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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:
2020-06-29 - 2020-08-18 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
yes
Remarks:
please refer to section "Any other information on materials and methods incl. tables"
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23 "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals"
Version / remarks:
2nd Ed., February 08, 2019
Deviations:
not applicable
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 33 and 105 µg/L, control and solvent control
- Sampling method: The samples were taken from the biological phase of the study. One sample from each replicate (aquarium) of the test media of all test concentrations and the controls were taken prior to the initiation of the test (equilibration phase). Afterwards once per week at day 0 (=start of the test), 1, 3, 10, 17, 24 and 30 day post hatch (DPH), one sample from each replicate (aquarium) of the test media of all test concentrations and the controls were taken. All test medium samples were taken from the approximate centre of the aquaria. All samples were diluted by a factor of 2 with acetonitrile. Additional samples of the control blank and the dilution solvent acetonitrile were taken at each sampling date without any sample treatment and filled directly into an HPLC vial.
- Sample storage conditions before analysis: Two of four replicate samples were analysed stand-by at the date of sampling. Any spare samples were stored in a freezer until delivery of the final report to enable additional analyses on request of the Sponsor.
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: 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.
Stock solutions for each test concentration were prepared by dissolving the test item in water-free DMF. From each stock solution a corresponding application solution in test water was prepared by mixing an appropriate amount in test water and intensive stirring. These application solutions were stirred continuously and pumped directly into the aquaria with a constant flow rate by peristaltic pumps.
A DMF stock solution of 1050 mg/L was prepared by dissolving the test item in water-free DMF, a DMF stock solution of 330 mg/L was prepared by adding 17.14 mL of water-free DMF to 7.86 mL of the 1050 mg/L stock solution.
The stock solutions were renewed twice weekly (after 3 or 4 days). Prior to the initiation of the test, the dosing system was calibrated through the use of appropriate analysis techniques.
100 µL of the DMF stock solutions were added to each L of test media twice daily to prepare the application solutions. Remains of the previous application solution were discarded once daily (in the morning). One application solution was used for two aquaria.
The dosage of the application solutions was performed with a tube pump. The accuracy of the dosage was checked 1 and 0 days prior the insertion of the eggs (DAI 0).
- 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 concentration separation factor: 3.18
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The appearance of the test item in the test medium was observed once every day in all test concentrations. All test media were clear and colorless.
- Other relevant information: Several pre-experiments were performed to determine the maintenance and reproducibility of test medium concentrations by concurrent analytical measurements and 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 procedure. According to these pre-experiments the test concentration of 105 µg/L in DMF represents the highest achievable and reproducible concentration of the registered substance for the performance of an aquatic long-term study.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio)
- Source: The eggs were obtained from the brood stock of ibacon GmbH.
- Age at study initiation (mean and range, SD): Freshly fertilised eggs (before beginning of gastrulation)
- Length at study initiation (length definition, mean, range and SD): not applicable due to insertion of eggs
- Weight at study initiation (mean and range, SD): not applicable due to insertion of eggs
- Method of breeding: not applicable due to insertion of eggs
- Feeding during test : From day 2 DPH (Days Post Hatch) onwards the fish were fed 3 times a day. The newly-hatched larvae were fed with conventionally finely ground flake food (mixture (1:1) of Sera Micron, Tetra Baby) and Paramecia ad libitum. The food was gradually supplemented with crushed 24 - 48 h-old brine shrimp nauplii. Juveniles were fed with 24 - 48 h-old brine shrimp nauplii (crushed if necessary) and conventionally flake food (TetraMin). Fish were fed ad libitum.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
31 d
Hardness:
89.0 to 178.0 mg CaCO3/L
Test temperature:
24.6 - 26.3 °C
pH:
7.2 - 7.8
Dissolved oxygen:
62 - 105% of the air saturation value (ASV)
Beside, the dissolved oxygen concentration was measured to be below 51% of the air saturation value on day 22 DPH in two replicates of the 33 µg test item/L group. For further information on this deviation please refer to section "Any other information on materials and methods incl. tables".
Nominal and measured concentrations:
nominal: 105 and 33 µg test item/L, a solvent control and a water control
time weighted average concentration: 95.8 and 27.5 µg test item/L, a solvvent control and a water control
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.
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 6 litre glass aquaria with 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 per day (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 stock solution and the dilution water were determined twice a week during the test. The flow rate of the test item solution was 7.9 ± 0.4 mL/min (mean over test duration ± SD).
- No. of organisms per vessel: At the start of the test 25 fertilised eggs were introduced into each aquarium (100 eggs per test concentration, except for the solvent control, where on replicate contained 26 eggs, i.e. 101 eggs per test concentration).
- 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: The loading rate was calculated by overall wet weight in each concentration at the end of the test divided by the volume of the test vessel and the 3 fold water exchange per 24 hours. Accordingly the loading rate was 32.80 to 39.32 mg/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: In deionised water (conductivity CaCl2 x 2H2O: 1.43 mmol/L (= 209.7 mg/L)
MgSO4 x 7H2O: 0.36 mmol/L (= 87.7 mg/L)
NaHCO3: 0.55 mmol/L (= 46.4 mg/L)
KCl: 0.055 mmol/L (= 4.13 mg/L)
Ratio of Ca:Mg = 4:1 (based on molarity)
Na:K = 10:1 (based on molarity)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark with a 30 min dawn/dusk period
- Light intensity: 700 to 880 lux

