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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 December 2017 to 15 March 2018
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))
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was directly pipetted into each test flask, which was filled with pure water. As a previous test revealed, that the test item has a large effect on the pH of the whole test approach, the pH was adjusted with 1M HCl to a pH between 7.4 and 7.6 in test item flasks with a test item concentration of 100 mg/L and 1000 mg/L.
- Test Concentrations: Nominal 10 mg test item/L (three replicates), 100 mg test item/L (three replicates) and 1000 mg/L (one replicate). A previous test revealed, that 1000 mg test item/L had toxic effects on the respiration rate, therefore only one replicate was used.
- Controls: Six controls (water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.
- Reference Item 3,5-Dichlorophenol: In parallel to the study with the test item, the reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4 and 16 mg/L (one replicate each) under otherwise identical test conditions. A stock solution of 3,5-dichlorophenol was prepared according to the OECD Guideline No. 209: 250 mg of 3,5-dichlorophenol was dissolved in 250 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it has cooled to room temperature. The final pH was determined to be 7.1. For the adjustment of the pH 1N NaOH was used.



Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by a municipal sewage treatment plant (Bensheim, Germany).
- Method of cultivation: The activated sludge used for this study was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and then the upper layer decanted. During holding prior to use for one day, the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at room temperature until use.
- Preparation of inoculum for exposure: An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gave a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.7 and therefore no adjustment was necessary.
- Initial biomass concentration: 1.5 g/L suspended solids in the test medium



Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Test temperature:
20°C ± 2°C
pH:
6.9-7.6 (pH values start)
Details on test conditions:
The influence of the test item on the activity of activated sludge was evaluated 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 three concentrations of the test item after an incubation period of 3 hours.
Proceudre: The test item was directly dosed into each test flask and pure water was added. Three test item solutions were tested for the 10 and 100 mg test item/L treatments and one test item solution was tested for the 1000 mg test item/L treatment, with a final volume of 500 mL per treatment in a glass flask.
For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer.
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations and controls. The water temperature was measured in one control medium at the start and the end of the incubation period.
For the determination of the EC10 and NOEC of the test item the Williams Multiple Sequential t-test Procedure (α = 0.05) was used.

- Parameters analysed / observed:
1) Measurement of Respiration Rate: For the measurement of the respiration rate a well-mixed sample of test medium from each flask was poured into a Karlsruher flask after exactly 3 hours incubation time and was not further aerated during measurement. The oxygen concentration was then measured with an oxygen electrode and recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption (in mg O2 L^-1 minute^-1) was determined over aperiod of 6.5 - 10 minutes in the range between 8.9 – 1.2 mg O2/L.
2) Measurement of pH, Dissolved Oxygen and Water Temperature: The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations and controls. The water temperature was measured in one control medium at the start and the end of the incubation period.

TEST SYSTEM
- Test vessel: Glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume. Each test unit was uniquely identified with the study number, treatment and replicate number.
- Aeration: With compressed air (approximately 0.5 - 1 litre per minute)
- Recording: The room temperature was constantly recorded by a software controlled temperature recording system (AMR Wincontrol).
- No. of vessels per concentration (replicates): nominal 10 mg test item/L (three replicates), 100 mg test item/L (three replicates and 1000 mg/L (one replicate). A previous test revealed, that 1000 mg test item/L had toxic effects on the respiration rate, therefore only one replicate was used.
- Sludge concentration (weight of dry solids per volume): 1.5 g/L susepended solids in test medium

TEST MEDIUM / WATER PARAMETERS
Synthetic sewage :
80 g peptone
55 g meat extract
15 g urea -> filled up to 5 litre with deionised water
3.5 g NaCl
2.0 g CaCl2 · 2H2O
1.0 g MgSO4 · 7H2O
14 g K2HPO4

The pH of this solution was 7.0

OTHER TEST CONDITIONS
- Adjustment of pH: as a previous test revealed, that the test item has a large effect on the pH of the whole test approach, the pH was adjusted with 1M HCl to a pH between 7.4 and 7.6 i

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Test concentrations: 10 mg test item/L; 100 mg test item/L and 1000 mg test item/L


Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
110.5 mg/L
Remarks on result:
other:
Remarks:
The 3-hour ECx values were calculated by cumulative distribution function for the test item and the reference item. The data set of the test item was tested for normal distribution by Shapiro-Wilk´s test (α=0.01), followed by the Levene´s test for variance homogeneity (α=0.01).
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Remarks on result:
other:
Remarks:
The 3-hour ECx values were calculated by cumulative distribution function for the test item and the reference item. For NOEC determination, the Williams Multiple Sequential t-test Procedure was used (α=0.05, one sided smaller). The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1., ® ToxRat Solutions GmbH.
Details on results:
Toxicity of Test Item
Inhibition of Respiration Rate: TThe mean respiration rates of the activated sludge treated with the test item at concentrations of 10 mg/L and 100 mg/L differed by less than 10% from control. The mean deviation in respiration rate was below 0% at a test item concentration of 10 mg/L (n=3) and 6.5% at a test item concentration of 100 mg (n=3) test item/L. A concentration of 1000 mg/L showed inhibiting effects in a previous failure-experiment, therefore only one replicat was tested which resulted in an inhibition of the respiration rate of 67.4%.
The test item at concentrations up to and including 100 mg/L has no biological relevant toxic effects on the respiration activity of activated sludge bacteria. The EC10 determined by statistical analysis is 110.5 mg/L, the statistical determined NOEC is established to be 10 mg/L (Williams Multiple Sequential t-test Procedure).
Although 100 mg test item/L showed a statistical significant difference to the control, the inhibition is very low at this concentration. Therefore it is assumed,
that 100 mg CA3324A/L has no biological relevant toxic effect on the respiration activity of activated sludge bacteria.
Results with reference substance (positive control):
Inhibition of Respiration Rate: The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 1, 4 and 16 mg/L. In comparison to the controls the respiration rate of the activated sludge was inhibited by 30.2% at the lowest nominal concentration of 1 mg/L. At the nominal concentrations of 4 and 16 mg reference item/L, the respiration rate was inhibited by 48.7% and 79.6%, respectively.
3-hour EC50 of 3,5-Dichlorophenol: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 3.5 mg/L. Due to mathematical reasons, no 95 % confidence limits could be determined.

