2,2'-azobis[2-methylbutyronitrile]

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

calculation (if not (Q)SAR)

Remarks:

calculation

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The scientific validity of the (Q)SAR model has been established in accordance with the OECD Principles for (Q)SAR Model Validation. The data provided for in this study in conjunction with that of the in vivo study with the read-across chemical, provide a weight of evidence for the hazard endpoint that is sufficient for the purpose of classification and labelling and/or risk assessment.

Justification for type of information:

QSAR prediction

Principles of method if other than guideline:

ECOSAR v1.11
QMRF and QPRF are attached in IUCLID

GLP compliance:

not specified

Test organisms (species):

other: Daphnid

Test type:

other: Q(SAR)

Dose descriptor:

NOEC

Effect conc.:

2.38 mg/L

Nominal / measured:

estimated

Remarks on result:

other: NOEC was derived by dividing the ECOSAR modelled ChV (4.753 mg/L) by 2.

Supporting documentation are provided in the attached QPRF and QMRF.

Validity criteria fulfilled:

not applicable

Conclusions:

The estimated NOEC for Daphnia is 2.38 mg/L. The data provided for in this study in conjunction with that of the in vivo study with the read-across chemical, provide a weight of evidence that fulfills the quality criteria (validity, reliability, repeatability).

Executive summary:

The NOEC to daphnids was estimated. A conservative approach for determining the NOEC is to divide the ECOSAR modeled ChV (4.753 mg/L) by 2. Therefore, the NOEC was estimated to be 2.38 mg/L.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Lack of some information; e.g. No C of A available. Concentration tested is far below the water solubility limit and one cannot safely conclude about the real toxicity potential. 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.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

conducted over 21 days (rather than 14)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Test solutions were sampled from all blocks (one test container per block) into stoppered glass centrifugal tubes, about 50 mL, on Days 7, 9, 19 and 20 of exposure. The test solutions sampled were analyzed by HPLC after filtration. Assay was conducted by the absolute calibration method from peak areas obtained with samples.
- Sample storage conditions before analysis: Not reported

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The necessary amount of the test substance was weighed using an electronic balance, and 10 parts of the solvent, acetone, were added.
After thorough mixing, dilution water was added to volume and stirred to prepare the stock test solution. The amount of the stock test solution necessary to prepare test solutions of nominal concentrations was measured using a measuring cylinder, dilution water was added to make 5000 mL or 4000 mL, and test solutions were prepared by stirring. Test solutions were dispensed into four test containers, 1000 per container. Dilution water was used without adding the test substance in the control block. In the solvent control block, acetone was weighed using an electronic balance, dilution water was added to volume, and the mixture was stirred to prepare a 100 mg/L solution. The 48-hr EC50 value was 10 mg/L, the highest concentration that makes even dispersion possible at the high solvent concentration of 100 mg/L, or greater in an acute immobilization study in Daphnia magna. Based on this finding, the high concentration for the reproduction inhibition study was set at 10 mg/L, followed by four lower concentrations with a common ratio of 2.2. The control block, which used only dilution water, and the solvent control block, which used acetone at 100 mg/L per block, were added. The study was therefore conducted in the following blocks: control, solvent control, 0.46, 1.0, 2.2, 4.6, and 10 mg/L (nominal concentrations).
- Controls: Dilution water; solvent control with dilution water and acetone to give concentration 100 mg/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Not stated
- Strain: Not stated
- Source: obtained from the National Institute for Environmental Studies, cultured in this laboratory.
- Age at study initiation (mean and range, SD): within 24 hrs after birth cultured for 2-4 weeks. When they become mature and started to produce juveniles, juveniles were eliminated at least twice a week. Adult female Daphnia magna with young were selected the day before exposure 2-4 weeks later, and juveniles obtained the following day (within 24 hrs) were used in the study. Deaths, dormant eggs, or males were not observed in a large number while culturing parental Daphnia magna.
- Weight at study initiation (mean and range, SD): N/A
- Method of breeding: Sufficiently large and macroscopically healthy, adult female Daphnia magna with young were selected from the successively cultured colony, transferred to a separate container, and juvenile Daphnia magna obtained the following day was transferred to another container. These juvenile Daphnia magna (parents) were cultured for 2-4 weeks
- Feeding during test
- Food type: Chlorella vulgaris - A commercial chlorella concentrate (brand name: Crude Chlorella V12; Chlorella Kogyo Co., Ltd.) was supplied after suspending it by substitution with dilution water and centrifugation
- Amount: Amount of feed given: As a rule, Chlorella vulgaris was supplied at 0.1-0.2 mg C (organic carbon content)/day per head. The organic carbon content in 1% suspension was 1050 mgC/L. The amount of feed to be added was determined based on this value.
- Frequency: Not stated

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

None

Hardness:

about 63 mg/L (converted to CaCO3)

Test temperature:

Temperature of test solutions ranged between 20.2°C and 20.9°C as targeted during the 21-day exposure period

pH:

between 7.4 and 8.1

Dissolved oxygen:

saturated dissolved oxygen concentration at 20°C: 8.8 mg/L

Salinity:

