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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-04 to 2011-10-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
adopted 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
adopted 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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 (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 weighted in a small inert plastic dish (plate). 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, first to the first blank control (CBA, thereafter CBB and CBC 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 small inert plastic dish containing the adequate amount of the test item was added shortly before the inoculum addition) and finally to the sixth blank control (CBD, after the CBE and CBF the “end” CB group).
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). The activated sludge was not used on the day of the collection but continuously aerated (~2L/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 after preparation and before use. The pH of the activated sludge inoculum after preparation was 7.69, before use: 7.71. The pH adjustment before use was considered as not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.1-22.0 °C
pH:
The pH of the activated sludge inoculum after preparation was 7.69, before use: 7.71. The pH adjustment before use was considered as not necessary.
Nominal and measured concentrations:
130, 216, 360, 600 and 1000 mg/L
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 organisms per vessel: 3 g/L activated sludge based on dry weight
- No. of vessels per concentration (replicates): five replicates
- No. of vessels per control (replicates): six blank controls, three nitrification controls, three refernce controls

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED:
respiration rate after 3 hours

Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
966.9 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.:
2 053.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
An additional nitrification control was examined in the main test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 67.41 mg/Lh, the heterotrophic respiration (RH) was 66.79 mg/Lh, the nitrification respiration (RN): 0.62 mg/Lh was calculated according to the following equation: RN= RT-RH. The obtained 0.62 mg/Lh was insignificant [lower than the 5 % of RT (3.37 mg/Lh) in blank controls]. According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 6.10 mg/L, (95 % confidence limits: 4.91-7.59).
Validity criteria fulfilled:
yes
Conclusions:
The EC10 was calculated to be 966.9 mg/L and the EC50 was calculated to be > 1000 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item TBPND 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. Following concentraions were tested 130, 216, 360, 600 and 1000 mg/L. At the concentrations of 130, 216 and 360 mg/L the observed-calculated specific respiration rates were within the range of the specific respiration rates of the blank controls, and within the biological variability of the applied system. The occurring negative values were considered and evaluated as zero inhibition. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls was 3.62 % at 130 mg/L, -0.31 % at 216 mg/L and -0.24 % at 360 mg/L. The test item showed slight inhibitory effect at the concentrations of 600 mg/L (7.70 %) and at 1000 mg/L (10.23 %). An EC10 of 966.9 mg/L was detected. The EC50 was calculated to be 2053.9 mg/L.

Description of key information

The toxicity to microorganisms was assessed according to OECD guideline 209, EU-method C.11 and EPS OPPTS 850.6800. After 3 hours of exposure an EC10 of 966.9 mg/L was detected.The EC50 was calculated to be 2053.9 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
2 053.9 mg/L
EC10 or NOEC for microorganisms:
966.9 mg/L

Additional information

The purpose of the 3-hour test was to evaluate the influence of the test item TBPND 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. Following concentraions were tested 130, 216, 360, 600 and 1000 mg/L. At the concentrations of 130, 216 and 360 mg/L the observed-calculated specific respiration rates were within the range of the specific respiration rates of the blank controls, and within the biological variability of the applied system. The occurring negative values were considered and evaluated as zero inhibition. The inhibition of the specific respiration rates compared to the respiration rates of the blank controls was 3.62 % at 130 mg/L, -0.31 % at 216 mg/L and -0.24 % at 360 mg/L. The test item showed slight inhibitory effect at the concentrations of 600 mg/L (7.70 %) and at 1000 mg/L (10.23 %). An EC10 of 966.9 mg/L was detected. The EC50 was calculated to be 2053.9 mg/L.