2,2'-azobis[2-methylpropionamidine] dihydrochloride

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

Study period:

26.2.-15.3.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus (water flea)
- Strain: Berlin
- Source: Umweltbundesamt Berlin
- Age at study initiation (mean and range, SD): less than 24 hours
- Weight at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Valve height at study initiation, for shell deposition study (mean and range, SD):
- Peripheral shell growth removed prior to test initiation:
- Method of breeding: similar to EN ISO 6341
- Feeding during test
- Food type: no feeding
- Amount: -
- Frequency: -

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food: green algae (Desmodesmus subspicatus)
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.502 mmol/L

Test temperature:

19.4 - 20.6 °C

pH:

7.7 - 7.8

Dissolved oxygen:

see Table 2

Nominal and measured concentrations:

nominal: control, 1.0, 2.2, 4.6, 10, and 22 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass, 20 ± 5 mL test solution
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): 24 ± 1 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 medium
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: none
- Light intensity: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

- Results with reference substance valid? yes; EC50 within demanded range of 0.6-1.7 mg/L.
- Mortality:
- EC50/LC50: 1.5 mg/L
- Other:

 

nominal concentration (mg/L)	Immobility 24 hours					Immobility 48 hours
	abs.				in %	abs.				in %
0	0	1	0	0	5	0	1	0	0	5
1.0	0	0	0	0	0	0	0	0	0	0
2.2	0	0	0	0	0	5	0	0	1	30
4.6	0	0	1	0	5	1	5	5	2	65
10	1	3	1	1	30	5	5	5	5	100
22	5	5	3	4	85	5	5	5	5	100

Table 1. Immobilities

 

nominal concentration (mg/L)	O2-Concentration in mg/L
	0 h	24 h old	24 h new	48 h
0	8.6	8.5	8.5	9.1
1.0	8.6	8.6	8.5	8.6
2.2	8.6	8.6	8.6	8.6
4.6	8.3	8.5	8.7	8.2
10	8.4	8.6	8.7	8.1
22	8.5	8.5	8.6	8.2

Table 2. O₂concentrations

 

nominal concentration (mg/L)	measured concentration (mg/L) 0 h	measured concentration (mg/L) 24 h old	measured concentration (mg/L) 24 h new	measured concentration (mg/L) 48 h old	geometric mean (mg/L)
0	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
1.0	1.28	1.15	1.20	1.12	1.18
2.2	2.59	2.24	2.61	2.11	2.38
4.6	5.18	4.39	4.87	4.86	4.81
10	10.96	9.61	10.50	9.10	10.02
22	23.96	20.21	22.81	20.51	21.75

Table 3. measured concentrations

 

Validity criteria fulfilled:

yes

Remarks:

all validity criteria are fulfilled

Conclusions:

Based on the experimental observations an effect concentration EC50 (48h) 3.5 mg/L was calculated. As all validity criteria were met in this study the results are considered to be adequate and were used in the further assessment of 2,2'-azobis(2-amidinopropane)-dihydrochloride.

Executive summary:

All validity criteria were met. For the estimation of the EC50s of the test item, the fits showed sufficient statistical correspondence of the data with the dose-response-equation.

Potassium dichromate K₂Cr₂O₇ (CAS No. 7778 -50 -9) was used as positive control in a current reference study (201207R201). The value was determined as 1.5 mg/L, lying within the demanded range of 0.6 -1.7 mg/L. No significant immobilisation occured in the control.

The recovery after 24 hours ranged from 85 % to 89 % of the start concentration. After 48 hours, recovery ranged from 81 % to 100 %. The correlation between nominal and measured concentration was good with exception the lowest concentrated treatment containing 1.0 mg/L (lower than LOQ). Therefore, the determination of the biological results was based on the geometric mean of the measured concentrations.

No observations were made which might cause doubts concerning the validity of the study outcome. The resukt of the test is considered valid.

Description of key information

Key value is observed in a semi-static study exposing Daphnia magna for 48 hours to 2, 2'-azobis(2-methylpropionamidine) dihydrochloride. The test solution was changed after 24 hours to maintain the concentration above 80 % of nominal concentration. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

3.5 mg/L