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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: mixtures contained 16.0 mL of synthetic sewage, 200 mL of inoculum, the appropriate amount of test substance and enough municipal water to bring the total volume up to 500 mL. All mixtures were aerated and stirred for three hours.
- Controls: two controls were prepared
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Cambridge Wastewater Treatment Plant, Cambridge, Maryland
- Preparation of inoculum for exposure: The sludge was sieved using a 2 mm screen and allowed to settle for approximately 30 minutes. The supernatant above the settled solids was removed and the total suspended solids (TSS) concentration of the settled sludge was determined. Total suspended solids in the settled sludge were adjusted to a nominal concentration of approximately 4000 mg/L by dilution with municipal water. 50 mL of synthetic sewage was added to each liter of adjusted sludge. The sludge was maintained at a temperature of 21 ± 1°C and continuously aerated overnight.
The initial total suspended solids (TSS) concentration of the activated sludge after removal of the supernatant layer measured 17 300 mg/L. The measured TSS concentration of the sludge on the day of testing was 4347 mg/L.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable
Hardness:
no data
Test temperature:
20.1 - 21.3 °C.
pH:
sludge- 7.3
Dissolved oxygen:
used to calculate respiration rates between 6.5 mg O2/L and 2.5 mg O2/L,
Salinity:
no data
Nominal and measured concentrations:
10, 30, 100, 300 and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: mixtures were prepared, mixed and aerated in 1000 mL Erlenmeyer flasks and then transferred to fill 300 mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements.
- Aeration: aerated for three hours
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): one/concentration
- No. of vessels per control (replicates): duplicate
- No. of vessels per vehicle control (replicates): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: municipal
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data. Direct reading of dissolved oxygen concentration using a using a YSI Model 50B-115 Dissolved Oxygen Meter

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : dissolved oxygen concentrations were measured every 10 seconds over a 15 minute period. Respiration rates and percent inhibitions were calculated from this.


Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no
- Substance was tested at far above water solubility limit, which is estimated to be 4.89 x 10E-7 mg/L. For this reason, direct addition of the test substance (by weight), and 3 hour contact time were used. Stimulation of respiration (6.1 - 181 % ) was observed over 4 of the 5 tested concentrations (11 % inhibition was observed for the other concentration), indicating that the test substance was being used as a substrate for growth/energy by the activated sludge microorganisms.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/l. The respiration rates for two controls were 45.3 and 40.1 mg O2/l/hr. The difference between the two control respiration rates was approximately 12 % and was within the 15 % difference limit established for the test. The validity of the test was supported by the results from the 3,5-dichlorophenol reference group, which resulted in an EC50 of 11.7 mg/l and was within the 5 to 30 mg/L range considered acceptable for the test.
Reported statistics and error estimates:
When the dose response pattern (percent inhibition versus test substance concentration) allowed for the calculation of an EC50 value, the data were analysed using the computer program of C.E. Stephan. The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average, or binomial probability with nonlinear interpolation.
Validity criteria fulfilled:
yes
Conclusions:
The highest percent inhibition value obtained in the study, for the test substance vessels, was 11.0 %. The EC50 value for the test substance in this study is therefore greater than 1000 mg/L, the highest concentration tested.
Executive summary:

The effect of the test substance on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with test substance at concentrations of 10, 30, 100, 300 and 1000 mg/L. After an exposure period of three hours, the respiration rates of the test solutions were measured using a YSI Model 50B-115 Dissolved Oxygen Meter. The respiration rates in the two controls were 45.3 and 40.1 mg O2/L/hr. The difference between the two control respiration rates was approximately 12 %, and was within the 15 % difference limit established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference group, which resulted in an EC50 of 11.7 mg/L. The EC50 for the reference substance was within the 5 to 30 mg/L range considered acceptable for the test. The highest percent inhibition value obtained in the study, for the test substance vessels, was 11.0 %. The EC50 value is therefore greater than 1000 mg/L, the highest concentration tested.

Description of key information

Activate sludge EC50 >1000 mg/L (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

The effect of the test substance on activated sludge microorganisms maintained in an aerobic environment was assessed by the Activated Sludge Respiration Inhibition Test Method (OECD Guideline 209). The test contained control, reference and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not dosed with the test or reference substance. The reference group was dosed with 3,5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15 and 50 mg/L. The treatment group was dosed with test substance at concentrations of 10, 30, 100, 300 and 1000 mg/L. After an exposure period of three hours, the respiration rates of the test solutions were measured using a YSI Model 50B-115 Dissolved Oxygen Meter. The respiration rates in the two controls were 45.3 and 40.1 mg O2/L/hr. The difference between the two control respiration rates was approximately 12 %, and was within the 15 % difference limit established for the test. The validity of the test was further supported by the results from the 3,5-dichlorophenol reference group, which resulted in an EC50 of 11.7 mg/L. The EC50 for the reference substance was within the 5 to 30 mg/L range considered acceptable for the test. The highest percent inhibition value obtained in the study, for the test substance vessels, was 11.0%. The EC50 value is therefore greater than 1000 mg/L, the highest concentration tested.