[No public or meaningful name is available]

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2017 to 28 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law)

Version / remarks:

Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint
ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-1-1

Version / remarks:

The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Duplicate samples were taken (2 mL) in plastic tubes at the beginning and at the end of the first and last renewal periods from the control and at the applied test concentration levels.
- After sampling, samples were frozen and kept approximately at -20 °C at the Test Facility. One set of the samples was sent to the Test Site for analysis and one set was retained as a back-up at the Test Facility, if required for any confirmatory analyses (discarded after satisfactory results were obtained on the first set of).

Vehicle:

no

Remarks:

(dilution water used as test medium)

Details on test solutions:

- Although the test material is well soluble in water, it precipitates in aquarium water. Precipitation was likely to be caused by the "encounter" of test material and dissolved calcium and magnesium content of the aquarium water (i.e. the hardness of the water). In case of filtration used, test material is not present in the "solution" practically. During the analytical method development micelle formation was observed at high concentration stock solution, so the concentration was above the critical micelle concentration (CMC) due to its chemical structure (anionic surfactant). Gel formation is predicted at high concentrations.
- Due to its chemical structure the above mentioned salt precipitation reaction occurs when the test material is placed into the aquarium water. For reasons detailed above the experiment was carried out in a different media, in which the test material could be kept in solution.
- Due to problems with preparation of test material stock solution (opalescent test solution and precipitation when trying to dissolve it in aquarium water) deionised water was used as a test medium instead of aquarium water.
- Prior to treatment, a stock solution with a concentration of 100 mg/L (nominal) was prepared with direct addition of the test material, mixed into the test medium using ultrasonic bath approximately for 5 minutes. The test solutions were prepared by appropriate diluting of this stock solution just before introduction of the fish.
- Because the test material is not stable over the 96h exposure period (based on the analytical method validation) the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test material solutions were prepared by the method described above.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Length at study initiation: 2.6 – 2.9 cm
- Weight at study initiation: 0.18-0.22 g

ACCLIMATION
- Acclimation period: 12 days
- Health during acclimation: Fish were bred in a well-known fish farm, under diseaseand parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- The fish were not fed during the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

12 mg/L (as CaCO3)

Test temperature:

20.5 - 22.3 °C

pH:

6.30 – 6.97

Dissolved oxygen:

- The dissolved oxygen concentration was in the range of 80 – 99 % of the air saturation value at the temperature used. Test solutions were aerated during the second renewal period to avoid decreasing of dissolved oxygen concentration below the accepted level.

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L
Measured: 5.9, 12.3, 25.9, 51.9 and 104.7 mg/L
As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, the biological results are based on the nominal test material concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: An aquarium
- Material, size, headspace, fill volume: 5 L test solution
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates):1
- Biomass loading rate: The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water was used as a test medium instead of aquarium water due to precipitation.
- Intervals of water quality measurement: The water temperature, oxygen concentration and pH were measured at the start and at the end of renewal periods in each test aquarium (where the fish were alive).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED: The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality and any sub-lethal effect were determined in each observation period.

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 0.1, 1, 10 and 100 mg/L (nominal)
Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions. The frequency of the water renewal periods was 24 hours.
- Results used to determine the conditions for the definitive study: yes the choice of the test concentrations was made on the basis of the results of the preliminary range-finding test.

Definitive test:
- Because toxic response was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control group were tested in the definitive study in a semi-static system. The frequency of the water renewal periods was 24 hours.
- The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Remarks:

and 24 h

Effect conc.:

17.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limits: 12.01 – 24.75 mg/L)

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

and 72 h

Effect conc.:

14.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (95 % conf. limits: 10.25 – 30.45 mg/L)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

VALIDITY
- The achievable validity criteria were within acceptable limits and therefore the study can be considered as valid.

MORTALITY DATA
- Cumulative mortality data from the exposure of zebrafish to the test material during the definitive test are given in Table 2.