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


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.18
- 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
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 105 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 105 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 105 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
> 105 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
> 105 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
length
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
> 105 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 95.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 95.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
30 d
Dose descriptor:
NOEC
Effect conc.:
>= 95.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
weight
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
> 95.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
> 95.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
> 95.8 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
- Mortality/survival at embryo, larval, juvenile, and adult stages: During the test several larvae died in all treatment groups. The survival in all treatment groups was comparable to the control and therefore within the biological variance. Hence, the LOEC for post hatch survival was determined to be > 105 µg test item/L nominal and the NOEC is ≥ 105 µg test item/L nominal, corresponding to 95.8 µg test item/L based on time-weighted arithmetic mean measured concentrations.
The observed sublethal effects were deformations, oedema, fish laying on side or back, tumbling during swimming, fish mainly on the bottom, apathy or fish mainly at the water surface.
The number of fish tumbling during swimming at a certain date was slightly higher in the test item treated groups, e.g. five fish on DPH 11 at 33 µg/L and 8 fish on DPH 12 at 105 µg/L compared to a maximum of one fish tumbling during swimming in the control or three fish tumbling during swimming in the solvent control (DPH 9). However, the effects were only temporary or did not lead to a statistically significantly increased mortality and were considered to be within in the range of the biological variance.
- Days to hatch or time to release of young: First larvae hatched from eggs 2 days after insertion in the control and all test item treatment groups.
- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: 87 % off eggs in the control and 94 % in the solvent control (91 % in the pooled control) were hatched at day 4 after insertion (start of post hatch period).
Hatching was completed after 7 days after insertion of eggs in the control and all test item treatment groups. The LOEC for hatchability was determined to be > 105 µg test item/L nominal and the NOEC is ≥ 105 µg test item/L nominal, corresponding to 95.8 µg test item/L based on time-weighted arithmetic mean measured concentrations.
- Observations on body length and weight of young and/or exposed parents at one or more time periods:
Length: Mean total length of fish (at age of 30 DPH) was 12.788 mm in the control, 13.489 mm in the solvent control and between 13.370 and 13.379 mm in the test item treated groups. There was no statistically significant difference between the test item treated groups and the pooled control (Dunnett's t-test, α = 0.05, one sided smaller). Therefore, the LOEC for body length was determined to be > 105 µg test item/L nominal and the NOEC was determined to be ≥ 105 µg test item/L nominal, corresponding to 95.8 µg test item/L based on time-weighted arithmetic mean measured concentrations.
Wet weight: Mean wet weight per fish for the control was 18.131 mg and 20.577 mg for the solvent control. The mean wet weight of larvae in the test item treatment groups ranged between 20.963 and 23.925 mg. There was no statistically significant difference between the test item treated groups and the pooled control (Williams t-test, α = 0.05, one sided smaller). Therefore, the LOEC for wet weight was determined to be > 105 µg test item/L nominal and the NOEC was determined to be ≥ 105 µg test item/L nominal, corresponding to 95.8 µg test item/L based on time-weighted arithmetic mean measured concentrations.
