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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 6 September 2018 to 19 March 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:
yes
Remarks:
Test water contained 1.08 mg/L DOC (slightly higher than the requirements, <1mg/L DOC). This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (validity criteria fulfilled).
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
Test water contained 1.08 mg/L DOC (slightly higher than the requirements, <1mg/L DOC). This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (validity criteria fulfilled).
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspection dates: 23-30-31 May 2018 / Signature date: 15 November 2018
Specific details on test material used for the study:
- Storage conditions: To be stored refrigerated (2-8°C) protected from light, stored under nitrogen after first opening.
- Vapour pressure: 1.3 Pa at 20°C and 2.4 Pa at 25°C (OECD Guideline 104, effusion method, LPL 2018)
- Water solubility: 39.7 mg/L (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Log Kow: 3.93 (iSafeRat v1.7 prediction). Experimental study on-going at the date of the present study
- Stability: Stable (less than 10% loss) from pH 2-12 (Firmenich internal data)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test vessels were 1 L cylindrical glass bottles closed with PTFE screw caps. A magnetic stirring bar was placed in each test vessel. Blank controls were prepared with 16 mL synthetic sewage feed and 234 mL test water. Test mixtures were prepared by addition of the required amounts of test item in pre-filled test vessels (with synthetic sewage feed and test water). The 3,5-DCP stock solution was diluted with test water as necessary to obtain the required test concentrations into reference mixtures after filling with synthetic sewage and water. Then, 250 mL of sludge suspension were sequentially added in all test vessels. After addition of the inoculum, the test vessels were immediately closed and the mixtures were stirred under aeration for 3 hours.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Origin: Activated sludge was collected (18/10/2018) from a wastewater treatment plant (Aureilhan, France) receiving predominantly domestic sewage.
Pre-treatment: Upon arrival in the test facility, the sludge was sieved (using a 1 mm2 mesh sieve), washed 4 times with chlorine-free tap water and resuspended in tap water. The activated sludge was aerated and fed with the synthetic sewage feed (35 mL per L of sludge suspension) until the next day (final test). The dry weight of the sludge suspension was determined, then the sludge was diluted in chlorine-free tap water to obtain the required sludge solids concentration of approximately 3.0 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
No data
Test temperature:
Temperatures were between 19.1 and 21.6°C throughout the test (average value: 20.6°C), and complied with the requirements (20°C ± 2°).
pH:
The pH of the reaction mixtures at the end of the test period ranged from 7.554 to 7.890
Dissolved oxygen:
Test water used during the study contained 1.08 mg/L DOC (DOC analysis not performed in compliance with the OECD GLP principles but in accordance with ISO 17025 and NF EN 1484), which
was slightly higher than the requirements laid down in the study plan (< 1 mg/L DOC). This minor deviation was considered not to affect the results of the test as no impact was observed on the blank controls (all validity criteria of the test have been fulfilled).
Salinity:
Not applicable
Conductivity:
No data
Nominal and measured concentrations:
- Nominal concentrations: 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass bottles of approximately 1 L capacity closed with PTFE screw caps, containing 500 mL of reaction mixture. At the end of incubation, samples were withdrawn to measure the rate of decrease of dissolved O2 in completely filled all-glass BOD (Biological Oxygen Demand) bottles of approximately 300 mL capacity.
- Type (delete if not applicable): closed
- Aeration: All suspensions were aerated and stirred during the exposure period.
- No. of vessels per concentration (replicates): 5 replicates bottles per test concentration containing synthetic sewage feed, test water, test item and inoculum (test mixtures)
- No. of vessels per control (replicates): 6 replicates bottles containing synthetic sewage feed, test water and inoculum (blank controls)
- No. of vessels per reference control (replicates): 1 replicate per reference substance concentration containing synthetic sewage feed, test water, reference substance and inoculum (reference mixtures). The 3,5-DCP stock solution was diluted to obtain the following nominal concentrations (spaced by a factor of approximately 1.68): 5.0, 8.4, 14.1, 23.7 and 40.0 mg/L.
- No. of vessels per abiotic control (replicates):
- Sludge concentration (weight of dry solids per volume): The activated sludge concentration in all test, reference and blank mixtures was approx. 1.5 g/L of suspended solids.

