2,2'-Azodi[2-methylbutyronitrile]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Remarks:

The study was conducted according to guideline in effect at time of study conduct.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

One sample plus a backup sample was received from each test concentration, including the dilution water control on day 0 of the test before the test solutions were poured into the replicate test chambers. One sample plus a backup sample was also received from 2 of the 4 replicate chambers (replicates B and D) at the nominal 120 mg/L test concentration on day 1 due to total immobility in the test concentration. One sample plus a backup sample was also received from 2 of the 4 replicate chambers (replicates B and D) at the nominal 7.5, 15, 30 and 60 mg/L test concentrations and the dilution water control at test end (day 2). All samples and backup samples were stored refrigerated when not in use.
Concentrations of the active substance in diluted samples were measured by high performance liquid chromatography with detection by ultraviolet (UV) spectroscopy (LC/UV) within 2 days of sample receipt.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
Test substance solutions were prepared by dilution from a stock solution in Haskell well water (HWW). A 120 mg/L stock solution was prepared by adding approximately 0.240 g of test material to HWW in a 2-L glass beaker (final volume 2000 mL), stirring for approximately 5 minutes, sonicating for approximately 13 minutes, and stirring for an additional 17 minutes. The stock solution was also the highest test concentration. Test solutions were prepared by adding the appropriate volume of the stock solution to the appropriate volume of HWW in 1-L glass beakers (final volume 1000 mL) and stirring for approximately 18 minutes. The nominal 120 mg/L stock solution (and highest test solution), and the 30 and 60 mg/L test solutions were clear and colourless with no visible precipitate and had a surface film. The dilution water control and the nominal 7.5 and 15 mg/L test solutions were clear and colourless with no visible precipitate or surface film.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM:
- Strain: Daphnia magna
- Age at study initiation: < 24 hours
- Feeding during test: None

ACCLIMATION
- Acclimation period: Neonates used in this test were less than 24 hours old and were collected from the 6th brood of 21-day old parent daphnids.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

- Total alkalinity: 73 to 82 mg/L as CaCO3
- EDTA hardness: 138 to 167 mg/L as CaCO3

Test temperature:

20.0 to 20.3 °C

pH:

8.3 to 8.4

Dissolved oxygen:

8.5 to 9.0 mg/L

Salinity:

freshwater

Nominal and measured concentrations:

Nominal concentrations: 0, 7.5, 15, 30, 60, and 120 mg/L
Measured concentrations: 0, 8.20, 16.7, 34.4, 78.4, and 132 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (250-mL) containing approximately 200 mL of test solution
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Conductivity: 278 to 299 µmhos/cm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: a photoperiod of 16 hours light and 8 hours darkness was employed, which included 30 minutes of transitional light preceding and following the 16-hour light interval.
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of test organisms were made daily. The criterion for the effect (immobility) was the inability to swim within 15 seconds after gentle agitation of the test vessel or test solution (even if the daphnids can still move their antennae).

TEST CONCENTRATIONS
- Range finding study: A range-finding study with 1 test chamber containing 10 organisms per test substance concentration was conducted using a dilution water control and nominal test substance concentrations of 0.12, 1.2, 12, and 120 mg/L. At the end of the 48 hours, the respective immobility values were 0, 0, 0, 0, and 100%. All test substance solutions except the nominal 120 mg/L concentration were clear and colourless with no visible precipitation at test start. The nominal 120 mg/L test substance concentration was clear and colourless with undissolved test substance present.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

51.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence intervals of 34.4 to 78.4 mg/L.

Details on results:

Six daphnids were lethargic at 24 hours in the mean, measured 78.4 mg/L concentration.
The highest mean, measured concentration causing no immobility at test end was 34.4 mg/L. The lowest mean, measured concentration causing 100% immobility at test end was 78.4 mg/L.
The 24-hour EC50, based on mean, measured concentrations of the test substance and immobility, was 83.4 mg/L with 95% confidence intervals of 68.8 to 99.4 mg/L.

Reported statistics and error estimates:

The 24- and 48-hour EC50 values were calculated by the moving average angle and the binomial methods respectively, based on mean, measured concentrations of the test substance and immobility. The highest mean, measured concentration causing no immobility at test end and the lowest mean, measured concentration causing 100% immobility at test end were assessed by visual observation.

Table 1: Immobility Data

Mean, Measured Concentration (mg/L)	Number Immobile / Number at Test Start
	24 Hours				48 Hours
	A	B	C	D	A	B	C	D
Dilution Water Control	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
8.20	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
16.7	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
34.4	0/5	0/5	0/5	0/5	0/5	0/5	0/5	0/5
78.4	0/5	4/5	0/5	0/5	5/5	5/5	5/5	5/5
132	5/5	5/5	5/5	5/5	5/5	5/5	5/5	5/5

Validity criteria fulfilled:

yes

Conclusions:

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
48-hour EC50 = 51.9 mg /L.

Executive summary:

The acute toxicity of the test substance to unfed Daphnia magna neonates, less than 24 hours old at test start, was determined in an unaerated, 48-hour, static test. The test was conducted in accordance with the OECD Guideline for the Testing of Chemicals 202.

The study was conducted with 5 concentrations and a dilution water control at a mean temperature of 20.2 ºC. Four replicates with 5 daphnids per replicate were used per test substance concentration and dilution water control. Exposure of daphnids to nominal concentrations of 7.5, 15, 30, 60, and 120 mg/L (which correspond to measured concentrations of 8.20, 16.7, 34.4, 78.4, and 132 mg/L) resulted in 0, 0, 0, 100, and 100% immobility, respectively, at the end of 48 hours.

The 48-hour EC₅₀, based on mean, measured concentrations of the active substance and immobility, was 51.9 mg/L.

Description of key information

A static, 48-hour study in Daphnia magna with the test substance was performed. The 48-hour LC₅₀ in Daphnia magna was 51.9 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

51.9 mg/L