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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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.
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Storage in original container, at room temperature (17 - 22ºC), in the darkinstead of 20-22°C. No impact.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not relevant
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item 2,2’-Azobis[isobutyronitrile] (AZDN) is not soluble in water. Therefore, the test item was directly dosed into each test flask and tap water was added. For complete suspension of the test item, this mixture was stirred for about 24 hours at 21°C in the dark / diffuse light before adding the inoculum. The test flasks were tightly closed to avoid water loss by evaporation.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Activated sludge, micro organisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Darmstadt, Germany. The activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. The latter procedure was repeated three times. The washed sludge was re-suspended in tap water and sewage feed (50 mL per L) was added. The sludge was kept at room temperature under continuous aeration until use overnight. Immediately before use, the dry weight of the activated sludge was determined, and diluted to 4 g/L with tap water. The pH of the activated sludge was 7.5 and therefore no adjustment was necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Not relevant
Test temperature:
21°C during dissolution phase (24 hours)
19 - 21°C during pre-incubation (3 hours)
18 - 19°C during evaluation period
pH:
7.3-8.3
Dissolved oxygen:
7.8-9.4
Nominal and measured concentrations:
Nominal 10, 32, 100, 320 and 1000 mg test item/L
Details on test conditions:
One test solution with a final volume of 500 mL was tested per treatment in a glass flask (>500 mL). Samples were prepared in about 15-minute intervals.
In parallel to the study with the test item, the reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 3.2, 10 and 32 mg/L under otherwise identical test conditions. 3,5-Dichlorophenol was used as a reference item (positive control): 0.25 g of 3,5-Dichlorophenol was dissolved according to the OECD Guideline No. 209 in 5 mL 1 mol/L NaOH and diluted to about 15 mL with deionised water. Excess of NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to 500 mL with deionised water. The final pH was measured to be 7.9 and the final concentration amounted to 500 mg/L. At least two controls (tap water, synthetic sewage and inoculum, but without addition of the test item) was tested in parallel.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Converted to 2,2'-azobis[2-methylbutyronitrile] with molecualr weight the NOEC is equal to or greater than 1171 mg/L.
Details on results:
Based on measured inhibition rates, the 3-hour EC20 and EC50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 20% inhibition was noted after three hours incubation. A dose response at all test concentrations could not be observed. The maximum deviation in respiration rates was 19.1% at a concentration of 1000 mg 2,2’-Azobis[isobutyronitrile] (AZDN) per litre. Nevertheless, the 3-hour EC20 and EC50 are clearly higher than 1000 mg/L under the present test conditions. Even the test was not designed for a NOEC determination, the NOEC of 2,2’-Azobis[isobutyronitrile] (AZDN) may be established above 1000 mg/L.
Results with reference substance (positive control):
The inhibition of the activated sludge treated with the reference item was in the range of 28% to 88% for the test concentrations of 3.2, 10 and 32 mg/L.

The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of 3,5-Dichlorophenol were calculated.
The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 1.2 2.3 7.5
lower 95%-cl 0.5 1.2 5.8
upper 95%-cl 2.0 3.3 9.6
Validity criteria fulfilled:
yes
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
3-hour NOEC >= 1000 mg/L test substance, converted to 2,2'-azobis[2-methylbutyronitrile] with molecular weight this value is 1171 mg/L.
Executive summary:

Based on measured inhibition rates, the 3-hour EC20 and EC50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 20% inhibition was noted after three hours incubation. A dose response at all test concentrations could not be observed. The maximum deviation in respiration rates was 19.1% at a concentration of 1000 mg 2,2’-Azobis[isobutyronitrile] (AZDN) per litre. Nevertheless, the 3-hour EC20 and EC50 are clearly higher than 1000 mg/L under the present test conditions. Even the test was not designed for a NOEC determination, the NOEC of 2,2’-Azobis[isobutyronitrile] (AZDN) may be established above 1000 mg/L.

Description of key information

No activated sludge respiration inhibition study with the test substance is available. An activated sludge respiration inhibition study with 2,2’-azobis(isobutyronitrile) was used to fulfill the data gap for the test substance.The underlying hypothesis for the read-across is based on the fact that the mode of action and chemical class specifics are relevant. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

A static, 3-hour respiration inhibition study in domestic activated sludge with 2,2‘-azobis(isobutyronitrile) was performed. The 3-hour NOEC and EC50 in activated sludge were >1000 mg/L, based on respiration rate. Converted to 2,2'-azobis[2-methylbutyronitrile] this value is 1171 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 171 mg/L
EC10 or NOEC for microorganisms:
1 171 mg/L

Additional information