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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 June to 12 July 2012
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 (incl. certificate)
Remarks:
June 2011
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance is poorly soluble in water. At test initiation weights of the test substance were added directly to the required test bottles followed by RO water (284 mL).
- Controls: control containing activated sludge only; positive control (3,5-DCP).
- An antifoam agent (silicone oil in water) was added to all bottles, 100 uL in 500 mL.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- A sample of activated sludge was obtained the day before the start of the test from Worlingworth Sewage Treatment Works (Suffolk, UK), which treats predominantly domestic waste.
- Laboratory culture: In the laboratory, the sample was maintained under aerobic conditions until required.
- Preparation of inoculum for exposure: On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105℃ for at least one hour, allowed to cool in a desiccator and reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
- Pretreatment: On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
At the end of 3-hour exposure period the aeration was discontinued and the headspace of each bottle was flushed with nitrogen for 15-minutes while oxygen measurements were made.
Test temperature:
20.6 - 20.9 ℃
pH:
Initial: 7.10 - 7.28
Final: 7.53 - 7.81
Nominal and measured concentrations:
Nominal concentrations: 10, 30, 100, 300 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No. of vessels per reference substance: 2 for control, 1 for each concentration.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: The results of this showed no significant inhibition of the mean respiration rate at 10 mg/L. At 100 and 1000 mg/L, mean respiration rates were decreased by 26% and 54% respectively.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
There was no significant inhibition of the respiration rates of activated sludge at nominal concentrations of 10 and 30 mg/L. The mean respiration rates of samples of sludge at nominal concentrations of 100, 300 and 1000 mg/L were decreased by 28%, 34% and 47%, respectively. The 20% effect concentration (EC20) was 64.5 mg/L (95% confidence intervals 19.6 and 212 mg/L). Statistical analysis of the data gave a 50% effect concentration (EC50) of 783 mg/L, however as the 95% confidence intervals for this were considered exceptionally wide (423 and 1148 mg/L) this value was not considered accurate. Therefore, the EC50 for BMI was considered to be approximately 1000 mg/L based on the level of inhibition (47%) observed at this concentration. The 80% effect concentrations (EC80) could not be calculated as it was higher than 1000 mg/L. The no observed effect concentration (NOEC) for BMI was 30 mg/L.
Results with reference substance (positive control):
The three-hour EC50 for 3,5-DCP (7.18 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 2 to 25 mg/L). The validity criterion relating to the respiration rates in the control (specific respiration rate ≥20 mgO2/gh and a coefficient of variation of ≤30%) were also satisfied.
Reported statistics and error estimates:
Where possible, the EC20, EC50 and EC80 (and 95% confidence intervals) and no observed effect concentration (NOEC) of the test substance were calculated with the Williams' Test, using SAFEstat programme SAS Proc NLIN (SAS Institute, 2002).

Nominal BMI concentration (mg/L)

Sample size

Mean specific respiration rate (Rs mgO2/gh)

% Inhibition

Control

4

25.1

0.00

10

5

27.0

-7.60

30

5

23.0

8.56

100

5

18.1

27.9*

300

5

16.5

34.2*

1000

5

13.3

47.0*

*: statistically significantly different from control, p<0.001

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' section for details on validity criteria.
Conclusions:
The EC50 was considered to be approximately 1000 mg/L. The no observed effect concentration (NOEC) for the test substance was 30 mg/L.
Executive summary:

An Activated Sludge Respiration Inhibition Test (ASRIT) was conducted to determine the effect of BMI on microorganisms. The test was conducted according to OECD test guideline 209, and in compliance with GLP.

Activated sludge was taken from a sewage treatment plant which treated predominantly domestic waste. Cultures were exposed to BMI at concentrations of 10, 30, 100, 300, and 1000 mg/L for 3 hours. There was no significant inhibition of the respiration rates of activated sludge at nominal concentrations of 10 and 30 mg/L. The mean respiration rates of samples of sludge at nominal concentrations of 100, 300 and 1000 mg/L were decreased by 28%, 34% and 47%, respectively. The 20% effect concentration (EC20) was 64.5 mg/L. The 50% and 80% effect concentrations (EC50 and EC80) could not be accurately calculated; based on the levels of inhibition observed, the EC50 is approximately 1000 mg/L. The no observed effect concentration (NOEC) for BMI was 30 mg/L. The test met all validity criteria and is considered reliable without restriction.

Description of key information

3 hr EC50 estimated to be 1000 mg/L

3 hr NOEC 30 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
30 mg/L

Additional information

An Activated Sludge Respiration Inhibition Test (ASRIT) was conducted to determine the effect of BMI on microorganisms. The test was conducted according to OECD test guideline 209, and in compliance with GLP.

Activated sludge was taken from a sewage treatment plant which treated predominantly domestic waste. Cultures were exposed to BMI at concentrations of 10, 30, 100, 300, and 1000 mg/L for 3 hours.There was no significant inhibition of the respiration rates of activated sludge at nominal concentrations of 10 and 30 mg/L. The mean respiration rates of samples of sludge at nominal concentrations of 100, 300 and 1000 mg/L were decreased by 28%, 34% and 47%, respectively. The 20% effect concentration (EC20) was 64.5 mg/L. The 50% and 80% effect concentrations (EC50 and EC80) could not be accurately calculated; based on the levels of inhibition observed, the EC50 is approximately 1000 mg/L.The no observed effect concentration (NOEC) for BMI was 30 mg/L.The test met all validity criteria and is considered reliable without restriction.