Registration Dossier

Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

1) Key_Short-term toxicity to fish: LC50(96h) = 5621 mg/L (nominal) for Danio rerio (static, freshwater, EU Method C.1) (ongoing)

2) Key_Long-term toxicity to fish: NOEC (30d) ≥ 33 µg/L for Danio rerio (flow-through, freshwater, OECD 210, GLP)

3) Key_Long-term toxicity to aquatic invertebrates: NOEC (21d) ≥ 105 µg/L (nominal), 72.5 µg/L (geometric mean measured); EC10 (21d) > 105 µg/L (nominal), > 72.5 µg/L (geometric mean measured) for Daphnia magna based on mortality, reproduction per introduced adult and reproduction per surviving adult (semi-static, freshwater, OECD 211, GLP)

4) Key_Toxicity to aquatic algae and cyanobacteria: ErC50(72h) > 100 mg/L (nominal), ErC50(72h) > 2.467 mg/L (geometric mean measured) for Desmodesmus subspicatus (static, freshwater, OECD 201, GLP)

Additional information

Due to the poor solubility and the very fast hydrolysis (DT50 = 37s) of the test substance, no standard application could be used for the preparation of test solution for aquatic toxicity testing. As recommended in the OECD Guidance Document No. 23 on aqueous phase aquatic toxicity testing of difficult test chemicals (second edition, February 2019) solubility pre-experiments including analytical dose verification were performed before initiation of any aquatic toxicity testing to determine the maximum dissolved concentration that can be achieved in the specific test solution under test conditions. Especially for the long-term studies the maintenance and reproducibility of test medium concentrations was determined by concurrent analytical measurements as well, to enable the performance of a valid main experiment. The results of these solubility pre-experiments were used to form a basis of, and justification for, the test solution preparation procedures adopted for the toxicity tests.

Short-term toxicity to fish

The acute toxicity of the test substance to fish was investigeted according to EU Method C.1 (which is in most parts equivalent to OECD guideline 203). No GLP compliance was cited but the test facility is known to work under standard good laboratory practice. Danio rerio (Zebra fish) was used as test organism and exposed to nominal test concentrations of 1000, 3160 and 10000 mg/L of the test substance for 96 hours under static conditions. A control was running in parallel containing dilution water only. The test was carried out using a static design with a temperature range of 21.2 °C - 22.0 °C (control: 21.6 °C - 21.9 °C) and a pH range of 7.2 - 7.8 (control: 7.4 - 7.9) depending on the substance concentrations (1000, 3160 and 10000 mg/L). The light-dark cycle was 16 hours light / 8 h dark. Direct weighing was the method of administration followed by stirring and filtration. Any mortality and / or visible sublethal effects were observed and recorded throughout the test.

The 96h LC0 and the 96h LC100 were determined directly from the raw data and reported to be 3160 and 10000 mg/L, respectively. The 96h LC50 was calculated as geometric mean of the 96h LC0 and the 96h LC100 and was reported to be 5621 mg/L.

Long-term toxicity to fish

The study is currently running under GLP according to OECD TG 210. The purpose of this study is to evaluate the toxicity of the test substance to the early-life stages of fish. For this purpose, fertilised eggs of Danio rerio (zebrafish) are exposed in a flow-through test to aqueous test media containing the test item at the nominal concentrations of 33 and 105 µg/L (each in 100 µL DMF/L) ) under defined conditions. In parallel a solvent control (100 µL DMF/L) and a control are running.

Following several independent pre-experiments to determine the solubility and stability of the test material in the test medium and the need to develop a reproducible analytical method, dimethylformamide (DMF) was used as solvent additive to achieve stable and reproducible concentrations in the test medium.

Due to the test item properties it was decided not to prepare the test media constantly by using a constant flow rate by peristaltic pumps or syringe pumps but to prepare the test media manually twice a day. In order to keep the test design in a manageable dimension the experiment will be running with two test concentrations only. According to OECD 210 this extended limit design is acceptable for the determination of a NOEC.

The test will last for 30 days after hatching. The recorded effects will be mortality, hatching success, growth and observed abnormal behaviour of the fish.

The purpose of the analytical part of this study is to verify the concentrations of the test item in the test medium. The quantification of the test material in the test samples will be performed using a HPLC method with MS/MS detection.

As soon as the final report is available, outstanding data, results and endpoints will be added to this study record. So far the lowest test concentration of 33 µg/L is considered as worst-case NOEC.