Dry weight: Mean dry weight per fish for the control was 4.235 mg and 4.707 mg for the solvent control. The mean dry weight of larvae in the test item treatment groups ranged between 4.754 and 6.460 mg. There was no statistically significant difference between the test item treated groups and the pooled control (Dunnett's t-test, α = 0.05, one sided smaller). Therefore, the LOEC for dry weight was determined to be > 105 µg test item/L nominal and the NOEC was determined to be ≥ 105 µg test item/L nominal, corresponding to 95.8 g test item/L based on time-weighted arithmetic mean measured concentrations.
- Other biological observations:
Number of coagulated eggs: The difference in the number of coagulated eggs occurred randomly and did not show any dose-response relationship.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
For hatching success no statistical difference between control and solvent control was observed (Chi² 2x2 Table Test with Bonferroni correction, α = 0.05, two-sided). Therefore, both controls were pooled.
For hatching success data a trend analysis was performed (Trend Analysis by Contrasts). As no trend was observed the NOEC and LOEC for hatching success was calculated by Chi² 2x2 Table Test with Bonferroni correction (α = 0.05, one sided greater).
For post-hatch survival a comparison between control and solvent control was performed by using a Chi² 2x2 Table Test with Bonferroni correction (α = 0.05, two-sided). No statistically significant difference between control and solvent control was detected so both controls were pooled for further statistical analysis.For post-hatch survival data a trend analysis was performed (Trend Analysis by Contrasts). As no trend was observed, the NOEC and LOEC for post-hatch survival was calculated by Chi² 2x2 Table Test with Bonferroni correction (α = 0.05, one sided greater).
For body fresh and dry weight and for body length a comparison between control and solvent control was performed by using a Student-t test. As no statistically significant effect was detected the controls were pooled for further statistical analysis.
The data for body fresh and dry weight and for body length were assessed for normal distribution and variance homogeneity with Shapiro-Wilk’s Test and Levene’s Test respectively. The data were normal distributed and variance homogenous.
For fresh weight a linear trend of concentration response was observed (trend analysis by contrast) so the Williams t-test (α = 0.05, one sided smaller) was used for NOEC and LOEC calculation.
For body dry weight and for body length no linear trend of concentration response was observed so the Dunnett's t-test (α = 0.05, one sided smaller) was used for NOEC and LOEC calculation.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 210 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the long-term toxicity of the test substance towards fish.
In a flow-through test fertilised eggs of the Zebrafish (Danio rerio) were exposed to aqueous test media containing the test item at concentrations of nominal 105 and 33 µg test item/L under defined conditions for a period of 30 days after hatching (DPH). Using LC-MS/MS detection time weighted average concentrations of 27.5 and 95.8 µg test item/L were determined, correspondingly. In parallel a solvent control (100 µL DMF/L) and a water control were tested. According to OECD 210 an extended limit design is acceptable for the determination of a NOEC. The recorded effects were mortality, hatching, growth and sublethal effects of the fish. Since no significant effects were observed throughout the test, the 30d NOEC was determined to be ≥105 µg/L with an associated LOEC of > 105 µg/L for the parameter hatching success, mortality, body length and body weight (wet and dry). The test concentration of 105 µg/L represents the highest achievable and reproducible concentration of the registered test substance for the performance of an aquatic long-term study.
Executive summary:

The study was performed under GLP according to OECD TG 210. Due to the fast hydrolysis of the test material (Neuland, 2020) the OECD Series on Testing and Assessment, No. 23 "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals" has also been taken into account.

The purpose of this study was to evaluate the toxicity of the registered substance to the early-life stages of fish. For this purpose, fertilised eggs of zebrafish (Danio rerio) were exposed in a flow-through test to aqueous test media containing the test item at nominal concentrations of 33 and 105µg/L under defined conditions. In parallel a solvent control (100 µL DMF/L) and a water control were tested. 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.

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 flow-through system.