TEST MEDIUM / WATER PARAMETERS
The medium was prepared by adding each of the following constituents at the stated amounts to each litre of ultrapure water:
- Peptone: 16g
- Meat extract: 11g
- Urea: 3g
- NaCl: 0.7g
- CaCl2, 2H2O: 0.4g
- MgSO4, 7H2O: 0.2g
- K2HPO4: 2.8g
The pH of this solution was 7.2 at 22.2°C in accordance with the requirements 7.5 ± 0.5. This nutrient solution was refrigerated and stored in the dark before use.

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of the pH.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After the exposure, samples from each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

TEST CONCENTRATIONS
Based on the results of the range-finding test and 3 previous invalid tests (see below), a limit test was performed at 100 and 1000 mg/L in order to determine a concentration with no significant effect and/or to demonstrate that EC50 was higher than 1000 mg/L.
Three other main tests were carried out before the final (valid) study. The first two tests (11/09/2018 and 14/09/2018) were repeated because the results could not be interpreted nor validated since oxygen uptake by the activated sludge was so fast that a meaningful calculation on effect concentration could not be obtained, notably in the blank controls. The third test (12/10/2018) could not be considered as valid, since the oxygen uptake rate in the blank controls were less than 20 mg O2/g/h.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (batch No. A0376465; purity 99%; expiry date 7 August 2020)
Key result
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
Duration:
3 h
Dose descriptor:
LOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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
Details on results:
See Table 6.1.7/1 in "Any other information on results incl. tables".
After 3 hours of exposure, the oxygen uptake observed at 100 mg/L was so fast for 3 replicates that it was not technically possible to plot a graph of oxygen concentration versus time (8 mg O2/L to <
2 mg O2/L were consumed in less than 40 s). Therefore, taking into consideration the results obtained from the first two replicates (average percentage of inhibition was approx. 4%), it was concluded by the Study Director that no significant inhibition on the respiration rate could be observed at 100 mg/L, which was thus considered as the highest test concentration without effect (= NOEC).
A slight effect on the respiration rate of activated sludge was observed at 1000 mg/L (average inhibition: 17.1%). Given these results, the 3-hour EC50 was therefore estimated to be > 1000 mg/L.
Results with reference substance (positive control):
EC50 of 10.1 mg/L for 3,5-DCP (within the prescribed range of 2 to 25 mg/L for total respiration).
Reported statistics and error estimates:
Statistical analyses were performed by the software ToxRat® Professional

Table 6.1.7/1: Test results (oxygen uptake rate, inhibition, and pH at the end of the test)

Content

Concentration

(mg.L-1)

Oxygen uptake rate

(mg O2.g-1.h-1)

Inhibition (%)

pH

Control (1)

0

31.436

-

7.874

Control (2)

0

31.671

-

7.870

Ref (1)

5.00

23.727

30.672

7.867

Ref (2)

8.40

23.230

32.126

7.813

Ref (3)

14.20

10.680

68.794

7.842

Ref (4)

23.80

4.576

86.628

7.890

Ref (5)

40.00

7.180

79.020

7.802

Control (3)

0

34.017

-

7.797

Control (4)

0

32.791

-

7.819

Test item

100.60

35.983

-5.135

7.627

Test item

100.16

29.830

12.843

7.583

Test item

100.54

n.d.

n.d.

7.554

Test item

100.10

n.d.

n.d.

7.570

Test item

100.50

n.d.

n.d.

