Di-tert-butyl 1,1,4,4-tetramethylbut-2-yn-1,4-ylene diperoxide

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:

from 2015-04-29 to 2015-06-09

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

Version / remarks:

(2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test (2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
One test solution with a final volume of 300 mL was tested per treatment in a glass flask. 9.6 mL synthetic sewage and an adequate volume of the stock solution of the reference item were filled up with water (with deionised water) to 150 mL before the start of the test. At the test item treatments 9.6 mL synthetic sewage was filled up with water to 150 mL before the start of the test. The adequate volume of the liquid test item was administered directly into the test containers. At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added to the test containers, first to the blank controls (CBA, thereafter CBB CBC and CBD the “start” CB group), then in appropriate time intervals to the nitrification controls, the test solutions of the reference item and the test item [the adequate amount (in a corresponding volume) of the test item was administered shortly before the inoculum addition] and finally to the “end” CB group (CBE, CBF, CBG and CBH). In time intervals of approximately 15 minutes (an arbitrary and convenient interval) four test vessels were started.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to 3 g per litre (on dry weight basis). At the concentration calculation the dilution resulted by the fed synthetic sewage was taken into consideration. The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and was fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked just before use: 7.85. pH adjustment of the inoculum was considered as not necessary.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

no data

Test temperature:

20.2 - 22.0 °C

pH:

The pH of the activated sludge inoculum was checked just before use: 7.85. pH adjustment of the inoculum was considered as not necessary.

Dissolved oxygen:

6.4 - 8.4 mg O2/L

Salinity:

no data

Nominal and measured concentrations:

Nominal: 1000 mg test item/L.
No measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer bottles of approximately 300 mL volume.
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): five replicates
- No. of vessels per control (replicates): eight blank controls, three reference controls

TEST MEDIUM / WATER PARAMETERS
- in accordance to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: not considered necessary

EFFECT PARAMETERS MEASURED: respiration rate after 3 hours

TEST CONCENTRATIONS
- Test concentrations: 1000 mg test item/L
- Justification for using less concentrations than requested by guideline: Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the concentration of 1000 mg/L as a limit concentration, only.
- Pre-Experiment: In the preliminary range-finding test three test item concentrations were tested (with two replicates at the lower and three replicates at the highest test item concentration). Defined volumes (2 x 3.5; 2 x 35; 3 x 345 μL liquid test item that corresponded to 2 x 3 mg; 2 x 30 mg and 3 x 300 mg *) of the test item (according to the concentrations of 10, 100 and 1000 mg/L: 3, 30 and 300 mg, respectively) were administered directly, just before the inoculation.

Reference substance (positive control):

yes

Remarks:

3.5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

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

Key result

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

Results with reference substance (positive control):

The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 16.09 mg/L (95 % confidence limits: 11.76 - 22.00 mg/L)

Reported statistics and error estimates:

No statistical significant differences were observed in the comparison with the blank control values, consequently based on the results of this study the NOEC can be statistically determined as ≥ 1000 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.

Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the concentration of 1000 mg/L as a limit concentration, only. Defined amounts (in a form of the corresponding volumes) of the test item were added directly into the test vessels.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed. Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of the test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

An activated sludge respiration inhibition test with the test item was performed according to OECD guideline 209 (United initiators 2015). The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on the preliminary information about the test item caused effect on the activated sludge inoculum, the test item was investigated at the concentration of 1000 mg/L as a limit concentration, only. Defined amounts (in a form of the corresponding volumes) of the test item were added directly into the test vessels.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect of the test item was observed. Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was ≥ 1000 mg/L.