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Ecotoxicological information

Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
Justification of not submitting the requested information by the deadline given by ECHA

A special analytical method for the determination of the substance in ecotoxicological medium was developed in an in-house laboratory by one of the co-registrants of the substance. As the water solubility of the substance is in the µg/L range and the substance is very adsorptive, a very sensitive method had to be developed. To achieve very low sensitivity of the LC-MS method, Tetrahydrofuran (THF) was used as a solvent for the samples and as a mobile phase for the LC-MS device. Whit this solvent and mobile phase an LOQ of 5 µg/L (the water solubility) was achieved. Using this method the Long-term toxicity test to Daphnia was conducted in the in-house laboratory of the co-registrant. For the Fish Early Life Stage (FELS) test a GLP CRO was commissioned. Unfortunately, after many pre-test measurements using two different LC-MS devices (Xevo TQ-XS LC/MS device from Waters, Inc. and LC/MS/MS ABSciex from Waters, Inc. and AB Sciex, Inc.) no sufficiently sensitive method for the detection of the substance in aqueous media could be validated. In the pre-tests THF and Acetonitrile (ACN) were used as solvents and mobile phases, and still the obtained LOQ was in the mg/L range.
Due to the unsuccessful analytical measurements and the approaching CCh deadline, all actions at this CRO had to be aborted and a new CRO had to be commissioned with the FELS study. Unfortunately, no established CRO was willing to validate an analytical method using THF as a mobile phase, due to the corrosive properties of THF and the high risk of damaging the devices. The producers of the devices (Waters, Inc. and AB Sciex, Inc.) were contacted for a consultation and all of them advised against using THF with their devices. At the end, no GLP CRO was able to replicate the sensitive analytical method developed by the co-registrant. Therefore, more time and effort is needed to develop an acceptable analytical method at the CRO to be able to perform the FELS study.
Due to the analytical struggles and the substances properties, the performance of the FELS study has been postponed in time and thus, the CCh deadline could not be met accordingly. As soon as any results from the CRO are available, the technical dossier will be accordingly updated.
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019 - 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
26 Jul 2013
Principles of method if other than guideline:
Dose range finder study.
GLP compliance:
no
Remarks:
The study was performed under non-GLP conditions from GLP compliant laboratory and personnel.
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions containing 5 g/L in acetone were prepared. These stock solutions were used for preparation of the lower concentrated stock solutions at the concentrations of 50 mg/L and 500 mg/L. These stock solutions were dosed with syringe pumps directly into the flowing dilution water. Flow of stock solution (1.4 µL/min) and dilution water (14 mL/min) was adjusted to reach the planned concentration of 5 µg/L and 50 µg/L in the treatments. The concentrations of the stock solutions were 10000-fold higher than the nominal concentrations in the treatments. For the solvent control acetone was used instead of stock solution.
- Controls: solvent control
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: 100 µL/L
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Strain: Wildtype Strain Westaquarium
- Source: University Heidelberg, Germany

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
For production of eggs, one day before the start of the test the spawn dishes were placed into the aquaria and charged with two adult males and one adult female, respectively. On the next morning, after spawning of the adult animals, the spawn dishes including the eggs were removed from the spawning aquaria and the eggs were checked with a binocular for fertilisation. Fertilised eggs can be clearly identified by the development of a blastula. Viable fertilized eggs were separated from unfertilized eggs and the scheduled number of fertilized eggs per replicate was transferred in a glass beaker, filled with dilution water.