7.592

Test item

1000.62

25.719

24.854

7.613

Test item

1002.48

26.208

23.424

7.646

Test item

1000.84

30.979

9.485

7.591

Test item

1001.44

29.508

13.782

7.565

Test item

1001.36

29.489

13.839

7.648

Control (5)

0

37.129

-

7.755

Control (6)

0

38.309

-

7.697

n.d.: not determined

Validity criteria fulfilled:
yes
Remarks:
See Overall remarks
Conclusions:
The possible effect of test substance on microorganisms from activated sludge was investigated in a respiration inhibition test. Under the experimental conditions and based on nominal concentrations, the 3-hour EC50 value was determined to be higher than 1000 mg/L. The NOEC value was 100 mg/L which was the second highest concentration tested. The LOEC was 1000 mg/L.
Executive summary:

A study was performed to assess the effects of test substance on microorganisms from activated sludge by performing a respiration inhibition test., according to OECD Guideline 209 and EU Method C.11 with GLP compliance.

A limit test was performed following the results of a range-finding test. Microorganisms from activated sludge were exposed to the test item at nominal concentrations of 100 and 1000 mg/L (five replicates for each concentration) and to six blank controls in order to determine a concentration with no significant effect and to demonstrate that EC50 was higher than 1000 mg/L. Furthermore, an exposure of the test organism to chemical 3,5-dichlorophenol (3,5-DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g/L of suspended solids. After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD

bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

The validity of the test was demonstrated by an oxygen uptake rate in the blank controls highernthan 20 mg O2/g/h, a coefficient of variation of oxygen uptake rate in control replicates below 30% and an EC50 of 10.1 mg/L for 3,5-DCP (within the prescribed range of 2 to 25 mg/L for total respiration).

After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 1000 mg test item/L (average inhibition: 17.1%). No effect on the respiration rate was observed at 100 mg/L.

Under the experimental conditions and based on nominal concentrations, the 3-hour EC50 value was determined to be higher than 1000 mg/L. The NOEC value was 100 mg/L with a LOEC at 1000 mg/L. An EC10 could not be estimated.

Description of key information

OECD 209, EU Method C11, GLP, key study, validity 1:

3h-NOEC (microorganisms), total respiration = 100 mg/L based on nominal concentration.

3h-EC50 (microorganisms), total respiration > 100 mg/L based on nominal concentration.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

One experimental study is available to assess the effects of the registered substance on microorganisms from activated sludge by performing a respiration inhibition test., according to OECD Guideline 209 and EU Method C.11 with GLP compliance. Microorganisms from activated sludge were exposed to the registered substance at nominal concentrations of 100 and 1000 mg/L (five replicates for each concentration) and to six blank controls in order to determine a concentration with no significant effect and to demonstrate that EC50 was higher than 1000 mg/L. Furthermore, an exposure of the test organism to chemical 3,5-dichlorophenol (3,5-DCP) was performed in order to check the sensitivity of the batch of activated sludge used on the day of exposure. All the mixtures contained an activated sludge concentration of about 1.5 g/L of suspended solids. After 3 hours of exposure under aeration and stirring, samples of each mixture were poured into BOD bottles to continuously measure the oxygen concentration using an oxygen electrode and determine the rate of decrease of dissolved O2. The measurement of total oxygen uptake inhibition was determined only (carbon and ammonium oxidation not separated).

The validity of the test was demonstrated by an oxygen uptake rate in the blank controls higher than 20 mg O2/g/h, a coefficient of variation of oxygen uptake rate in control replicates below 30% and an EC50 of 10.1 mg/L for 3,5-DCP (within the prescribed range of 2 to 25 mg/L for total respiration).

After 3 hours of exposure, a slight effect on the respiration rate of activated sludge was observed at 1000 mg test item/L (average inhibition: 17.1%). No effect on the respiration rate was observed at 100 mg/L. In conclusion, the 3-hour EC50 value was determined to be higher than 1000 mg/L. The NOEC value was 100 mg/L with a LOEC at 1000 mg/L. An EC10 could not be estimated.