Long-term toxicity to aquatic invertebrates

The purpose of this study was to evaluate the influence of the test material on mortality and reproduction of Daphnia magna during an exposure period of 21 days. Therefore, female Daphnia (< 24 hours old) taken from an in-house laboratory culture of the test facility, were exposed in a semi-static test to aqueous test media containing the test item at concentrations of 105, 34, 11, 3.4, 1.1 and 0.34 µg test item/L (each in 100 µL DMF/L) ), a solvent control (100 µL DMF/L) and a control, corresponding to geometric mean measured concentrations of 72.5, 24.7, 8.28, 2.11, 1.32 and 1.32 µg test item/L, for a period of 21 days under defined conditions. Following several independent pre-experiments to determine the solubility and stability of the test material in the test medium and the need to develop a reproducible analytical method, dimethylformamide (DMF) was used as solvent additive to achieve stable and reproducible concentrations in the test medium. The mortality of adults, the number of offspring per introduced adult and the number of offspring per surviving adult were compared with corresponding parameters in the controls.

The quantification of the test material in the test samples was performed using HPLC MS/MS detection.

Based on the obtained results no toxic effects against daphnia were observed up to the highest concentration of nominal 105 µg/L, corresponding to a geometric mean measured concentration of 72.5 µg/L, under exposure conditions. The following endpoints were determined referring to the geometric mean measured concentrations of the test material:

EC50 (21d) mortality > 72.5 µg/L

EC10 (21d) mortality > 72.5 µg/L

NOEC (21d) mortality 72.5 µg/L

 

EC50 (21d) reproduction per introduced adult > 72.5 µg/L

EC10 (21d) reproduction per introduced adult > 72.5 µg/L

NOEC (21d) reproduction per introduced adult 72.5 µg/L

 

EC50 (21d) reproduction per surviving adult > 72.5 µg/L

EC10 (21d) reproduction per surviving adult > 72.5 µg/L

NOEC (21d) reproduction per surviving adult 72.5 µg/L

Toxicity to aquatic algae and cyanobacteria

The acute toxicity of the test substance to aquatic algae was tested according to EU Method C.3, which is equivalent to OECD TG 201, in a static freshwater test with Desmodesmus subspicatus (former name:Scenedesmus subspicatus) as test organism. The study was conducted under certificated GLP compliance.

Due to the test substance properties, a limit test was performed at the limit of water solubility under test conditions in order to demonstrate that the EC50values (based on yield as well as on growth rate) are greater than this concentration. Pre-experiments were conducted, which provided the concentration, as well as the biological and analytical design to be used in the main test. The final test concentration was chosen as nominal 100 mg/L, whereby the test medium was prepared by direct weighing, prolonged stirring and a subsequent settling period before withdrawal of the solved fraction in the middle.

For the test item concentration as well as for the control 6 replicates were prepared. The algal inocula for the experiment were taken from an exponentially growing pre-culture and were mixed with the nutrient medium to make up to a final cell density of about 5000 cells per milliliter in the test medium. The algae were exposed for a period of 72 hours and the cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate and yield, relative to control cultures grown under identical conditions. All calculations were carried out using a statistical software programme. The maintenance of test item concentrations was proven by analytical measurements (HPLC MS/MS).

The following results were determined for yield and growth rate of the algal population within 72 h exposure period based on nominal and geometric mean measured concentrations of the test substance:

ErC50 (72h) > 100 mg/L

ErC10 (72h) > 100 mg/L

NOErC (72h) 100 mg/L

ErC50 (72h) > 2.467mg/L

ErC10 (72h) > 2.467 mg/L

NOErC (72h) 2.467 mg/L

Acute toxicity tests towards freshwater as well as marine algae were investigated for the read-across substance m-tolylidene diisocyanate (TDI) according to OECD Guideline 201 also. The rapid hydrolysis rate of the test substance was taken into consideration when presenting the test results.

The freshwater algal species Chlorella vulgaris was tested for a period of 96 hours under defined conditions, resulting in an 96h EC50of 4300 mg/L and a NOEC of 1000 mg/L (Tadokoro et al., 1997).

The marine algal species Skeletonema costatum was tested for a period of 96 hours under defined conditions, resulting in an 96h EC50 of 3230 mg/L (Tadokoro et al., 1997).

Toxicity to aquatic microorganisms

The acute toxicity of the test substance to microorganisms was investigated in accordance with OECD Guideline 209 (Kanne, 1989). Activated sludge was used in the static freshwater experiment with an exposure time to the test substance of 3 hours. The EC50 value of greater or equal to 10000 mg/L is given as result, with the remark of water insolubility for the substance.

Toxicity to aquatic plants (other than algae) and other aquatic organisms

In accordance to REACH Regulation (EC) 1907/2006 this information is not mandatory for a registration of a chemical at a tonnage band of 100 - 1000 tons/year.

Considering all available data on short- and long-term toxicity to aquatic organisms, no acute toxicity was determined for the aquatic environment. However, based on the available long-term results (NOEC </= 0.1 mg/L), the test substance need to be classified as Aquatic Chronic Category 1 according to Regulation (EC) No 1272/2008 as worst case assumption.