POST-HATCH FEEDING
- Start date: 1. after hatching, day 6; 2. from the 3rd week until the end of the study, day 35
- Type/source of feed: 1. Powdered flake food; 2. Artemia nauplii
- Frequency of feeding: 1. 1 - 2times per working day; 2. once a day
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Test temperature:
25.0 - 27.0
pH:
7.1 - 7.4
Dissolved oxygen:
7.0 - 8.1 mg O2/L
Nominal and measured concentrations:
Nominal concentrations: 5, 50 µg/L
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type: open, covered with a plastic covering to avoid dust and particles antering the system
- Material, size, headspace, fill volume: stainless steel, 12 L
- Aeration: no
- Type of flow-through: syringe pumps pumping the stock solutions directly in the flowing dilution water from the flow meters to the aquaria
- Renewal rate of test solution (frequency/flow rate): 2 medium renewals per aquarium per day, 14 mL/min
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted freshwater is used in the test. Tap water is desalinated by reverse osmosis. The conductivity and pH value are then adjusted by adding electrolyte solutions (sea salt and sodium bicarbonate).

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED : survival, daily on working days

VEHICLE CONTROL PERFORMED: yes

POST-HATCH DETAILS
- Begin of post-hatch period: day 6
Reference substance (positive control):
no
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
ca. 50 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality

The surviving rate is presented in the following table:


Table 1. Hatching Data















































































Nominal


Concentration


 in µg/L



Replicate



Number of Eggs at the Start



Number of Fish


survived



Surviving rate



Solvent Control



1



20



15



76 %



2



20



16



3



20



16



4



20



14



5



1



20



15



75 %



2



20



15



3



20



14



4



20



16



50



1



20



18



81 %



2



20



17



3



20



16



4



20



14



No dead larvae or eggs from which nothing hatched could be detected. None of the surviving fish showed abnormal appearance or abnormal behaviour.


The NOEC is determined to be 50 µg/L.

Validity criteria fulfilled:
yes
Conclusions:
The NOEC under the conditions of the FELS test was determined to be 50 µg/L.
Executive summary:

The toxicity of the test item in the Early-Life Stage Toxicity Test with Danio rerio was tested under flow-through conditions according to OECD 210. The water solubility of the test item was estimated as 1 – 5 µg/L. Therefore, this study was performed with the concentrations 5 µg/L and 50 µg/L. Because of the poor water solubility of the test item, acetone was used to spike the test solutions. Dilution water was added with flow meters. Stock solutions of the test item were added with syringe pumps. The stock solutions were dosed with the syringe pumps directly into the flowing dilution water. 20 fertilised eggs were added to each replicate of the solvent control and each replicate of the treatments. After 35 days the surviving fishes were counted. The tested concentrations showed no toxicity. The surviving rate in the highest concentration was even slightly higher than in the solvent control. No abnormal appearance or behavior of the surviving fishes was observed. The validity criteria of the guideline, hatching success in the controls greater than or equal to 70 % and post-hatch success greater than or equal to 75 % were fulfilled. The NOEC is expected to be around 50 µg/L.

Description of key information

The main test on the endpoint Long-term toxicity to Fish will be submitted later based on ECHA communication/decision number CCH-D-2114453333-55-01/F.

Key value for chemical safety assessment

Additional information

FELS Pre-test


The toxicity of the test item in the Early-Life Stage Toxicity Test with Danio rerio was tested under flow-through conditions according to OECD 210 in a pre-test. The water solubility of the test item was estimated as 1 – 5 µg/L. Therefore, this study was performed with the concentrations 5 µg/L and 50 µg/L. Because of the poor water solubility of the test item, acetone was used to spike the test solutions. Dilution water was added with flow meters. Stock solutions of the test item were added with syringe pumps. The stock solutions were dosed with the syringe pumps directly into the flowing dilution water. 20 fertilised eggs were added to each replicate of the solvent control and each replicate of the treatments. After 35 days the surviving fishes were counted. The tested concentrations showed no toxicity. The surviving rate in the highest concentration was even slightly higher than in the solvent control. No abnormal appearance or behavior of the surviving fishes was observed. The validity criteria of the guideline, hatching success in the controls greater than or equal to 70 % and post-hatch success greater than or equal to 75 % were fulfilled. The NOEC is expected to be around 50 µg/L.