2,2'-Azodi[2-methylbutyronitrile]

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. This study is used for read-across.

Justification for type of information:

see 13.2 for attached read across rationale

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Remarks:

Conducted according to guideline in effect at time of study conduct

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION: On the basis of the results of the preliminary test the following nominal concentrations were used in the definitive test: 62.5, 125, 250, 500 and 1000 mg/L. These concentrations were obtained by addition of the following amounts of test substance in each vessel (3 Litre): 188.5, 374.3, 749.8, 1500.6 and 3000.1 mg test substance. After renewal of the test medium the following concentrations were made at: 187.3, 375.1, 750.4 and 1499.2 mg test substance in 3 Litre test medium. One control group was included in the test.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Size at study initiation: The fish had an average size of 3.1 cm and an average wet-weight of 0.31 g.
- Feeding during test: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22.5°C to 23.5°C

pH:

7.9 to 8.2

Dissolved oxygen:

8.0 to 8.9 mg/L

Salinity:

frestwater

Nominal and measured concentrations:

Nominal concentrations: 62.5, 125, 250, 500 and 1000 mg/L
Measured concentrations: not reported

Details on test conditions:

TEST SYSTEM
- Test vessel: 5 L glass aquaria
- Renewal rate of test solution: renewed at 48 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The diluting water used for the preparation of the test concentrations was a synthetic water, Dutch Standar Water, having a pH of approximately 8.2 and a hardness of 12°dH, containing per litre of deionized water:
100 mg of NaHCO3
20 mg of KHCO3
200 mg of CaCI2.2H20
180 mg of Mg504.7H20.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortalities were recorded at least at 24, 48, 72 and 96 hours. Furthermore, sub-lethal effects, such as changes in the behaviour or the appearance of the fish were recorded. The fish were considered dead if touching of the caudal peduncle produced no reaction and no breathing movements were visible. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Range finding study results: The preliminary test was performed using the following nominal concentrations: 0.1, 1, 10, 100, and 1000 mg/L (3 fish per test concentration and control). No mortality at less than or equal to 100 mg/L; 2 of 3 dead at 96 hours in 1000 mg/L

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

580 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: This concentration is higher than the water solubility limit of 318 mg/L.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 318 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: This concentration is the maximum water solubility.

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test substance, which is insoluble in the test medium, disappeared (visibly) during the course of the test, mainly at the lower concentrations. A possible explanation for this phenomenon is that the test substance particles were ingested by the fish during the test. However, no mortalities or sub-lethal effects were observed as a result of this. Another clear observation was that the test substance was very sticky and was attached to the fish and possibly also to the gills, which eventually may lead to oxygen depletion in the fish. This phenomenon may be a possible explanation for the mortality at the higher concentrations in the test.

Sublethal observations / clinical signs:

The maximum water solubility of this test substance is 318 mg/L. The LC50 determined was greater than this limit and therefore 96h-LC50 > max. water solubility.

Validity criteria fulfilled:

yes

Conclusions:

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
96-hour LC50 = 580 mg/L
This is greater than the water solubility limit of this test substance, therefore 96h-LC50 > max. water solubility.

Executive summary:

The test substance was examined in an acute toxicity test with the fresh water fish Brachydanio rerio (zebra fish) under semi-static conditions in accordance with EEC Method C.1 and OECD Test Guideline 203. The fish were exposed to concentrations of the test substance ranging from 62.5 to 1000 mg/L for 96 hours in a semi-static test, and mortalities and other biological observations, such as deviations in the behaviour or appearance of the fish were recorded at least at 24, 48, 72 and 96 hours. The 96-hour LC₅₀ value calculated from the results is 580 mg/L (458 - 735mg/L 95% confidence limits) based on nominal concentrations of the test substance. The highest concentration causing no mortality (no observed effect concentration, NOEC) after 96 hours derived from the results was 250 mg/L.

The test substance, which is insoluble in the test medium, disappeared (visibly) during the course of the test, mainly at the lower concentrations. A possible explanation for this phenomenon is that the test substance particles were ingested by the fish during the test. However, no mortalities or sub-lethal effects were observed as a result of this. Another clear observation was that the test substance was very sticky and was attached to the fish and possibly also to the gills, which eventually may lead to oxygen depletion in the fish. This phenomenon may be a possible explanation for the mortality at the higher concentrations in the test.

Description of key information

The 96h-LC50 for 2,2'-azobis[2-methylbutyronitrile] is greater than the maximum water solubility.

Additional information

No short-term toxicity to fish study with the test substance is available. A short-term fish study with 2,2’-azobis(isobutyronitrile) was used as a read across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across is that based on the structural and functional group similarity, the presence of the nitrile group in both substances is likely driving aquatic toxicity. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

A semi-static, 96-hour test was conducted in Danio rerio with 2,2‘-azobis(isobutyronitrile). The 96-hour LC₅₀ in Danio rerio was reported as 580 mg/L in the original study report. The reported 96-hour NOEC was 250 mg/L. Since the reported LC₅₀ of 580 mg/L is above the substance water solubility of ~318 mg/L the 96h-LC50 for 2,2'-azobis[2-methylbutyronitrile] is also greater than the maximum water solubility.