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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2019 to 06 June 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 (Carbon and Ammonium Oxidation))
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
14 February 2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No correction for purity of the test material was applied.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test material was added by directly weighing the test material into the bottles (and using ultrasonic bath for 20 minutes). The test formulation was freshly prepared at the beginning of the experiment, in the testing laboratory.
The pH of the solutions was checked but not adjusted due to the direct application of the test material.

Test organisms (species):
activated sludge of a predominantly domestic sewage
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 Veszprém, Hungary
- Method of conditioning: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use.
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 chlorine-free tap water and again centrifuged.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.35. 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:
19.2 - 21.4 °C
pH:
7.21 - 8.19
Nominal and measured concentrations:
Preliminary Test: 0, 10, 100, 1 000 mg/L
Definitive Test: 0, 6.14, 15.36, 38.4, 96.0, 240.0, 600.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
- Aeration: With compressed air (about 1 L/min).
- No. of vessels per concentration: The test was performed at six nominal concentrations of the test material and 5 replicates per concentration.
- No. of vessels per reference control: In parallel with the test material, the reference item 3,5-Dichlorophenol (REF1, REF2, REF3, REF4) was tested (at nominal test concentrations of 1.0, 3.2, 10 and 32 mg/L) under identical test conditions.
- No. of vessels per untreated control: Seven replicates (B1 – B7) of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the test material) were tested in parallel with all of the test series.
- No. of vessels per abiotic control: The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.
- Sludge concentration (weight of dry solids per volume): 3 g/L

PREPARATION OF THE TEST FLASKS
One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask.
A volume of 12.8 mL synthetic sewage and an adequate amount of the test material given into the flask by direct addition was filled up with deionised water to 200 mL before the start of the incubation. At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids was added.

EFFECT PARAMETERS MEASURED
For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after about 3-hours incubation time, and was not further aerated. The oxygen concentration was measured for about fifteen minutes with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 /L/minute) was determined from the most linear part of the respiration curve.
The pH of the test mixtures was determined at the start and at the end of the incubation period in all test vessels. The temperature was measured in the laboratory with a min/max thermometer during the experiment.

TEST CONCENTRATIONS
- Range finding study: Three test material concentration series and one abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures four reference item concentrations without replicates were also prepared with one blank control.
- Test concentrations: 0, 10, 100, 1 000 mg/L
- Results used to determine the conditions for the definitive study: Yes. Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test, a further definitive test was conducted. Test concentrations for the definitive test were selected based on the findings from the preliminary test.

RESULT EVALUATION
- Calculation of oxygen uptake rates
The oxygen uptake rates (R) was calculated from the measured values, based on the linear part of the graphs of oxygen concentration (values taken in consideration between 2.0 mg/L and 7.0 mg/L only) versus time.

R = (Q1 – Q2)/Δt X 60

Where:
Q1: Oxygen concentration at the beginning of the selected section of the linear phase (mg/L).
Q2: Oxygen concentration at the end of the selected section of the linear phase (mg/L).
Δt: Time interval between the two measurement (min).
The R value was composed for all of the parallels and the mean R value was calculated from them, to determine the inhibition effects.

- Calculation of percentage of inhibition
The percentage inhibition of the total oxygen consumption (IT) was calculated:

IT = [1-(RT – RTA)/RTB] X 100 % (preliminary test)
or
IT = [1-(RT/RTB)] X 100 % (definitive test)

Where:
RT: Rate of total oxygen consumption (mg/L/hour)
RTA: Rate of total oxygen consumption (mg/L/hour) due to abiotic processes
RTB: Rate of total oxygen consumption (mg/L/hour) due to untreated (blank) controls

- Calculation of specific respiration rate
The specific respiration rate (Rs) is the ratio of the consumed oxygen and the dry weight (g) of sludge per hour (mg/g/h)

Rs = R/SS

Where:
SS: Concentration of suspended solids in the test mixture (g/L).
R: Oxygen uptake rate (see above)
The different indicates of R which may be combined are:
S specific respiration rate
T total respiration rate
A rate due to abiotic processes
B rate based on blank assays (mean)
The oxygen uptake rates, percentage of inhibition and specific respiration rates were calculated by using Excel for Windows Software.

- Determination of percentage inhibition of oxygen uptake
The 3-hour EC20, EC50 and EC80 values of the test item and the 3-hour EC50 values of the reference item with their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

VALIDITY CRITERIA
- The blank controls (without the test material or reference substance) oxygen uptake rate should be > 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.
- The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of definitive test.
- The EC50 of the positive control substance 3,5-dichlorophenol (3,5-DCP) should be in the range of 2 mg/L to 25 mg/L for total respiration using activated sludge derived from domestic sewage.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
101.76 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % confidence limits of 83.22 - 124.43 mg/L.
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
24.27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % confidence limits of 18.64 - 31.61 mg/L.
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
426.61 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95 % confidence limits of 312.25 - 582.86 mg/L.
Details on results:
PRELIMINARY TEST
In comparison to the inoculum controls the inhibition of the total respiration rate in the case of the activated sludge was between 10.3 % and 81.65 % in the examined test material concentration range of 10 - 1 000 mg/L.
Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test further testing was required.

