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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2017-03-22
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:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OCSPP 850.3300 (Modified Activated Sludge, Respiration Inhibition Test)
Version / remarks:
January 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
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 amount of the test item (in a form of adequate volumes) 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 12 minutes (an arbitrary but convenient interval) a maximum of four test vessels were started.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Preparation of inoculum for exposure:
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 48 hours (2 days) and was fed once with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.59) and before use (pH: 7.90).
- Initial biomass concentration: 3 g/L (on dry weight basis)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
No data
Test temperature:
20.8 - 21.6 °C.
pH:
The pH of the synthetic sewage was checked after preparation (pH=7.62) and additional pH adjustment was not performed.
Nominal and measured concentrations:
Nominal: 100; 40; 16; 6.33 and 2.67 mg/L.
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: Glass flasks of approximately 300 mL volume.
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): Eight blank controls (containing water, synthetic sewage and inoculum, but without addition of the test or reference item), three reference controls (3,5-Dichlorophenol at nominal test concentrations of 2, 7 and 24.5 mg/L) and three nitrification controls (same as the blank controls, however containing 11.6 mg/L N-allylthiourea)
- No. of vessels per abiotic control (replicates): The main test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed.

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
- Range finding study:
In the preliminary range-finding test three test item concentrations were tested: 10, 100 and 1000 mg/L (with one test vessel at the lower and three replicate vessels at the highest test item concentration). Blank, abiotic, reference and nitrification controls were tested in parallel with the test item.
- Results used to determine the conditions for the definitive study: In the preliminary concentration range-finding experiment the test item showed significant inhibitory effect: ~100% inhibition when compared to the blank control values, at the nominal concentration of 1000 mg/L. ~87 % inhibition was noticed at the nominal concentration of 100 mg/L and ~11 % at the lowest examined concentration of 10 mg/L. Based on the results of the performed preliminary range-finding test, in the main test five concentrations in a geometric series with five replicates were tested. The five concentrations (with spacing factor of ~2.5) examined were: 100, 40, 16, 6.33 and 2.67 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
12.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 10.5–15.6
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 32.4–42.2
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
74.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 61.7–89.1
Details on results:
Inhibitory effect of the test item was not observed at the lowest examined concentration of 2.67 mg/L. The observed-calculated oxygen consumption rates and corresponding inhibitory tendencies showed a dose-related tendency in the examined concentration range of 6.33-100 mg/L. At the concentration of 6.33 mg/L 4.51 %, at 16 mg/L 13.2 % inhibitions were calculated. At the concentration of 40 mg/L 49.4 %, at 100 mg/L 92.0 % inhibitions were obtained.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 13.3 mg/L.
Reported statistics and error estimates:
The 3-hour EC50 values of the test and reference items (and the EC10 and theoretical EC80 values of test item) and their 95 %-confidence limits were calculated by appropriate statistical test (Probit analysis by TOXSTAT software).
For the determination of the NOEC the calculated specific respiration rates were tested on significant differences to the control values by Bonferroni t-Test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Hartley’s and Bartlett’s Test.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of test item was determined as 37.0 mg/L (95% confidence limits: 32.4–42.2 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 6.33 mg/L.
Executive summary:

The influence of the test item Methyl-isobutyl-ketone-peroxide (MIKP) on the activity of the activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD guideline 209 and EU method C.11. 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 Methyl-isobutyl-ketone-peroxide (MIKP) was investigated at the nominal concentrations of 2.67; 6.33; 16; 40 and 100 mg/L, without pH adjustment. Defined amounts of the test item (in a form of the corresponding volumes) were added directly into the test vessels.

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of test item was determined as 37.0 mg/L (95% confidence limits: 32.4–42.2 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 6.33 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as 12.8 mg/L (95% confidence limits: 10.5–15.6 mg/L) and 37.0 mg/L (95% confidence limits: 32.4–42.2 mg/L), respectively. Based on the statistical and biological evaluation in this test the NOEC was determined as 6.33 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
37 mg/L
EC10 or NOEC for microorganisms:
12.8 mg/L

Additional information

The influence of the test item Methyl-isobutyl-ketone-peroxide (MIKP) on the activity of the activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD guideline 209 and EU method C.11. 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 Methyl-isobutyl-ketone-peroxide (MIKP) was investigated at the nominal concentrations of 2.67; 6.33; 16; 40 and 100 mg/L, without pH adjustment. Defined amounts of the test item (in a form of the corresponding volumes) were added directly into the test vessels.

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of test item was determined as 37.0 mg/L (95% confidence limits: 32.4–42.2 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 6.33 mg/L.