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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Jan 2018 to 18 Apr 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))
Version / remarks:
July 2010
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): NDBC
- Batch No.: 70900202
- Source: Performance Additives Italy S.p.A.
- Date of receipt: 3 Oct 2017
- Storage conditions: Upon receipt, the NDBC was stored at ambient room temperature in designated test substance storage room.
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Easton Wastewater Treatment Facility, Easton, Maryland
- Preparation of inoculum for exposure: Prior to use, the sludge was allowed to settle for 30 minutes and the supernatant above the settled solids was removed. Total suspended solids (TSS) concentration of the sludge was determined. The inoculum was prepared by diluting of an aliquot of the settled sludge with an aliquot of deionized (reverse osmosis) water to achieve target TSS concentration of ~3000 mg/L. The inoculum was maintained at an average temperature of 20 ± 2 °C, continuously aerated, and used for testing the same day. The pH and TSS concentration of the inoculum were determined prior to use on the day of the study.
- Pretreatment: The sludge was sieved using a 2 mm screen and was kept under aeration in temperature controlled room. 50 mL of synthetic sewage per liter of the sludge was added at the end of each day.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19.6 - 21.3 °C
pH:
7.38 at t=0
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 62.5, 125, 250, 500, and 1000 mg/L
Details on test conditions:
TEST PREPARATION
Test mixtures were prepared at 15 minute intervals starting with the first control. Each control vessel contained 16.0 mL of synthetic sewage, 234 mL of deionized water, and 250 mL of inoculum for a total volume of 500 mL. The mixture was promptly aerated at a rate sufficient to keep the solids in suspension. Subsequent reference and test substance dosed biotic mixtures were prepared by combining 16.0 mL of synthetic sewage, the appropriate volume of reference substance stock solution or appropriate amount of test substance and an appropriate volume of deionized water needed to achieve total volume of 250 mL. After the addition of deionized water, 250 mL of inoculum was added into each test vessel to bring the total volume to 500 mL and the vessel was placed under aeration for three hours prior to start DO measurements.

TEST SYSTEM
- Test vessel: The mixtures were prepared and aerated in 1000-mL Erlenmeyer flasks and then transferred to fill 300-mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements.
- Fill volume: 500 mL
- Aeration: Yes
- No. of vessels per concentration: 3
- No. of vessels per control : 2
- No. of vessels per abiotic control: 3
- No. of vessels per reference control: 3
- Total suspended solids concentration: 3273 mg/L (average of three replicates at t=0)
- Sludge concentration (weight of dry solids per volume): 1.64 mg/L

NUTRIENT SOLUTION
The synthetic sewage was prepared by dissolving the following amounts of substances in 1 liter of deionized water:
- Peptone: 16.0
- Meat extract: 11.0
- Urea: 3.0
- NaCl: 0.7
- CaCl2.2H2O: 0.4
- MgSO4.7H2O: 0.2
- K2HPO4: 2.8
The constituents of the synthetic sewage are not known to contain any contaminants that are reasonably expected to be present and are known to be capable of interfering with the study.

EFFECT PARAMETERS MEASURED: oxygen consumption
After 3 hours of aeration, the contents of the first control vessel was transferred to a BOD bottle and dissolved oxygen concentration was measured every 10 seconds over a 10 minute period or until the DO measurement dropped below 2.0 mg O2/L using a YSI Model 5000 Dissolved Oxygen Meter. The respiration rate in all other vessels was determined in an identical manner at 15 minute intervals. The contact time for each concentration of reference or test substance with the inoculum was three hours.

REFERENCE SUBSTANCE STOCK SOLUTION
502 mg/L stock solution of the reference substance, 3,5-dichlorophenol, was prepared by dissolving 0.5022 g of the reference substance in 10 mL of 1N NaOH and then adding 20 mL of deionized (GenPureTM) water. While stirring, approximately 10 mL of 1N H2SO4 was added to reach the point of incipient precipitation. The solution of 3,5-dichlorophenol was then diluted to the final volume of 1 L with GenPureTM water. The pH of the solution was adjusted by adding 1N H2SO4 and the flask was filled up to the volume with GenPureTM water. The final pH of the reference substance stock solution was 7.22. The reference substance solution was administered to the reference group by volumetric addition. Volumetric addition is the most appropriate route of administration of aqueous solutions.

