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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2015-09-30 to 2015-09-30
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:
(July 2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OCSPP 850.3300:Modified Activated Sludge, Respiration Inhibition Test (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 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 (0.5 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: 8.01. pH adjustment of the inoculum was considered as not necessary.
- 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
Post exposure observation period:
None
Hardness:
No data
Test temperature:
20.1 - 22.0 °C
pH:
The pH of the activated sludge inoculum was checked just before use: 8.01. pH adjustment of the inoculum was considered as not necessary.
Salinity:
No data
Nominal and measured concentrations:
Nominal: 10.24, 25.6, 64, 160, 400 and 1000 mg/L.
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles
- Type (delete if not applicable): closed
- Material, fill volume: Glass flasks with a final volume of 300 mL
- Aeration: With compressed air (0.5 litre per minute)
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Eight blanc controls (containing water, synthetic sewage and inoculum, but without addition of the test or reference item), three reference controls (3,5-Dichlorophenol at the nominal 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)

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
- Spacing factor for test concentrations: 2.5
- Range finding study: In a preliminary range-finding study concentrations of 10, 100 and 100 mg/L were tested (with three replicates at the highest test item concentration, with two replicates at the lower concentrations). Blank controls, nitrification controls and a abiotic control group were inculded in the test.
- Results used to determine the conditions for the definitive study: In the preliminary experiment the test item showed significant inhibitory effect (~65 % inhibition when compared to the blank control values) at the nominal concentration of 1000 mg/L, slight inhibition (~13 %) was noticed at the nominal concentration 100 mg/L and no inhibition was found at 10 mg/L. In the preliminary test the nitrification respiration was insignificant and it was assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification is occurred. Therefore the main test was performed in one set of test vessels, without ATU addition.The abiotic control group in the preliminary experiment did not show oxygen uptake therefore in the main test abiotic control group was not included.
Reference substance (positive control):
yes
Remarks:
(3,5-Dichlorophenol)
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
68.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (52.3 - 89.3)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
407.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (333.0 - 497.7)
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
1 314.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: (955.1 - 1808.1)
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The observed-calculated oxygen consumption rates remained nearly within the range of the rates of the blank controls at the concentration range of 10.24-64 mg/L. The observed 2 %, 5 % and 3 % inhibitions did not show any dose relationship, and were considered rather as reflecting the biological variability of the applied test system than a test item effect. At the concentration of 160 mg/L 16 %, at 400 mg/L 62 %, at the highest examined concentration of 1000 mg/L 73 % inhibitions were obtained. The inhibition of the oxygen consumption rates showed clear dose-related tendency.
Results with reference substance (positive control):
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 15.2 mg/L (95 % confidence limits: 12.5-18.5 mg/L).
Reported statistics and error estimates:
The 3-hour EC50 values of the test and reference items 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 EC10 and EC50 values were determined as 68.3 and 407.1 mg/L, respectively. Based on the statistical and biological evaluation in this test the NOEC was determined as 64 mg/L
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item TBPA-50-AL1 on the activity of 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, in the main test the test item TBPA-50-AL1 was investigated at the nominal concentrations of 10.24; 25.6; 64; 160; 400 and 1000 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 407.1 mg/L (95% confidence limits: 333.0–497.7 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 64 mg/L.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC50 value of test item was determined as 407.1 mg/L (95% confidence limits: 333.0–497.7 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 64 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
407.1 mg/L
EC10 or NOEC for microorganisms:
64 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item TBPA-50-AL1 on the activity of 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, in the main test the test item TBPA-50-AL1 was investigated at the nominal concentrations of 10.24; 25.6; 64; 160; 400 and 1000 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 407.1 mg/L (95% confidence limits: 333.0–497.7 mg/L). Based on the statistical and biological evaluation in this test the NOEC was determined as 64 mg/L.

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