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Reference
Endpoint:
toxicity to microorganisms, other
Remarks:
activated sludge respiration/nitrification inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 15 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test item was directly weighed into test vessels to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours. For the nitrification setup 2.32 g N-allylthiourea (ATU-solution) were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period.
- Controls: 6 replicates without ATU and 4 replicates with ATU
- Other: Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8. 8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C. To determine the heterotrophic oxidation four additional controls and two replicates with the test item concentration 100 mg/L, all containing 1.25 mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L, were prepared.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture: no
- Name and location of sewage treatment plant where inoculum was collected: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim), date of collection: 06 Feb 2017
- Method of cultivation: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- Preparation of inoculum for exposure: Sludge was settled and the supernant was decanted. The sludge was centrifuged and the supernant was decanted. A portion of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and diluted with deionised water.
- Storage: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
19.3 - 19.9 °C
pH:
8.3 - 8.4
Nominal and measured concentrations:
nominal: control and 100 mg/L
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: closed (with air inlet and outlet)
- Material, size, headspace, fill volume: 300 mL glass Erlenmeyer flasks, fill volume: 250 mL, headspace: 50 mL
- Aeration: permanent aeration (at 50-100 L/h with clean oil-free air)
- No. of vessels per concentration (replicates): 100 mg/L without ATU: 3 replicates; 100 mg/L with ATU, 2 replicates
- No. of vessels per control (replicates): 6 replicates without ATU; 4 replicates with ATU
- No. of vessels per abiotic control (replicates): yes, 1 replicate
- Sludge concentration (weight of dry solids per volume): 800 mg/L suspended solids
- Nutrients provided for bacteria: For each liter of water - 16.0 g peptone, 11.0 g meat extract , 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O , 0.2 g MgSO4 x 7H2O, and 2.8 g K2HPO4
- Nitrification inhibitor used: N-allylthiourea

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: without ATU: to pH 7.2 - 7.4; with ATU: to pH 7.2 - 7.3
- Details on termination of incubation: For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode)

EFFECT PARAMETERS MEASURED: microbial activity was measured based on the oxygen consumption of the total, hetertrophic and nitrifying microbial community after aeration time of 3 h

TEST CONCENTRATIONS: control and 100 mg/L

- Range finding study: no range-finding test was performed
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Blank controls respiration rate: 28.290 [mg/L*h] (with ATU 29.772 [mg/L*h])
- Coefficient of variation of oxygen uptake rate in control replicates: 3.7%
-Other: Results are summarized within the tables 1-2 in section "Any other information on results incl tables"
Results with reference substance (positive control):
- Results with reference substance valid? The EC50 of the reference compound 3,5-Dichlorophenol should be in the range of 2 - 25 mg/L for total respiration. In the present test the EC50 of the reference compound was 20.171 mg/L; thus the test is valid.
Reported statistics and error estimates:
Student-t test for Homogeneous Variances

Results of ATU set-up for oxygen consumption based on nitrification:

As no inhibition was observed for the total oxygen consumption at 100 mg/L no differences between the heterotrophic and the nitrification oxygen uptake rates have to be calculated.

Results of abiotic control:

As nearly no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations.

Table 1: Test without ATU (total respiration): Respiration rates after 3 hours incubation period, percentage inhibition, temperature and pH values

Treatment [mg/L]

Respiration rate

[mg/L · h]

Mean Temp.

[°C]

pH-

value

Inhibition

[%]

Control 1

--

28.789

19.8

8.4

--

Control 2

--

27.332

19.5

8.4

--

Control 3

--

27.193

19.3

8.4

--

Control 4

--

28.601

19.3

8.4

--

Control 5

--

27.823

19.4

8.4

--

Control 6

--

29.999

19.4

8.4

--

Control, mean

28.290

--

--

--

 

Test item

100

31.133

19.9

8.3

0.000

Test item

100

26.576

19.7

8.4

6.057

Test item

100

28.183

19.4

8.4

0.377

Test item, mean

100

28.631

--

--

2.145

Physico-chemical oxygen consumption control

100

0.497

19.5

7.3

--

Reference compound

2.5

30.591

19.1

8.4

0.000

Reference compound

5

25.165

19.0

8.5

11.046

Reference compound

10

20.013

19.3

8.4

29.257

Reference compound

20

10.994

19.5

8.3

61.138

Reference compound

40

8.097

19.4

8.4

71.378

 

Table 2: Test with ATU (heterotrophic respiration): Respiration rates after 3 hours incubation period, percentage inhibition, temperature and pH values

Treatment [mg/L]

Respiration rate

[mg/L · h]

Mean Temp.

[°C]

pH- value

Inhibition [%]

Control 1

 

29.657

19.4

8.4

--

Control 2

 

32.330

19.3

8.4

--

Control 3

 

27.897

19.3

8.4

--

Control 4

 

29.205

19.5

8.4

--

Control, mean

 

29.772

--

--

--

Test item

100

29.010

19.4

8.4

2.561

Test item

100

28.551

19.1

8.4

4.102

Test item, mean

100

28.780

--

--

3.331

 

Validity criteria fulfilled:
yes

Description of key information

EC50 (3 h) > 100 mg/L (activated sludge, OECD 209)

NOEC (3 h) ≥ 100 mg/L (activated sludge, OECD 209)

Key value for chemical safety assessment

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

Additional information

One experimental study was conducted in accordance with OECD Guideline 209 and EU Methode C.11 under GLP conditions (M-585194-01-1). The activated sludge was exposed to the test item at a limit test item concentration of 100 mg/L without ATU (N-allylthiourea) representing the total microbial community and with ATU representing the heterotrophic part of the microbial community. By subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification are calculated. The respiration rate of each mixture was determined after aeration periods of 3 hours. The test item showed 2.1% respiration inhibition of activated sludge at a test item concentration of 100 mg/L. No physico-chemical oxygen consumption was observed at that test item concentration. As no inhibition was observed for the total oxygen consumption at 100 mg/L no differences between the heterotrophic and the nitrification oxygen uptake rates have to be calculated. The EC50 (3 h) is higher than 100 mg/L. The NOEC (3 h) is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.