2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.04.04.05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: directly weighted
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc.):

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: ESN (Stadtentsorgung Neustadt) D-67435 NW-Lachen-Speyerdorf
- Method of cultivation:
- Preparation of inoculum for exposure: washing 3x tap water, re-suspension in tap water
- Pretreatment: aeration, feeding daily 50 mL synthetic sewage/L
- Initial biomass concentration: Test 1: dry matter sludge: 3.14 g suspended solids/L; dry matter in test: 1.57 g suspended solids/L
Test 2: dry matter sludge: 2.60 g suspended solids/L; dry matter in test: 1.30 g suspended solids/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Test 1 (range finding): 19.2 - 20.3 °C
Test 2: 19.2 - 20.6 °C

pH:

check tables on results

Nominal and measured concentrations:

Test 1 (range finding): 1000, 100, 10, 1 mg/L (nominal)
Test 2: 3200, 1000, 320, 100, 32 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass,
- Aeration: purified air via Pasteur pipettes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 1 (positive), 2 (system)
- No. of vessels per vehicle control (replicates): -
- No. of vessels per abiotic control (replicates):
- Sludge concentration (weight of dry solids per volume): Test 1 (range finding): 1.57 g/L; Test 2: 1.30 g/L
- Weight of dry solids per volume of reaction mixture per unit of time:-
- Nutrients provided for bacteria: 16 mL standard nutrient solution / vessel
- Biomass loading rate:
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Particulate matter:
OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:
- Details on termination of incubation:
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using fewer concentrations than requested by guideline:-
- Range finding study
- Test concentrations: see above
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

3,5-DCP

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

2 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: Test 1 (range finding): EC50 8.8 mg/L (95 % c.i. 7.0-11 mg/L); Test 2: EC50 6.9 mg/L (95 % c.i. 5.2-8.4 mg/L)
- Other:

O₂ consumption, first experiment (range finding)

Vessel No.	Content	Concentration in mg/L nominal	O2consumption in mg/(L*min)	O2consumption in mg/(L*h)	Inhibition in %	pH
1	Control	0	0.7222	43.330	0.0	7.7
2	Control	0	0.7091	42.545	0.0	7.7
3	Positive Control	5	0.5043	30.261	29.8	7.9
4	Positive Control	10	0.3257	19.541	54.6	7.9
5	Positive Control	20	0.1462	8.775	79.6	7.9
6	Positive Control	40	0.0674	4.044	90.6	7.9
7	Control	0	0.7104	42.624	0.0	7.7
8	Control	0	0.7014	42.083	0.0	7.7
9	Test Item	1000	0.4564	27.382	36.5	7.8
10	Test Item	1000	0.4582	27.493	36.2	7.8
11	Test Item	1000	0.4855	29.127	32.4	7.8
12	Test Item	1000	0.4636	27.816	35.4	7.8
13	Test Item	1000	0.4587	27.525	36.1	7.8
14	Test Item	100	0.6546	39.277	8.8	7.7
15	Test Item	10	0.7055	42.331	1.8	7.7
16	Test Item	1	0.6971	41.827	2.9	7.6
17	Control	0	0.6954	41.724	0.0	7.7
18	Control	0	0.7704	46.222	0.0	7.7

