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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2018 to 06 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with international guidelines in a GLP testing laboratory.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection: 28 November - 6 December 2017 Date of Signature on Certificate: 22 January 2018
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature, in the dark.
- Stability under test conditions:
Stable
- Solubility and stability of the test substance in the solvent/vehicle:
7.9 mg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Weighted amounts of sample were added to test bottles to which 200 mL Milli-RO water was added. The mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added.
- Preliminary purification step (if any):
No
- Final dilution of a dissolved solid, stock liquid or gel:
See above.
- Final preparation of a solid:
N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Solution

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
: N/A

OTHER SPECIFICS: N/A
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- The test item was not sufficiently soluble to allow preparation of an aqueous solution at all a concentration of 10 g/L. Therefore, weighed amounts were directly added to the test bottles to prevent any loss of test item. Subsequently 200 mL Milli-RO water was added (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item - Milli-RO water mixtures were magnetically stirred for a short period and, subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required concentrations (see Table 1 and 2 in Section 'Any more information on materials and methods incl. tables' below). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

Any residual volumes were discarded.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Waterschap Aa en Maas, Hertogenbosch, The Netherlands
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collections; therefore 50 mL of synthetic medium (sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
10 minutes
Hardness:
Not reported
Test temperature:
20 ± 2°C
pH:
Before the addition of sludge: 7.6-7.7
After the three hour exposure period: 8.0-8.4
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentrations: 10, 100, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass
- Aeration: during exposure time
- No. of vessels per concentration (replicates): 5 (final test)
- No. of vessels per control (replicates): 6 (final test)
- No. of vessels per vehicle control (replicates): -
- No. of vessels per abiotic control (replicates): 1 (limit/range finding test)
- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
- Nitrification inhibitor used (delete if not applicable): none
- Biomass loading rate: -
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis: Millipore Corp., Bedford, Mass., USA
- Particulate matter: -
OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment made
- Photoperiod: -
- Light intensity: -
- Details on termination of incubation: -
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of oxygen was performed with multiple oxygen sensors conencted to a BlueBox (GO-Systemelektronic GmbH, Germany), a multichannel measuring and controlling system.
- pH was determined
- The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using fewer concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (4 replicates in final test)
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
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 total respiration
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: 2.8 mg/L in the final test
Reported statistics and error estimates:
Calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.

Calculations were performed with ToxRat Professional v3.2.1 (ToxRat Solutions GmbH, Germany).

Combined Limit/Range-Finding Test

The combined limit/range-finding showed no inhibition, 13% and 2% inhibition of the respiration rate at a concentration of 10, 100 and 100 mg/L respectively. Therefore, the EC50 was above 1000 mg/L.

There was no oxygen uptake from abiotic processes.

All test conditions and acceptability criteria prescribed by the study plan were met.

The results of the combined limit range finding test showed no effect on the respiration rate of the activated sludge. Although the test procedure intended to transfer the test item quantitatively to the test vessels, a slight discolouration of the weighing bottle and cap was observed. To confirm the results of the combined limit range finding test, a limit test was performed with direct weighing of test item in the test vessel.

Table 3: Combined Limit/Range-Finding Test - Respiration Rate/Inhibition, pH Values

Replicate

Concentration (mg/L)

pH

Respiration Rate

% Inhibition respiration rate (mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)1

C 1

0

7.3

7.7

29.95

19.97

 

C 2

0

7.3

7.5

32.83

21.89

 

C 3

0

7.3

7.4

42.50

28.33

 

C 4

0

7.3

7.3

52.92

35.28

 

C 5

0

7.3

7.4

36.74

24.49

 

C 6

0

7.3

7.4

41.61

27.74

 

C Mean

 

 

 

39.43

26.28 (Rc)

 

SD

 

 

 

8.21

5.47

 

CV (%)

 

 

 

21

21

 

 

 

 

 

 

 

 

R 1

1.0

7.3

7.7

36.43

24.29

7.60

R 2

3.2

7.3

7.9

23.88

15.92

39.43

R 3

10

7.3

7.7

11.91

7.94

69.79

R 4

32

7.3

7.7

1.95

1.30

95.05

 

 

 

 

 

 

 

T 1

10

7.3

7.4

40.85

27.23

-3.61

T 2

100

7.3

7.5

34.46

22.97

12.59

T 3a

1000

7.4

7.4

41.74

27.83

-5.87

T 3b

1000

7.4

7.5

36.68

24.45

6.96

T 3c

1000

7.4

7.4

37.87

25.25

3.94

T3 Mean

 

 

 

38.76

25.84 (Rt)

1.68 (It)

 

 

 

 

 

 

 

TA

1000

7.4

7.4

0.00#

0.00

100.00

C: Control

R: Reference item, 3,5-dichlorophenol

T: Test item

TA: Abiotic control

SD: Standard deviation

CV: Coefficient of variation

Rr: Total respiration of test item

Rc: Total respiration in the control

It: % inhibition of total respiration relative to Rc

1: The amount of suspended solids in the final test mixture was 1.5 g/L

#: No respiration, therefore expressed as 0 mg O2/L.h

Limit Test

Inhibition of Respiration Rate

The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and EC50.

