assay in two in vitro bioassays. The first assay assess cytotoxicity and the second one assess the aryl hydrocarbon receptor agonism, so called dioxin-like activity. The permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss) was used and cells were exposed 48 hours and 72 hours for the NR assay and EROD assay respectively. Furthermore, established reference fuels were used in order to compare the toxicological effects of the substance to those of the fuels; NR50-value = 7.95 g/l. With an NR50-value of 7.95 g/l, the substance appears to have a low toxic potential. The Effective chemical Activity of the substance- EA-50NR is well above 0.01, showing a lower EA-50NR (0.052). EA-50NR values well above 0.01 indicate a baseline toxicity and no excess toxicity. Neither the potential biofuels nor theWAFs of the reference fuels revealed any significant dioxin-like activity.

According to the authors, bioassays detect adverse effects of single compounds and complex chemical mixtures on a variety of test organisms, including animals, plants, fungi and bacteria. They allow evaluating effects of complex samples even though none or only few compounds are known or present at very low concentrations. With regard to their limit of detection and their sensitivity, bioassays, such as the 7-ethoxyresorufin-O-deethylase (EROD) assay, can even compete with GC-MS analytical procedures. Thus, they are ideally suited for the investigation of newly developed biofuel candidates as well as complex fossil fuels. Within the interdisciplinary German Cluster of Excellence “Tailor-made fuel from Biomass” (TMFB) of the German Research Foundation (DFG), ecotoxicological bioassays were applied for the first time as part of the development process of novel biofuel molecules. These first experiments should give insight into requiredmodifications of the bioassay protocols, and deliver a first assessment of the ecotoxicological hazard potential of these potential biofuels. Moreover, as part of an environmentally conscious and sustainable design process within the TMFB, they allow the identification of potential harmful fuel candidates at an early stage of the development and can be useful for an early selection of low hazard biofuel candidates for further development. In vitro cytotoxicity tests are commonly applied as rapid and reproducible biotests that can be used in a preliminary screening for the identification of toxic samples, or for defining required modifications of the test design. They can give a first insight into the acute toxic potential of a sample and are often applied prior to mechanism-specific bioassays. In this regard, the Neutral Red retention (NR) assay is ideally suited for this study and the investigation of nearly unknown substances, such as biofuel candidates, due to its relatively simple and easily modifiable test design. It is commonly applied for the detection of cytotoxic effects before investigating a sample in mechanism-specific bioassays. These assays, such as the EROD assay, give insight into the toxic mechanisms of the investigated biofuel samples, such as the induction of the aryl-hydrocarbon receptor (AhR). These cellular changes that can be detected by the EROD assay are often first warning signals for an environmental disturbance. Both bioassays were applied using the liver-derived cell line RTL-W1 (Oncorhynchus mykiss), which is considered as a sensitive tool for the investigation of the toxic potency of environmental contaminants or mechanism specific endpoints, such as EROD activity.

A comparison between the findings of the two literature articles was made by the authors of the in vitro bioassays. Acute embryotoxicity of the substance was found to be nearly two orders of magnitude and approximately 4 times higher than cytotoxicity, respectively. Danio rerio embryos appear to be very sensitive to an exposure with the substance. However, the specific mode-of-action is not known. Moreover, many fish cell lines are known to be several orders of magnitude less sensitive than fish. The increased toxicity of the substance to D. rerio is most likely caused by species-specific differences between D. rerio embryos and RTL-W1 cells or differences between the level of complexity (cell vs.embryo). Applying a different and more sensitive test system, such as the RTgill-based toxicity assays, might result in an effect concentration more similar to the LC-value determined for D. rerio embryos. Thus it could be examined if other cell line than RTL-W1 would be favourable for a screening regarding the cytotoxic potential of the substance in the future.

As per the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7b: Endpoint specific guidance (Version 4.0 June 2017) a possible alternative, the fish embryo toxicity test, is currently under evaluation in the OECD Guideline program. OECD 236 is not included in the adopted OECD Guidelines required for the evaluation of aquatic pelagic toxicity (Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7b: Endpoint specific guidance, Version 4.0 June 2017). However if alternative and adequate test methods are available for acute fish toxicity they may be considered to be used instead for classification (e.g. a proposal to use the fish embryo test (FET) as an alternative to the acute fish toxicity test has been made and is currently under evaluation in the OECD Guideline program.

In addition, it is mentioned that, at present, there are no EU / OECD guidelines for in vitro tests of relevance to aquatic toxicity. There are ongoing efforts to develop and validate in vitro methods, which in future might be useful in a testing strategy for acute aquatic toxicity (e.g. ECVAM study on optimisation of cytotoxicity tests and CEFIC LRi study ECO 8 aiming to replacing the acute fish toxicity test using fish cell lines and fish embryos). Most of the tests with permanent cell lines (monolayers or suspension cultures) measure the basal cytotoxic effects of chemical substances. Although the extrapolation of in vitro data to in vivo data is discussed in literature further research in this area is needed and there is currently not enough information available to give guidance for the extrapolation from in vitro data to in vivo data. Various publications show that, for the correlation with in vivo results the in vitro bioavailability of the substances tested should be considered. Currently, there are no validated fish cell systems available. Nevertheless, information from in vitro studies might be considered in a Weight of Evidence approach provided that they fulfil certain data quality aspects and comply with the Annex XI criteria. Cytotoxicity tests using fish cell lines are more likely to indicate acute toxic effects although it is necessary to consider that they might lack of realistic toxicokinetics including metabolism.

By accounting the above considerations, the two literature studies (FET and in vitro bioassays) are considered as supporting data to the key study on Similar Substance 01. The study on Similar Substance 01 is conducted accoring to an Intenational standard method and respects the exposure conditions and the species/size of fish reccomended in the respective standard method, OECD 203 Guideline and classification as per the CLP Regulation (EC) No.1272/2008. Moreover, the effect concentration (LC50) is lower than the one obtained in the FET toxicity study with the substance and thus it considers the worst-case scenario. Considering that the extrapolation from in vitro to in vivo data is not possible at the moment, the in vitro bioassays' findings are not the ones to predominantly be used for the safety assessement and classification of the substance. Notwithstanding the short comings of the two literature studies, their findings support the evaluation of the toxic potential of the substance to fish.