PRELIMINARY TEST
- Method: Preliminary test was performed using procedure similar to the procedure of definitive test specified below but at only 3 test substance concentrations, 10, 100, and 1000 mg/L. Testing at 10 and 100 mg/L was performed with one replicate and testing at 1000 mg/L was conducted in triplicate with an abiotic control.
- Results: In preliminary test, respiration rates in the two controls were 36.38 and 51.81 mg O2/L/h corresponding to 22.1 mg O2/g sludge solids/h and 31.5 mg O2/g sludge solids/h, respectively, based on concentration of the sludge solids in the text mixtures of 1.64 g/L. Both values were greater than 20 mg O2/g sludge solids/h, which is considered as the low limit of validity of the test.
The respiration inhibition rates for the 3,5-dichlorophenol reference group provided EC50 value of 24.95 mg/L, with 95% confidence limits of 21.7 mg/L and 28.4 mg/L. This EC50 value was within the 2 to 25 mg/L range considered acceptable for the test.
The respiration rates in biotic test mixtures dosed with the test substance at concentrations of 10 and 100 mg/L were 42.9 and 37.3 mg O2/L/h, respectively. Compared to the untreated control test mixtures, respiration inhibition of the test substance treated test vessels at concentrations of 10 and 100 mg /L were 2.6% and 15.5%, respectively. Average (of 3 replicates) respiration rate in biotic test mixture dosed with the test substance at 1000 mg/L was 35.7 mg O2/L/h and average respiration inhibition was 19%.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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
Remarks on result:
other: derived by the registrant
Duration:
3 h
Dose descriptor:
other: EC15
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The respiration rates in biotic test mixtures dosed with the test substance at concentrations of 62.5 to 1000 mg/L ranged from 31.2 to 41.8 mg O2/L/h. Compared to the untreated control test mixtures, respiration inhibition of the test substance treated test vessels at all tested concentrations ranged from -20.4% to 9.8%. The average value of respiration inhibition over all studied range of concentrations was -2.0% ± 8.45%. Thus, the test substance provided no inhibition at the dose levels from 62.5 to 1000 mg/L dose levels. A detailed overview of the results is tabulated in 'Any other information on results incl. tables'.
Results with reference substance (positive control):
For the reference substance, an 3-h EC50 value of 12 mg/L, with 95% confidence limits of 8.7 mg/L and 22.7 mg/L.

Table: Respiration Rates and Percent Inhibition

Treatment

Replicate

Nominal concentration (mg/L)

Respiration rate (mg O2/L/h)

Percent Respiration Inhibition (%)

Average Respiration Rate (mg O2/L/h)

Average Respiration Inhibition (%)

Control 1

 

0

29.40

NA

34.5

NA

Control 2

 

0

39.60

NA

Reference

 

3

26.89

22.0%

 

 

Reference

 

15

15.81

54.2%

 

 

Reference

 

50

5.17

85.0%

 

 

Test substance

1

62.5

33.51

5.5%

33.8

5%

Test substance

2

62.5

33.00

7.0%

Test substance

3

62.5

34.84

1.6%

Test substance

Abiotic Treatment

62.5

0.90

 

 

 

Test substance

1

125

35.61

-2.5%

38.0

-9%

Test substance

2

125

36.64

-5.5%

Test substance

3

125

41.80

-20.4%

Test substance

Abiotic Treatment

125

0.26

 

 

 

Test substance

1

250

36.99

-7.0%

35.0

-1%

Test substance

2

250

36.85

-6.6%

Test substance

3

250

31.20

9.8%

Test substance

Abiotic Treatment

250

0.06

 

 

 

Test substance

1

500

31.46

9.6%

33.2

4%

Test substance

2

500

33.03

5.0%

Test substance

3

500

35.26

-1.4%

Test substance

Abiotic Treatment

500

0.26

 

 

 

Test substance

1

1000

37.00

-6.3%

37.7

-8%

Test substance

2

1000

37.95

-9.1%

Test substance

3

1000

38.23

-9.9%

Test substance

Abiotic Treatment

1000

0.32

 

 

 

Calculations were performed in Excel in full precision mode.

Calculations based on rounded numbers presented in the table may provide slightly different results.

NA = Not Applicable.

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. tables'.

Description of key information

The 3-h EC50 and 3-h EC15 values are > 1000 mg/L as observed in a respiration inhibition study with activated sludge of a domestic sewage. The derived 3-h NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of the substance to microorganisms was investigated in an activated sludge respiration inhibition study according to OECD TG 209 and in compliance with GLP criteria (EAG, Inc., 2018). In this study, non-adapted activated sludge from predominantly domestic sewage was exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/L for 3 hours. Test concentrations were not analytically verified. The effects on microorganisms was determined by measuring total respiration rates and comparing these to an unexposed control. No inhibition of respiration rates was observed up to the highest concentrations. Therefore, the 3-h EC50 and 3-h EC15 values were both determined to be > 1000 mg/L. The derived 3-h NOEC was 1000 mg/L.