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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:
2008-09-03 to 2008-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1988
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Before the start of the test defined amounts of the test item were directly weighed into the test flasks and mechanically dispersed using short ultrasonic bath treatment (< 10 min) and orbital shaker for 10 min.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprem, Hungary.
- Preparation of inoculum for exposure: The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and a ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.37. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.0 - 20.2 degree Celius (during the incubation) and
19.0 - 2 1.1 degree Celsius (during oxygen measurement)
pH:
7.42-7.82
Dissolved oxygen:
Oxygen consumption: 0.5 mgO2/L/min
Nominal and measured concentrations:
Nominal concentrations:
10, 31, 100, 313 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer bottles and BOD bottles with special neck
- Size, fill volume: 350 mL (Erlenmeyer bottles) and 300 mL (BOD bottles); fill volume: 330 mL
- Aeration: With compressed air (approximately 1 litre per minute)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 2
- Sludge concentration: 4 g/L (on dry weight basis)

TEST MEDIUM / WATER PARAMETERS
- According to guideline

OTHER TEST CONDITIONS
- Details on termination of incubation: For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after exactly 3 hours incubation time, and was not further aerated.

EFFECT PARAMETERS MEASURED:
- Respiration rate was measured after exactly 3 hours incubation time.
- pH and oxygen concentrations were determined at the start and at the end of the incubation period.
- Temperature was measured during the incubation period.
- Water temperature was recorded during oxygen measurement in all BOD bottles.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~ 3.2

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
Details on results:
- Blank controls oxygen uptake rate: 0.490 mg O2/L/min (mean)
- Coefficient of variation of oxygen uptake rate in control replicates: 0%
- Other: Slight stimulation of respiration rates was observed (-2.0 - -6.1 %), with obtained values in the range of biological variability of the test at the concentr,ations of 31, 100 and 3 13 mg/L. Slight inhibition of-8.2 % was observed at the concentration of 1000 mg/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes (The EC50 was in the acceptable the range of 5 to 30 mg/L)
- Relevant effect levels: In comparison to the controls the respiration rate of the activated sludge was inhibited by 24.5 % at the lowest nominal concentration of 5 mg/L.
At the nominal concentrations of 16 and 32 mg/L , the respiration rate was inhibited by 69.4 % and 81.6 %, respectively.
Reported statistics and error estimates:
Probit analysis (EC50 and 95% CL of the reference item)
Validity criteria fulfilled:
yes
Conclusions:
Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.
Executive summary:

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item TODI on the activity of activated sludge by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations (10, 31, 100, 313 and 1000 mg/L) of the test item after an incubation period of 3 hours. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of two controls. 3,5-Dichlorophenol served as positive control and two inoculum controls were used in parallel. The validity criteria of the study were met.

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the examined concentration range of 10-313 mg TODI /L. Slight stimulation of respiration rates was observed (-2.0 - -6.1 %),with obtained values in the range of biological variability of the test at the concentrations of 3 1, 100 and 3 13 ma.Slight inhibition of 8.2 % was observed at the concentration of 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were not inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (8.2 %) at the highest examined concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.450 (at 1000 mg/L) was in the historical control data range (0.514 +/- 0.081), and the deviation from the control was within +/- 15 %, can be considered as a biological variability of the test system.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

 

Description of key information

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item TODI on the activity of activated sludge by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations (10, 31, 100, 313 and 1000 mg/L) of the test item after an incubation period of 3 hours. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of two controls. 3,5-Dichlorophenol served as positive control and two inoculum controls were used in parallel. The validity criteria of the study were met.

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited in the examined concentration range of 10-313 mg TODI /L. Slight stimulation of respiration rates was observed (-2.0 - -6.1 %),with obtained values in the range of biological variability of the test at the concentrations of 3 1, 100 and 3 13 ma.Slight inhibition of 8.2 % was observed at the concentration of 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were not inhibited and influenced dose dependently in the whole concentration range, and the observed slight inhibition (8.2 %) at the highest examined concentration level of 1000 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.450 (at 1000 mg/L) was in the historical control data range (0.514 +/- 0.081), and the deviation from the control waswithin +/- 15 %, can be considered as a biological variability of the test system.

Based on measured inhibition rates it can be stated that the 3-hour EC20, EC50 and EC80 were higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.