In the limit test no inhibition of the respiration rate of the sludge was recorded at a concentration of 1000 mg test item per litre. Therefore the NOEC was >1000 mg/L ad the EC50 was > 1000 mg/L.

Experimental Conditions

The pH in all test vessels, before addition of sludge, was between 7.6 and 7.7. After the 3 hour exposure period the pH was between 8.0 and 8.4.

The temperature continuously measured in the temperature controlled vessels ranged between 20 and 22°C during the test.

Table 4: Final Test - Respiration Rate/Inhibition, pH Values

 

Replicate

Concentration (mg/L)

pH

Respiration Rate

% Inhibition respiration rate (mean value)

Start

End

(mg O2/L.h)

(mg O2/g.h)1

C 1

0

7.6

8.1

27.07

18.05

 

C 2

0

7.6

8.1

30.32

20.21

 

C 3

0

7.6

8.0

35.10

23.40

 

C 4

0

7.6

8.0

36.56

24.37

 

C 5

0

7.6

8.0

33.80

22.53

 

C 6

0

7.6

8.0

33.92

22.61

 

C Mean

 

 

 

32.80

21.86 (Rc)

 

SD

 

 

 

3.48

2.32

 

CV (%)

 

 

 

11

11

 

 

 

 

 

 

 

 

R 1

1.0

7.6

8.3

22.03

14.69

32.83

R 2

3.2

7.6

8.4

14.77

9.85

54.96

R 3

10

7.6

8.4

11.63

7.75

64.54

R 4

32

7.6

8.4

3.91

2.61

88.08

 

 

 

 

 

 

 

T 1

10

7.7

8.1

33.73

22.49

-2.85

T 2

100

7.6

8.1

38.92

25.95

-18.68

T 3

1000

7.6

8.1

31.09

20.73

5.20

T 4

1000

7.6

8.1

26.06

17.37

20.54

T 5

1000

7.6

8.0

35.32

23.55

-7.70

T1-5 Mean

 

 

 

33.02

22.02 (Rt)

-5.44 (It)

C: Control

R: Reference item, 3,5-dichlorophenol

T: Test item

SD: Standard deviation

CV: Coefficient of variation

Rr: Total respiration of test item

Rc: Total respiration in the control

It: % inhibition of total respiration relative to Rc

1: The amount of suspended solids in the final test mixture was 1.5 g/L

#: No respiration, therefore expressed as 0 mg O2/L.h

Acceptability of the Test

1) The mean control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (22 mg oxygen per one gram of activated sludge in the final test). The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test (11%).

2) The EC50 of 3,5 -dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (2.8 mg/L in the final test).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Validity criteria fulfilled:
yes
Conclusions:
The EC50 of the test item was determined to be > 1000 mg/L and the NOEC was determined to be ≥ 1000 mg/L.
Executive summary:

The ability of the test item to adversely affect microbial treatment plants was investigated based on the OECD guideline No. 209 (2010).

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were directly added to the test bottles to prevent any loss of test item. Subsequently Milli-RO water was added. The test item - Milli-RO water mixtures were magnetically stirred for a short period and, subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen concentration was recorded for approximately 10 minutes.

The limit test was performed based on the result of a preceding combined limit/range-finding test. Five replicates of 1000 mg/L and six replicates for an untreated control group were tested.

In the limit test no inhibition of the respiration rate of the sludge observed at a concentration of 1000 mg test item per litre. Therefore, the NOEC was > 1000 mg/L and the EC50 was > 1000 mg/L.

The batch of activated sludge was tested for sensitivity with the reference item 3,5 -dichlorophenol and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The test item was determined to be not toxic to waste water (activated sludge) bacteria at a concentration of ≥ 1000 mg/L (NOEC).

Description of key information

EC50 >1000 mg/L, NOEC ≥ 1000mg/L; OECD 209; Buitenweg S (2019)

Key value for chemical safety assessment

Additional information

The ability of the test item to adversely affect microbial treatment plants was investigated based on the OECD guideline No. 209 (2010).

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were directly added to the test bottles to prevent any loss of test item. Subsequently Milli-RO water was added. The test item - Milli-RO water mixtures were magnetically stirred for a short period and, subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen concentration was recorded for approximately 10 minutes.

The limit test was performed based on the result of a preceding combined limit/range-finding test. Five replicates of 1000 mg/L and six replicates for an untreated control group were tested.

In the limit test no inhibition of the respiration rate of the sludge observed at a concentration of 1000 mg test item per litre. Therefore, the NOEC was ≥ 1000 mg/L and the EC50 was > 1000 mg/L.

The batch of activated sludge was tested for sensitivity with the reference item 3,5 -dichlorophenol and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The test item was determined to be not toxic to waste water (activated sludge) bacteria at a concentration of ≥ 1000 mg/L (NOEC).