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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.10.17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

yes

Remarks:

deviations were stated as uncritical

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

224-583-8

EC Name:

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

Cas Number:

4419-11-8

Molecular formula:

C14H24N4

IUPAC Name:

2-[(E)-2-(1-cyano-1,3-dimethylbutyl)diazen-1-yl]-2,4-dimethylpentanenitrile

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Test organisms

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: sludge was taken from
the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NWLachen-
Speyerdorf.
- Method of cultivation:
- Preparation of inoculum for exposure:
- Pretreatment: filtrated, washed with tap water 3 times and re-suspended in tap water. The activated
sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.
- Initial biomass concentration: #01 (pre-test): 2.76 g suspended solids/L dw, respectively, 1.38 g suspended solids/L
dw for the test; #02 (main test): 2.92 g suspended solids/L dw, respectively, 1.46 g suspended solids/L
dw for the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

0.71 mmol/L

Test temperature:

#01: 20.2 – 21.4 °C (first experiment (pre-test))
#02: 19.0 – 21.4 °C (second experiment (main test))

pH:

8.26

Conductivity:

174 µS/cm (25 °C)

Nominal and measured concentrations:

#01: 1000, 100, 10, 1 mg/L
#02: 3200, 1000, 320, 100, 32 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: glass, 500 mL
- Aeration: purified air, using Pasteur pipettes, flow approx. 0.75 L/min
- Type of flow-through (e.g. peristaltic or proportional diluter): -
- Renewal rate of test solution (frequency/flow rate): -
- No. of organisms per vessel: 250 mL inoculum
- No. of vessels per concentration (replicates): #01: positive control & lowest test concentration: one
replicate, five replicates (highest concentrations); #02: 1 rep/treatment (positive control), 5 rep/treatment (test item)
- No. of vessels per control (replicates): two replicates before and two after measuring positive control
and test item, respectively
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate:
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: 0.6 mg/l
- Particulate matter:
- Metals:< 0.007 mg/L
- Pesticides: < LOD
- Chlorine: 9 mg/L
- Alkalinity: not stated
- Ca/mg ratio: 12
- Conductivity: 174 μS/cm
- Culture medium different from test medium:
- Intervals of water quality measurement:
OTHER TEST CONDITIONS
- Adjustment of pH: -
- Photoperiod: -
- Light intensity: -
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : inhibition of respirati
on rate
TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
Test concentrations:1000, 100, 10, 1 mg/L (nominal)
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

3,5- DCP (CAS# 591-35-5)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

280 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 3

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 3 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 7.6 mg/L (95% confidence interval: 5.5 – 9.5 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

Two experiments were performed.

 

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 2.76 g suspended solids/L, giving a concentration of 1.38 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 7.6 mg/L (95 % confidence interval: 5.5 – 9.5 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline.

Inhibition at the concentration 1000 mg/L did not rise above 19 %. Nevertheless, at 1000 mg/L clearly toxicity occurred. Therefore one additional experiment was performed under the same test conditions.

 

In the second experiment (main test) 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 2.92 g suspended solids/L, giving a concentration of 1.46 g suspended solids/L in the test.

For the positive control an EC50 of 8.7 mg/L (95 % confidence interval: 5.0 – 12 mg/L) was determined. The value lies within the recommended range of the OECD guideline.

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

 

3h NOEC=320 mg/L

3h EC10 = 280 mg/L (Confidence Interval: 220 – 350 mg/L)

3h EC50>3200 mg/L (Confidence Interval: not determinable)