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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 February 2019 - 13 February 2019
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant, Hildesheim, Germany
- Pretreatment: washing twice with chlorine free tap water and adjusted to a dry sludge concentration of 3 g/L (+/-10%).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
19.6 °C
pH:
7.5 ± 0.5
Dissolved oxygen:
60 - 70%
Nominal and measured concentrations:
Nominal concentraion: 1000 mg a.i./ L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask 500 mL, DIN 12380
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 1.45 g/L
- Biomass loading rate: 50 vol% inoculum

ATMOSPHERE QUALITY MEASUREMENTS
- pH: The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use and adjustment to 7.5 ± 0.5 was not
necessary.
- Oxygen: After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.
- Temperature: The temperature was determined in the first replicate at test start with the pH-meter. The room temperature during the test was recorded continuously.
- Atmospheric pressure: The atmospheric pressure was determined with a barometer before the first replicate was measured (needed for measurement of oxygen concentration).

EFFECT PARAMETERS MEASURED: oxygen uptake rate
The oxygen uptake rates of all samples were determined following an incubation period of 3 h. From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L/h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/L/h. The inhibition of the oxygen uptake rates of the test item concentrations in % was calculated in comparison to the oxygen uptake rates of the control.

TEST CONCENTRATIONS
- Range finding study : based on the results of the preliminary test (10, 100 and 1000 mg/L), it was concluded that the heterotrophic respiration and the nitrification are not inhibited.
- Test concentrations: 1000 mg active ingredient / L used in final test (limit concentration).
Reference substance (positive control):
yes
Remarks:
copper (II) sulphate pentahydrate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Details on results:
The mean oxygen update rate was 31.0 mg O2/(Lxh) in the control. While the mean oxygen update rate was 29.2 mg O2/(Lxh) in the treatment group. The specific respiration rate in the control was 21.4 mg O2/g·h. In comparison to the control, 6% inhibition of the respiration was found at 1000mg/L of the test substance after 3h exposure. See Table 2 and Table 3 in "Any other information in results incl. tables" for an overview of the results.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 105 mg/L
Reported statistics and error estimates:
The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
The EC-value of the reference item was calculated by linear regression using software Graph Pad Prism. Calculation of the confidence intervals for the EC50-value was carried out using
standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

Table 2. Oxygen update rates of the control replicates

Treatment

Replicate

Oxygen concentration* (mg O2/L)

Oxygen update rate R (mg O2/(Lxh))

Specific Respiration rate (mg O2/g·h)

Control

1

7.18

27.0

18.6

 

2

7.24

29.3

20.2

 

3

7.44

29.3

20.2

 

4

7.19

32.7

22.6

 

5

7.52

34.0

23.4

 

6

7.17

33.9

23.4

 

 

Mean value

31.0

21.4

 

 

Standard deviation

2.65

 

 

 

CV (%)

8.55

 

*) determined at start of the measurement

CV = Coefficient of variation

 

Table 3. Oxygen update rates and inhibition of the respiration of the test item concentration

Treatment

Replicate

Oxygen concentration* (mg O2/L)

Oxygen update rate R (mg O2/(Lxh))

Inhibition (%)

Mean inhibition (%)

1000

1

6.18

29.1

6

6

 

2

6.63

28.5

8

 

3

6.63

29.9

4

*) determined at start of the measurement

Validity criteria fulfilled:
yes
Remarks:
See Table 1 in 'Any other information on materials and methods incl. tables'
Conclusions:
The NOEC of the test item Reactive Red 45:1 is ≥ 1000 mg a.i./L. Reactive Red 45:1 caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg a.i./L.
Executive summary:

The toxicity of the test substance to microorganisms was investigated in an activated sludge respiration inhibition Test according to OECD TG 209 and in compliance with GLP-criteria. In this study, non-adapted activated sludge from a predominantly municipal sewage treatment was exposed to a limit concentration of 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference substance were determined in comparison to the control respiration rates. The validity criteria of the test guideline were fulfilled and thus the study is considered valid for assessment.

After 3 hours, the mean inhibition of respiration for the test substance was 6%. As the effects of the test substance on the microorganisms was considered to be negligible, the 3-h NOEC was determined to be≥ 1000 mg/L.

 

Description of key information

The NOEC of the the test substance is 1000 mg/L. The test substance caused no inhibiting effects on activated sludge of a municipal sewage treatment plant at the limit test item concentration of 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of the test substance to microorganisms was investigated in an activated sludge respiration inhibition Test according to OECD TG 209 and in compliance with GLP-criteria. In this study, non-adapted activated sludge from a predominantly municipal sewage treatment was exposed to a limit concentration of 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference substance were determined in comparison to the control respiration rates. The validity criteria of the test guideline were fulfilled and thus the study is considered valid for assessment.

After 3 hours, the mean inhibition of respiration for the test substance was 6%. As the effects of the test substance on the microorganisms was considered to be negligible, the 3-h NOEC was determined to be ≥ 1000 mg/L.

 

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