O₂ consumption, second experiment

Vessel No.	Content	Concentration in mg/L nominal	O2consumption in mg/(L*min)	O2consumption in mg/(L*h)	Inhibition in %	pH
1	Control	0	0.5135	30.810	0.0	8.0
2	Control	0	0.4884	29.305	0.0	8.0
3	Positive Control	5	0.3139	18.833	40.6	8.1
4	Positive Control	10	0.2104	12.625	60.2	8.1
5	Positive Control	20	0.1205	7.228	77.2	8.2
6	Positive Control	40	0.0713	4.277	86.5	8.2
7	Control	0	0.5302	31.813	0.0	8.0
8	Control	0	0.5161	30.968	0.0	8.0
9	Test Item	3200	0.3208	19.248	39.3	8.1
10	Test Item	3200	0.2644	15.863	50.0	8.1
11	Test Item	3200	0.2727	16.359	48.4	8.1
12	Test Item	3200	0.2664	15.984	49.6	8.1
13	Test Item	3200	0.2710	16.262	48.7	8.1
14	Test Item	1000	0.2927	17.561	44.6	8.1
15	Test Item	1000	0.3017	18.101	42.9	8.1
16	Test Item	1000	0.3052	18.313	42.3	8.1
17	Test Item	1000	0.3067	18.403	42.0	8.1
18	Test Item	1000	0.3002	18.011	43.2	8.1
19	Test Item	320	0.4007	24.044	24.2	8.0
20	Test Item	320	0.4062	24.374	23.2	8.0
21	Test Item	320	0.4031	24.186	23.7	8.0
22	Test Item	320	0.4073	24.439	23.0	8.0
23	Test Item	320	0.3908	23.449	26.1	8.0
24	Test Item	100	0.4854	29.126	8.2	8.0
25	Test Item	100	0.4838	29.029	8.5	8.0
26	Test Item	100	0.4975	29.850	5.9	7.9
27	Test Item	100	0.4880	29.280	7.7	7.9
28	Test Item	100	0.4922	29.533	6.9	7.9
29	Test Item	32	0.5300	31.799	-0.3	7.9
30	Test Item	32	0.5814	34.884	-10.0	7.9
31	Test Item	32	0.5611	33.668	-6.1	7.9
32	Test Item	32	0.5491	32.945	-3.9	7.9
33	Test Item	32	0.5507	33.044	-4.2	7.9
34	Control	0	0.5640	33.842	0.0	7.9
35	Control	0	0.5596	33.575	0.0	7.9

Validity criteria fulfilled:

yes

Conclusions:

In the first experiment, which was performed as the range finding test, the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. Because signifi-cant inhibition was observed, an additional experiment was performed under the same test conditions.
In the second experiment, the test item was tested using 5 concentrations ranging from 3200 to 32 mg/L nominal concentration. The inhibition in the highest concentrated treatment 3200 mg/L was only marginal higher than in the treatment 1000 mg/L. For the estimation of the EC50 the fits showed acceptable statistical correspondence of the data with the dose-response-equation. For the test item an EC50 of 2500 mg/L was calculated. The difference between trea-tment 32 mg/L and the control can be considered as not significant. Therefore, the concentrati-on 32 mg/L is stated as NOEC.

The pre-test and the main test show good correlation of the inhibitory effects of the test item. The result of the test can be considered valid.
No observations were made which might cause doubts concerning the validity of the study outcome.

Executive summary:

Two valid experiments were performed.

In the first experiment, the test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 3.14 g suspended solids/L, giving a concentration of 1.57 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 8.8 mg/L (95% confidence interval: 7.0 – 11 mg/L) was determined, which lies within the range of 2 – 25 mg/L.

Because significant inhibition was observed, an additional experiment was performed under the same test conditions.

 

In the second experiment, the test item was tested using 5 concentrations ranging from 3200 to 32 mg/L nominal concentration.

The dry matter of the inoculum was determined as 2.60 g suspended solids/L, giving a concentration of 1.30 g suspended solids/L in the test.

For the positive control an EC50 of 6.9 mg/L was determined. The value lies within the recommended range.

The following results for the test item2,2'-Azobis (4-methoxy-2,4-dimethyl valeronitrile)were determined:

 

3h NOEC = 32 mg/L
3h EC50=2500 mg/L

3h EC10=110 mg/L

Description of key information

The key values for the chemical safety assessment are determined based on the findings in an OECD 209 study exposing activated sludge for 3 hours with the test item.

Key value for chemical safety assessment

EC50 for microorganisms:

2 500 mg/L

EC10 or NOEC for microorganisms:

110 mg/L