Table 2: Influence of the test item on oxygen consumption of activated sludgeina 3-hour respiration inhibition test

Nominal concentration
(mg CA3324A/L)

Oxygen consumption rate (mg O2/L*h)

Oxygen consumption rate (mg O2/g*h)

Inhibition (%)

pH values

 

Oxygen concentration

(mg O2/L)

Start*

End*

Start*

End*

Control 1

35.14

23.43

-

6.9

7.3

7.0

7.0

Control 2

34.15

22.77

-

6.9

7.3

7.2

6.0

Control 3

33.00

22.00

-

6.9

7.2

6.8

7.5

Control 4

32.40

21.60

-

6.9

7.2

7.1

7.7

Control 5

33.60

22.40

-

7.0

7.3

7.0

7.7

Control 6

30.60

20.40

-

6.9

7.3

7.3

8.0

CA3324A 10 (1)

34.20

22.80

-3.2

6.9

7.3

6.9

7.7

CA3324A 10 (2)

34.80

23.20

-5.0

6.9

7.3

7.3

7.4

CA3324A 10 (3)

35.40

23.60

-6.8

6.9

7.3

7.0

7.8

CA3324A 100 (1)

30.60

20.40

7.7

7.4

7.5

7.1

7.9

CA3324A 100 (2)

32.40

21.60

2.3

7.4

7.5

6.7

8.1

CA3324A 100 (3)

30.00

20.00

9.5

7.4

7.5

6.9

7.6

CA3324A 1000 (1)

10.80

7.20

67.4

7.6

8.2

7.2

8.9

3,5-dichlorophenol 1

23.14

15.43

30.2

6.9

7.7

7.2

6.4

3,5-dichlorophenol 4

17.00

11.33

48.7

6.9

7.7

7.1

7.7

3,5-dichlorophenol 16

6.75

4.50

79.6

6.9

7.3

7.0

8.4

Result Evaluation

Definitions: Respiration rate: the oxygen consumption of waste-water microorganisms in aerobic activated sludge, expressed as mg O2 per litre per hour.

ECx: the calculated concentration of the reference item or test item which results in a x % inhibition of the respiration rate.

NOEC: No Observed Effect Concentration

Determination of the Inhibitory Effects: The inhibitory effect of the test item or the reference item at a particular concentration on the total respiration rate is expressed as percentage of the mean value of the

respiration rates of the six controls according to:

((1 -(R1/meanRc))*100 = % inhibition

where

Rt = total oxygen consumption rate at tested

concentration of test item or reference item

Rc = oxygen consumption rate of controls (mean)

Statistical Analysis (Limit Study): The 3-hour ECx values were calculated by cumulative distribution function for the test item and the reference item. The data set of the test item was tested for normal distribution by Shapiro-Wilk´s test (α=0.01), followed by the Levene´s test for variance homogeneity (α=0.01). For NOEC determination, the Williams Multiple Sequential t-test Procedure was used (α=0.05, one sided smaller). The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1., ® ToxRat Solutions GmbH.

Validity Criteria of the Study

Inoculum Control: The respiration rates of the six controls did not differ by more than 30%. The coefficient of variation was 4.7%. The oxygen uptake in the blank controls was 22.1 mg oxygen/g/h.

Reference Item: The 3-hour EC50 of the reference item 3,5 -Dichlorophenol was determined to be 3.5 mg/L and was thus in the range of 2 to 25 mg/L for the activated sludge batch which was used.

Validity criteria fulfilled:
yes
Conclusions:
The test substance at concentrations up to and including 100 mg/L has no biological relevant toxic effects on the respiration activity of activated sludge bacteria. The EC10 determined by statistical analysis is 110.5 mg/L, the statistical determined NOEC is established to be 10 mg/L (Williams Multiple Sequential t-test Procedure).
Executive summary:

1.1 Study Design

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item on the activity of activated sludge by measuring the respiration rate under defined conditions. The test was conducted in accordance with the OECD Guideline for Testing of Chemicals, No.209: "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)", adopted July 22, 2010.

1.2 Results

The mean respiration rate of activated sludge treated with the test itemat concentrations of 10 mg/L and 100 mg/L differed from the mean respiration rate in the control by less than 10%. The mean inhibition of the respiration rate was -5.0% for 10 mg/L and 6.5% for 100 mg/L.

1.3 Conclusion

The test substance at concentrations up to and including 100 mg/L has no biological relevant toxic effects on the respiration activity of activated sludge bacteria. The EC10 determined by statistical analysis is 110.5 mg/L, the statistical determined NOEC is established to be 10 mg/L (Williams Multiple Sequential t-test Procedure).

Description of key information

Isobutylamine toxicity to activated sludge in a respiration inhibition test was determined according to OECD 209 guideline. The test was conducted undr GLP conditions.

Isobutylamine at concentrations up to and including 100 mg/L has no biological relevant toxic effects on the respiration activity of activated sludge bacteria. The EC10determined by statistical analysis is 110.5 mg/L, the statistical determined NOEC is established to be 10 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information