The test duration was 30 days after hatching. The eggs and larvae were observed daily for survival, hatching, abnormal appearance and behaviour. Additionally, at the end of the test, the individual length of all surviving fish was determined using a digital caliper. Also, at the end of the test all surviving fish were weighed (wet weight after blotting dry and dry weight) in groups by test vessel (replicate). Dead larvae were removed at least once daily and discarded. The test item concentrations in the test water taken at test start and after DAI 7, 14, 21, 28 and 34 were chemically analysed by LC-MS/MS.

Since TRIDI is hydrolytically unstable with a t(1/2) of 37 seconds at a temperature of 20 °C, it will be degraded during toxicity studies in aqueous media. Based on the result of a hydrolysis study according to OECD 111, 1,3,5-triisopropyl-2,4-diaminobenzene (TRIDA) will be formed as major hydrolysis product. Therefore, the quantification of the hydrolysis product 1,3,5-triisopropyl-2,4-diaminobenzene (TRIDA) was performed using liquid chromatography with MS/MS detection and the obtained results refer to the theoretical amount of TRIDA which can be built from TRIDI considering equimolar conversion.

Accordingly time weighted arithmetic mean measured concentrations of 27.5 and 95.8 µg test item/L were determined. However, as the mean test recoveries of the nominal test concentration varied between 83 and 91% (all test concentrations considered) the test item concentrations were determined to be stable during the run of the test and results were based on nominal test concentrations:

Hatching success: NOEC ≥ 105 µg test item/L

Mortality/Survival: NOEC ≥ 105 µg test item/L

Body Length: NOEC ≥ 105 µg test item/L

Body Weight (wet): NOEC ≥ 105 µg test item/L

Body Weight (dry): NOEC ≥ 105 µg test item/L

Description of key information

Key_Long-term toxicity to fish: NOEC (30d) ≥ 105 µg/L nominal (95.8 µg/L based on time weighted average concentrations) for Danio rerio (flow-through, freshwater, OECD 210, GLP)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
>= 105 µg/L

Additional information

The study was performed under GLP according to OECD TG 210. Due to the fast hydrolysis of the test material (Neuland, 2020) the OECD Series on Testing and Assessment, No. 23 "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals" has also been taken into account.

The purpose of this study was to evaluate the toxicity of the registered substance to the early-life stages of fish. For this purpose, fertilised eggs of zebrafish (Danio rerio) were exposed in a flow-through test to aqueous test media containing the test item at nominal concentrations of 33 and 105µg/L under defined conditions. In parallel a solvent control (100 µL DMF/L) and a water control were tested. 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.

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 flow-through system.

The test duration was 30 days after hatching. The eggs and larvae were observed daily for survival, hatching, abnormal appearance and behaviour. Additionally, at the end of the test, the individual length of all surviving fish was determined using a digital caliper. Also, at the end of the test all surviving fish were weighed (wet weight after blotting dry and dry weight) in groups by test vessel (replicate). Dead larvae were removed at least once daily and discarded. The test item concentrations in the test water taken at test start and after DAI 7, 14, 21, 28 and 34 were chemically analysed by LC-MS/MS.

Since TRIDI is hydrolytically unstable with a t(1/2) of 37 seconds at a temperature of 20 °C, it will be degraded during toxicity studies in aqueous media. Based on the result of a hydrolysis study according to OECD 111, 1,3,5-triisopropyl-2,4-diaminobenzene (TRIDA) will be formed as major hydrolysis product. Therefore, the quantification of the hydrolysis product 1,3,5-triisopropyl-2,4-diaminobenzene (TRIDA) was performed using liquid chromatography with MS/MS detection and the obtained results refer to the theoretical amount of TRIDA which can be built from TRIDI considering equimolar conversion.

Accordingly time weighted arithmetic mean measured concentrations of 27.5 and 95.8 µg test item/L were determined. However, as the mean test recoveries of the nominal test concentration varied between 83 and 91% (all test concentrations considered) the test item concentrations were determined to be stable during the run of the test and results were based on nominal test concentrations:

Hatching success: NOEC ≥ 105 µg test item/L

Mortality/Survival: NOEC ≥ 105 µg test item/L

Body Length: NOEC ≥ 105 µg test item/L

Body Weight (wet): NOEC ≥ 105 µg test item/L

Body Weight (dry): NOEC ≥ 105 µg test item/L