OBSERVATIONS
- Behaviour of fish was found as normal in the control group and at the lowest tested concentration level of 6.25 mg/L (nominal).
- In the test material group of 12.5 mg/L (nominal) localization close to the surface of the water was observed 72 hours after the start of the experiment, while decreased activity, localisation at the bottom of the aquaria or close to the surface of the water, fast motility of operculum and immobility was noted at the test group of 25.0 mg/L (nominal) during the first 6 hours of the experiment and all fish died 24 hours after the start of the test.
- All fish were observed as dead 3 hours after the start of the experiment at the two highest concentration levels of 50.0 and 100.0 mg/L (nominal).

BODY WEIGHT
- There was no considerable difference observed concerning body weights between the groups.

Reported statistics and error estimates:

The 24, 48, 72 and 96 hours LC50 values were calculated by Probit analysis using SPSS PC+ software.
The NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 2: Cumulative mortality data in the Definitive Test

Nominal concentration (mg/L)	Mortality [number of fish] (initial population = 7 fish)
	3 h	6 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0	0
6.25	0	0	0	0	0	0
12.5	0	0	0	0	2	2
25.0	0	0	7	-	-	-
50.0	7	-	-	-	-	-
100.0	7	-	-	-	-	-

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:
The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)
The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)
The 96h LC100 value: 25.0 mg/L (nominal)
The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)
The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

Executive summary:

The acute toxicity of the test material was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. The test was performed in accordance with the standardised guidelines OECD 203, EU Method C.1 and OCSPP 850.1075, under GLP conditions.

Due to problems with preparation of test material stock solution (opalescent test solution and precipitation when trying to dissolve it in aquarium water) deionised water was used as a test medium instead of aquarium water.

Because significant toxicity was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under semi-static conditions. The frequency of the water renewal periods was 24 hours.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods. Concentration of the examined test groups of 25.0, 50.0 and 100.0 mg/L (nominal) was only analysed during the first renewal period, when all animals were observed as dead. The corresponding measured geometric mean test material concentrations were: 5.9; 12.3; 25.9; 51.9 and 104.7 mg/L. As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal test material concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litre test solution. The 24, 48, 72 and 96 hours LC50 values of the test material were calculated using Probit analysis by SPSS PC+ software. The NOEC and LOEC and LC100 values were determined from the raw data.

All achievable validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:

The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)

The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)

The 96h LC100 value: 25.0 mg/L (nominal)

The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)

The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

Description of key information

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:

The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)

The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)

The 96h LC100 value: 25.0 mg/L (nominal)

The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)

The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

14.21 mg/L

Additional information

The acute toxicity of the test material was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. The test was performed in accordance with the standardised guidelines OECD 203, EU Method C.1 and OCSPP 850.1075, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Due to problems with preparation of test material stock solution (opalescent test solution and precipitation when trying to dissolve it in aquarium water) deionised water was used as a test medium instead of aquarium water.

Because significant toxicity was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment under semi-static conditions. The frequency of the water renewal periods was 24 hours.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L. The test concentrations were analytically determined at the start and at the end of the first and at the last renewal periods. Concentration of the examined test groups of 25.0, 50.0 and 100.0 mg/L (nominal) was only analysed during the first renewal period, when all animals were observed as dead. The corresponding measured geometric mean test material concentrations were: 5.9; 12.3; 25.9; 51.9 and 104.7 mg/L. As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, biological results are based on the nominal test material concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litre test solution. The 24, 48, 72 and 96 hours LC50 values of the test material were calculated using Probit analysis by SPSS PC+ software. The NOEC and LOEC and LC100 values were determined from the raw data.

All achievable validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of the test material were the following:

The 24h and 48h LC50 value: 17.85 mg/L (nominal) (95 % conf. limits: 12.01 – 24.75 mg/L)

The 72h and 96h LC50 value: 14.21 mg/L (nominal) (95 % conf. limits: 10.25 – 30.45 mg/L)

The 96h LC100 value: 25.0 mg/L (nominal)

The 96h No-Observed Effect Concentration (NOEC): 6.25 mg/L (nominal)

The 96h Lowest Observed Effect Concentration (LOEC): 12.5 mg/L (nominal)