Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

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:

02 March-13 June, 2018

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:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

30 March 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Date of production: May 22, 2015
Expiry date: May 22, 2020

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For determination of the test item concentration, samples were taken from the treatment group and the control at the start and at the end of the renewal period.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). Appropriate amount of test item was suspended in the dilution water (ISO Medium; see 4.4) in order to give the loading rate of 100 mg test item/L. The solution was handled by ultrasonic bath for 10 minutes thereafter stirred for a period of approximately 24 hours to achieve equilibrated concentration. The solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Untreated control group (ISO Medium without test item) was included in the test. As the test item was not stable for the duration of 96 hours in ISO Medium, a semi-static test was performed with a renewal on day 2.
- Controls: Negative control (dilution water only)

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium Magazin Kft. (Pasaréti Gyula), 1222 Budapest, Dévény u. 36., Hungary
- Number of animals: 10 animals at each group (no replicates were used)
- Age and size of animals: Juveniles; size within a range of 2.0 ± 1 cm. The loading of the test aquaria did not exceed 1.0 g/L.
- Sex: Both female and male (not separated).

ACCLIMATION
- Acclimation period: Fish were held for at least 12 days before test initiation
- Acclimation conditions: Same as test
- Type and amount of food during acclimation: During holding, fish were fed with appropriate, commercial diet.
- Feeding frequency during acclimation: At least three times per week until 24 hours before the test started.
- Health during acclimation: Apparently healthy animals were used in the test.

FEEDING DURING TEST : No

Test type:

semi-static

Water media type:

other: reconstituted water (ISO medium, prepared according to Annex 2 of the OECD guideline 203)

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

Fish were exposed to aqueous test media containing the test item for 96 hours in a semi-static test (renewal on day 2). Ten fish were exposed to the test concentration and ten fish were included as control (no replicates were used).

Post exposure observation period:

Fish were observed at approximately 4, 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. Measurements of pH, dissolved oxygen and temperature were carried out daily.

Hardness:

The measured hardness of the ISO Medium used in the study was 242.08 mg/L (as CaCO3).

Test temperature:

The measured temperatures were within a range of 21.6 - 22.5°C in the test aquariums (the maximum deviation did not exceed ± 1°C).

pH:

The test was carried out without adjustment of pH. The measured pH values were in the range of 7.18 - 7.80 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 78.0 - 97.2 % of the air saturation value at the temperature used.

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Limit test; using a single nominal concentration of 100 mg/L and a concurrent control.
The measured concentrations deviated more than 20 % from the nominal concentration at the start and at the end of the renewal periods, therefore the geometric mean of the measured concentration was calculated to determine exposure concentration. The corresponding calculated concentration was 0.040 mg/L (based on total product).

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Aeration: Yes
- Renewal rate of test solution: Renewal on day 2
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per negative control: 1
- Biomass loading rate: < 1.0 g fish / L test solution

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: ISO Medium according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light: 8 h dark

EFFECT PARAMETERS MEASURED: The fish were observed at 24, 48, 72 and 96 hours after start of the test for signs of intoxication and mortality. Fish were considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produces no reaction. Dead fish were removed when observed and mortalities were recorded. All sub-lethal effects were recorded.

NON-GLP RANGE FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, non-GLP preliminary tests were performed at the nominal concentration of 100 mg/L. The first pre-test was a static test while in the second pre-test the test solutions were renewed daily. In both preliminary range-finding tests the test solution was prepared by dissolving an amount of 0.1 g test item in 1000 mL ISO Medium to obtain the 100 mg/L nominal test concentration. This solution was stirred rigorously for about 18 hours, thereafter filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. Three fish were exposed for 96 hours in the test item treated and control group. No replicates were included in the pre-tests. No mortality or any sub-lethal effects were detected in the treatment group or in the control group during the 96-h exposure period in both preliminary range finding tests.

The preliminary range-finding tests were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance.

- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: corresponding to > 0.04 mg/L mean measured

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks:

or sublethal effect

Remarks on result:

other: the test item Yellow 22 had no toxic effect on fish at aquatic saturation (i.e. limit test concentration)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 0.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks:

or sublethal effect

Remarks on result:

other: the test item Yellow 22 had no toxic effect on fish at aquatic saturation (i.e. limit test concentration)

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: No sub-lethal effects
- Other biological observations: No
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: No
- Any observations that might cause a difference between measured and nominal values: No (WAF)
- Effect concentrations exceeding solubility of substance in test medium: Nominal loading rate of 100 mg/L exceeded the water solubility

Results with reference substance (positive control):

Not performed.

Reported statistics and error estimates:

Since a limit test was performed (and mortality is not expected), no statistical analysis is necessary. The endpoints will be determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

In this 96-hour acute toxicity test on Zebrafish (Danio rerio) the test item Yellow 22 had no toxic effect on fish at aquatic saturation (i.e. limit test concentration). Accordingly, the 96-h NOEC was determined to be 0.040 mg/L and the LOEC > 0.040 mg/L (based on total product), equivalent to 100 mg/L and > 100 mg/L nominal loading rate respectively.

Executive summary:

The acute toxicity of the test item to Danio rerio was determined in a 96-hour semi-static test according to the Commission Regulation (EC) No 440/2008, Annex Part C, C.1 and OECD Guideline for Testing of Chemicals, Section 2, No. 203. The 96 -h NOEC was determined to be 0.040 mg/L (equivalent to 100 mg/L nominal loading rate) and the LOEC > 0.040 mg/L (equivalent to > 100 mg/L nominal loading rate).

Description of key information

In the 96 -hour test according to OECD Guideline 203 on Danio rerio the read across substance Yellow 22 (CAS 90268 -98 -7) had no toxic effect on fish at aquatic saturation (i.e. limit test concentration). Accordingly, the 96 -h NOEC was determined to be 0.040 mg/L and the LOEC > 0.040 mg/L (based on total product), equivalent to 100 mg/L and > 100 mg/L nominal loading rate, respectively.

Additional information

No data on toxicity to fish was available for the registration substance itself. Therefore, a read across from the structural analogue Yellow 22 (CAS 90268 -98 -7) is conducted.

The acute toxicity of Yellow 22 (CAS 90268 -98 -7) to Danio rerio was determined in a 96 -hour semi-static test according to the Commission Regulation (EC) No 440/2008, Annex Part C, C.1 and OECD Guideline for Testing of Chemicals, Section 2, No. 203. The 96-h NOEC was determined to be 0.040 mg/L (equivalent to 100 mg/L nominal loading rate) and the LOEC > 0.040 mg/L (equivalent to > 100 mg/L nominal loading rate)