DEFINITIVE TEST
In comparison to the inoculum controls the inhibition of the total respiration rate in the case of the activated sludge was between 4.00 % and 80.26 % in the examined test material concentration range of 6.14 - 600.0 mg/L.
The average specific total respiration rate (RTS) of the untreated (Blank) controls (B1-B7) was 30.50 mg O2/L/h/g solid, so the validity criteria was met.
The variation coefficient of oxygen uptake rate in above untreated replicates was 2.46 % and thus, the validity criteria was met.
Results with reference substance (positive control):
A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209 (1.0 g of powdered 3,5-Dichlorophenol dissolved in 1 000 mL of water). Warm deionised water and ultrasonic bath was used to help the dissolution of the reference substance and the volume of the solution was made up to 1.0 L with deionised water and then cooled to room temperature. The pH of the solution was checked and adjusted, with NaOH solution (2 mol/L) to pH 7.32.

The following nominal concentrations of the reference control 3,5-dichlorophenol were tested on the same activated sludge and under identical conditions as in the case of the test item: 1.0, 3.2, 10.0 and 32.0 mg/L.
In comparison to the controls the inhibition of the total respiration rate of the activated sludge was 79.01 % at the highest nominal concentration of 32.0 mg/L.
At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 18.46 %, 31.49 % and 40.16 % inhibition of the respiration rate was calculated respectively.
The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 9.31 mg/L with 95 % confidence limits of 6.92 - 12.52 mg/L.
The EC50 values of the positive control substance 3,5-dichlorophenol (3,5-DCP) were in the recommended range for total respiration using activated sludge derived from domestic sewage as well.

Influence of Test Material on the Total Oxygen Consumption of Activated Sludge During the Definitive Test

Test Group

Conc. of Test Material in Test Mix

(mg/L)

Total Oxygen Consumption Rate (RT)

(mg O2/L/h)

Specific Respiration Rate (RTS)

(O2/L/h)

Inhib.

(%)

Variation Coefficient of O2 Consumption Rate

(%)

ID

Name

B1

Blank

0.00

46.97

31.31

-

2.46

B2

0.00

46.50

31.00

-

B3

0.00

45.60

30.40

-

B4

0.00

45.24

30.161

-

B5

0.00

44.55

29.70

-

B6

0.00

44.30

29.53

-

B7

0.00

47.07

31.38

-

REF1

Ref. item

1.00

37.30

24.87

18.46

-

REF2

3.20

31.34

20.89

31.49

-

REF3

10.00

27.34

18.25

40.16

-

REF4

32.00

9.60

6.40

79.01

-

TI1

Test material

6.14

43.92

29.28

4.00

-

TI2

15.36

41.20

27.47

9.95

-

TI3

38.4

30.79

20.53

32.69

-

TI4

96.0

22.68

15.12

50.43

-

TI5

240.0

12.11

8.07

73.53

-

TI6*

600.0

9.03

6.02

80.26

-

* Data recorded of two replicates (the second and third ones) were out of the range between 7.0 – 2.0 mg O2/L during the measured period. Therefore, these replicates were excluded from evaluation of the results. It has no impact on the study results, mainly id it does not change the magnitude of the expected EC50 values.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study the 3-hour EC50 value of the test material based on total respiration rate was 101.76 mg/L (with 95 % confidence limits of 83.22 - 124.43 mg/L).
Executive summary:

In order to evaluate the effect of the test material to microorganisms by measuring the respiration rate, an activated sludge respiration inhibition study was conducted in accordance with the standardised guidelines OECD 209, EU Method C.11 and US EPA OPPTS 850.6800, under GLP conditions.

Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test, a further definitive test was conducted with the following concentrations: 6.14, 15.36, 38.4, 96.0, 240.0, and 600.0 mg/L.

The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

The purpose of the test was only to assess the effects on total oxygen uptake, the inhibition effect in respect of nitrification process was not determined.

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

Under the conditions of the study the 3-hour EC50 value of the test material  based on total respiration rate was 101.76 mg/L (with 95 % confidence limits of 83.22 - 124.43 mg/L).

Description of key information

Under the conditions of the study the 3-hour EC50 value of the test material  based on total respiration rate was 101.76 mg/L (with 95 % confidence limits of 83.22 - 124.43 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:
101.76 mg/L

Additional information

In order to evaluate the effect of the test material to microorganisms by measuring the respiration rate, an activated sludge respiration inhibition study was conducted in accordance with the standardised guidelines OECD 209, EU Method C.11 and US EPA OPPTS 850.6800, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test, a further definitive test was conducted with the following concentrations: 6.14, 15.36, 38.4, 96.0, 240.0, and 600.0 mg/L.

The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

The purpose of the test was only to assess the effects on total oxygen uptake, the inhibition effect in respect of nitrification process was not determined.

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

Under the conditions of the study the 3-hour EC50 value of the test material  based on total respiration rate was 101.76 mg/L (with 95 % confidence limits of 83.22 - 124.43 mg/L).

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