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Ecotoxicological information

Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
study ongoing
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, Section 2, No. 210 "Fish, Early-life Stage Toxicity Test", adopted July 26, 2013
Deviations:
not applicable
Remarks:
study ongoing
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 33 and 105 µg/L, control and solvent control
- Sampling method: At least one sample from the freshly prepared stock solutions and at least one sample from the test media of each replicate (aquaria) of all test concentrations and the controls will be taken prior to the start of the test. Afterwards at least once per week one sample from the test media of each replicate (aquaria) of all test concentrations will be taken. All test medium samples will be taken from the approximate centre of the aquaria. All samples will be diluted by a factor of 2 with acetonitrile. Additional samples of the control blank and the dilution solvent acetonitrile will be taken at each sampling date without any sample treatment and filled directly into an HPLC vial.
- Sample storage conditions before analysis: All samples will be analysed stand-by at the date of sampling. Any spare samples will be stored in a freezer until delivery of the final report to enable additional analyses on request of the Sponsor. After delivery of the final report, all samples will be discarded.
Vehicle:
yes
Remarks:
Dimethylformamid (DMF) at a concentration of 100 µL/L
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions for each test concentration were prepared by dissolving the test item in dimethylformamide (DMF). From each stock solution a corresponding application solution in test water was prepared by mixing an appropriate amount in test water and stirring for approximately 30 minutes. These application solutions were stirred continuously and pumped directly into the aquaria with a constant flow rate by peristaltic pumps. The application solutions will be prepared twice daily. The flow rate of the application solution and the dilution water will be determined at least twice weekly during the test.
- Controls: Control (pure test water), solvent control (test water with the solvent DMF at a concentration of 100 µL/L)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: The fertilized eggs will be obtained from in-house breeding or a commercial fish breeder (e.g. University of Heidelberg, Germany) and will be specified in the final report.
- Age at study initiation (mean and range, SD): Freshly fertilised eggs
- Length at study initiation (length definition, mean, range and SD): will be specified after receipt of the final report.
- Weight at study initiation (mean and range, SD): Will be specified after receipt of the final report.
- Method of breeding: Will be specified after receipt of the final report.
- Feeding during test :
The newly-hatched larvae will be fed with conventionally finely ground flake food (for example Sera Micron, Tetra Baby) and Paramecia ad libitum. The food will be gradually supplemented with crushed 24 - 48 h-old brine shrimp nauplii.
Juveniles will be fed with 24 - 48 h-old brine shrimp nauplii (crushed if necessary) and conventionally flake food. Fish will be fed ad libitum.
If necessary (e.g. in tests with high flow rates), water flow respectively the pumps can be stopped for approximately 30 min. in order to improve food supply.
The information will be updated after receipt of the final report.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
31 d
Test temperature:
26 + 1.5 °C, not differing by more than ±1.5 °C between the test vessels or between days during the test.
pH:
6.5 - 8.5
Dissolved oxygen:
At least 60 % of the air saturation value (ASV).
Nominal and measured concentrations:
nominal: 105 and 33 µg test item/L, a solvent control and a water control
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: at least 4 litre test medium
- Aeration: yes
- Type of flow-through (e.g. peristaltic or proportional diluter): 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.
- Renewal rate of test solution (frequency/flow rate): Threefold (a 2-3 fold water exchange is accepted by the OECD 210, if the loading rate of < 0.5 g/L per 24 h is respected). The flow rate of the application solution and the dilution water will be determined at least twice weekly during the test.
- No. of organisms per vessel: at least 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: < 0.5 g/L per 24 h

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Defined amounts of four analytical grade salts (see below) will be added in defined amounts of deionised water to produce reconstituted water. Details will be updated after receipt of the final report.
CaCI2 x 2H20 MgS04 x 7H20 NaHC03 KCI
Ratio of Ca : Mg = 4:1 (based on molarity) Na : K = 10 :1 (based on molarity)

OTHER TEST CONDITIONS
- Adjustment of pH: Information will be updated after receipt of the final report.
- Photoperiod: 12 -16 h light: 8-12 h dark
- Light intensity: Information will be updated after receipt of the final report.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : During the test period, the eggs and larvae will be observed daily for survival, hatching, abnormal appearance and behaviour. Additionally, at the end of the test the individual length of all surviving fish will be determined. Also, at the end of the test all surviving fish will be weighed (dry weight and wet weight (blotted dry)) in groups by test vessel. Dead embryos and fish will be removed directly after observation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: 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 was running with two test concentrations only. According to OECD 210 this extended limit design is acceptable for the determination of a NOEC.

RANGE-FINDING STUDY
- Test concentrations: 100, 10, 1.0 and 0.10 µg/L
- Results used to determine the conditions for the definitive study: No effect on mortality was observed at all test concentrations in the rang-fnding study.
Reference substance (positive control):
not required
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 33 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: worst-case assumption
Remarks:
true results outstanding
Details on results:
Will be given after receipft of the final report.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Will be given after receipft of the final report.
Validity criteria fulfilled:
not applicable
Remarks:
Study ongoing
Conclusions:
The study is running under GLP according to OECD TG 210 on the registered substance itself. The method is to be considered scientifically reasonable and no deficiencies in documentation or deviations from the guidelines are expected. Hence, the outstanding results can be considered as reliable to assess the long-term toxicity of the test substance towards fish. Currently outstanding results and additional relevant study data will be added to complete the study record as soon as the final report is available.
Executive summary:

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.

Description of key information

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

Key value for chemical safety assessment

Additional information

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.