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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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Remarks:
The study was conducted according to the test guidelines in effect at the time of study conduct.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The dilution water used to prepare solutions of test mixtures, synthetic sewage and the reference substance was reverse osmosis (RO) water. The method of preparation used during the test was based on the results of a formulation trial. Information provided by the Sponsor indicated that the test substance was soluble in water. Therefore, 2.5 g of the test substance was dissolved in 1 Litre of RO water to give a stock concentration of 2500 mg/L. Appropriate volumes were added to vessels to give correct concentrations.
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. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test. 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°C 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. 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:
yes
Total exposure duration:
3 h
Test temperature:
Initial: 19.9-20.8
Final: 19.8-20.7
pH:
Initial: 7.46-7.68
Final: 7.67-8.38
Dissolved oxygen:
Initial: 5.56-7.75 mg O2/L
Final: 2.74-6.75 mg O2/L
Salinity:
freshwater
Nominal and measured concentrations:
Nominal: 0, 10, 100, 1000 mg/L
Details on test conditions:
The test was carried out using 2 L flasks containing 500 mL of liquor placed on a reciprocating shaker. Measurements of dissolved oxygen were taken after 3 hours incubation for a period of 15 minutes. Mixtures of the test substance were prepared at nominal concentrations of 10, 100 and 1000 mg/L with three replicates at the highest concentration. Two controls and single replicates of the reference substance were employed. In order to suppress foaming, 100 µL of a silicone based antifoam agent was added to each culture at the start of the test. The final volume in each mixture was 500 mL and the concentration of suspended solids 1.6 g/L. The control, test and reference mixtures were prepared in 2 L flasks and placed on a reciprocating shaker in a room maintained at a temperature of 20±2ºC.
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
Remarks on result:
other: highest concentration tested
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 - 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:
The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 20 mg O2/gh with a coefficient of variation (CV) of 16.3%. There were insufficient replicates containing test substance for a credible statistical analysis to be performed, however at the end of the incubation period, levels of inhibition were 9%, 7% and 22% (mean) at nominal test concentrations of 10, 100 and 1000 mg/L respectively, the latter being considered significant. Therefore the test substance notably inhibited the respiration rates of the samples of activated sludge at a nominal concentration of 1000 mg/L.
Results with reference substance (positive control):
Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP, with 24%, 50% and 81% inhibition obtained at 3, 10 and 32 mg/L respectively. The EC50 of 3,5-DCP was approximately 10 mg/L and as such was within the acceptable range of 2 to 25 mg/L. These results demonstrated that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.
Reported statistics and error estimates:
There was insufficient samples for credible statistical analysis to be performed.

Measurement Times and Specific Respiration Rates

Group

Nominal conc. (mg/L)

Bottle No.

Measurement time (minutes)

Specific respiration rate (Rs mg O2/gh)

Mean Rs (mg O2/gh)

% inhibition

Mean % inhibition

Control

1

6

17.6

19.9

10

4

22.2

Test

10

2

5

18.2

18.2

9

9

100

3

5

18.5

18.5

7

7

1000

4

8

15.4

15.6

23

22

1000

5

8

15.4

23

1000

6

7

16.1

19

Reference

3

7

7

15.2

24

10

8

6

10.0

50

32

9

10

3.8

81

The data presented were calculated using unfounded values. Minor numerical differences may be observed in the respiration rate calculation if founded values are used to calculate the data. This minor discrepancy is not considered to be significant.

The Coefficient of variation for the control group was 16.3%.

Validity criteria fulfilled:
yes
Conclusions:
3-hour EC50 >1000 mg/L

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

The study was to assess the effect of the test substance on the respiration rate of activated sludge using the methods detailed in OECD Procedure 209 of the “Guidelines for Testing of Chemicals”: Activated Sludge, Respiration Inhibition Test (carbon and ammonium oxidation), adopted 22 July 2010.

Samples of activated sludge (suspended solids 1.6 g/L), fed with synthetic sewage, were exposed to dilutions of the test substance for 3 hours. Their rates of oxygen consumption were determined using oxygen electrodes and were compared with those of two replicate controls that contained activated sludge and synthetic sewage alone. The nominal the test substance concentrations used in the study were 10, 100 and 1000 mg/L; a single replicate was tested at 10 mg/L and 100 mg/L and three replicates at 1000 mg/L. The control consisted of two replicates. Single replicates of the reference inhibitor 3,5-dichlorophenol (3,5-DCP) were tested at 3, 10 and 32 mg/L, as a positive control.

The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 20 mg O2/gh with a coefficient of variation (CV) of 16.3%. Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP, with 24%, 50% and 81% inhibition obtained at 3, 10 and 32 mg/L respectively. The EC50 of 3,5-DCP was approximately 10 mg/L and as such was within the acceptable range of 2 to 25 mg/L. These results demonstrated that the sample of activated sludge employed was sensitive to inhibition and that the test was valid.

There were insufficient replicates containing test substance for a credible statistical analysis to be performed, however at the end of the incubation period, levels of inhibition were 9%, 7% and 22% (mean) at nominal test concentrations of 10, 100 and 1000 mg/L respectively, the latter being considered significant. Therefore the test substance notably inhibited the respiration rates of the samples of activated sludge at a nominal concentration of 1000 mg/L. Based on these results it can be assumed that the NOEC lies between 100 and 1000 mg/L and the EC50 can be considered to be greater than the highest level tested, 1000 mg/L.

Description of key information

3-hour EC50 >1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information