N/A

Nominal and measured concentrations:

Nominal: Control (0.0), Solvent control (0.0), 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Measured Day 7: 0.44, 0.98, 2.1, 4.4 and 9.2 mg/L ; Day 19: 0.43, 0.88, 2.0, 4.7 and 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 1000-mL glass beaker; 1000 mL/container
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): : The entire volume of the test solution was replaced 3 times a week (on Monday, Wednesday and Friday) until Day 8 of exposure and every day on Day 9 and onward taking into account decreases in dissolved oxygen concentrations.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates):4 replicates/block
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated water, obtained by treating tap water supplied by the city of Takarazuka with activated carbon
and thoroughly aerating after eliminating residual chlorine, etc., was used.
- Total organic carbon: Mean: 1050 (mg/L)
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Reported as eliminated
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: once per week, before and after test solution replacement using one container per block, during the
exposure period.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: room light (16 hrs light/8 hrs dark)
- Light intensity: Not stated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
. Calculation of the 50% lethal concentration (LC50) in parental Daphnia magna
. Calculation of the 50% reproduction inhibition concentration (ErC50)
. Maximum no-observed-effect concentrations (NOECr) and lowest-observed-effect concentrations (LOECr)

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Corrected for molecular weight of 2,2'-azobis[2-methylbutyronitrile] the NOEC is 2.58 mg/L

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

7.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 4.6 -10 mg/L

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

- Behavioural abnormalities: None reported
- Observations on body length and weight: N/A
- Other biological observations: None
- Mortality of control: None in water controls but 1 death in the solvent control group at day 18
- Abnormal responses: None reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

Because the mortality in the highest concentration block (10 mg/L) was 5%, statistical analysis (by the binomial, moving average or Probit method) was impossible.

Because the number of juveniles produced in the control block was apparently small compared to the solvent control block and the low concentration block, data from the solvent control block were used for analysis.

Validity criteria fulfilled:

yes

Conclusions:

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The maximum no-observed-effect concentration (NOEC) was 2.2 mg/L and the lowest-observed-effect concentration (LOEC) was 4.6 mg/L. When converted to 2,2'-azobis[2-methylbutyronitrile] with correction for molecular weight the NOEC is 2.58 mg/L.

Executive summary:

A 21 day Daphnia magna reproduction inhibition study was conducted at Sumika Technos Corp., Japan, in order to determine the maximum concentration of the test substance 2,2’-azobis (2-methylpropanitrile) at which no significant differences are observed in the reproduction performance between the treated and control blocks (NOECr) and the calculated concentration at which the number of juveniles produced is expected to be reduced by 50%. The study was conducted to GLP and according to the OECD Guideline No. 202, except that rather than a 14 day reproduction study, the exposure period was for 21 days. Sufficiently large and macroscopically healthy, adult female Daphnia magna with young were selected from a successively (in-house) cultured colony, transferred to a separate container, and juvenile Daphnia magna obtained the following day were transferred to another container. These juvenile Daphnia magna (parents) were cultured for 2-4 weeks under the same conditions as the test. Adult female Daphnia magna with young were selected the day before exposure 2-4 weeks later, and juveniles obtained the following day (within 24 hrs) were used in the study.

The exposure period was for 21 days and consisted of concentrations of the test substance at nominally 0.46, 1.0, 2.2, 4.6 and 10 mg/L in the dilution water. Controls were run concurrently consisting of the same number of organisms in dilution water and a further group in dilution water with the solvent (solvent control; acetone at a concentration of 100mg/L).

Parental Daphnia magna in the control block showed no particular changes, but the number of juveniles produced in the control block showed a tendency to decrease compared to the solvent control block and the low concentration block from around Day 12 of exposure, with the cumulative number of juveniles produced showing statistically significant decreases at the end of the exposure period. This was thought to be attributable to some inhibitory effect on the reproductive physiology of Daphnia magna. Data from the solvent control block was used as control in this study because data from the control block was thought to be inappropriate for use as control.

The 21 day 50% lethal concentration (LC₅₀) in parental Daphnia magna was found to be>10 mg/L, whilst the 21 day 50% reproduction inhibition concentration (EC₅₀) was 7.5 mg/L (95% confidence interval 4.6 -10 mg/L). The maximum no-observed-effect concentration (NOEC) was 2.2 mg/L and the lowest-observed-effect concentration (LOEC) was 4.6 mg/L.

Description of key information

No measured long-term toxicity to aquatic invertebrates with the test substance is available. A long-term study in aquatic invertebrates with 2,2’-azobis(isobutyronitrile) was used to fulfill the data gap for the test substance along with ECOSAR calculation. The underlying hypothesis for using 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.

The 21-day NOEC and LOEC in a semi-static study with Daphnia magna, based oncumulative number of juveniles producedwere 2.2 and 4.6 mg/L, respectively. Converted with molecular weight to 2,2'-azobis[2-methylbutyronitrile] the NOEC becomes 2.58 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.58 mg/L