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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 December 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:
OECD Guidelines for Testing of Chemicals, No.: 209 Adopted: 22 July 2010.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part C, C.11
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Version / remarks:
Product Properties Test Guideline OCSPP 850.3300 (Public Draft OPPTS 850.6800, April 1996) of the United States Environment Protection Agency (EPA) "Modified Activated Sludge, Respiration Inhibition Test", June 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
not specified
Details on sampling:
Not specified
Vehicle:
no
Details on test solutions:
Because of the low solubility, the test item was added by directly weighing the test item into the bottles (and using ultrasonic bath for 10 minutes). The test formulation was freshly prepared at the beginning of the experiment, in the testing laboratory.

The pH of the solutions was checked but not adjusted due to the direct application of the test substance.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage (see 3.7.2.) per litre and kept aerated at 20 ± 2°C until use.
The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged.
An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined.
Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.32. The activated sludge was used directly after conditioning.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
Not specified
Test temperature:
19.8 – 20.7 °C
pH:
7.05 - 7.91
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
The test was performed at three concentrations of the test item: 10, 100 and 1000 mg/L were used.
Details on test conditions:
CONTROLS
Abiotic Control (A)
In parallel to the Study, abiotic control flasks (A1, A2, A3, A4 and A5) were tested without inoculum, but with the highest concentration of the test item (1000 mg/L nominal concentration), under identical test conditions.

Untreated Control (B)
1-1 member of five replicates (B1, B2, B3, B4 and B5) of the untreated control group (deionised water, synthetic sewage and inoculum, without addition of the test item) was tested in parallel with all of the test series.

Reference Control (REF)
In parallel with the test item, the reference item 3,5-Dichlorophenol (REF1, REF2, REF3, REF4) was tested (at nominal test concentrations of 1.0; 3.2; 10 and 32 mg/L) under identical test conditions.
A stock solution of 3,5-Dichlorophenol was prepared according to the OECD Guideline No. 209 (1.0 g of 3,5-Dichlorophenol dissolved in 1000 mL of water). Warm deionised water and ultrasonication was used to help the dissolution of the reference substance and the volume of the solution was made up to 1.0 L with deionised water, then cooled to room temperature. The pH of the solution was checked and adjusted with NaOH solution (2 mol/L) to pH 7.41.

Test units
Type and size: Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
Identification: Each test flask was uniquely identified with study code, treatment and replicate codes.

Test conditions
Surrounding type: Temperature controlled laboratory
Temperature: 19.8 – 20.7 C
Aeration: With compressed air (about 1 L/min)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.

Administration of the test item
Test Concentrations
The test was performed at three concentrations of the test item: 10 (TI11, TI12, TI13, TI14, TI15), 100 (TI21, TI22, TI23, TI24, TI25) and 1000 mg/L (TI31, TI32, TI33, TI34, TI35) were used.

Performance of the test
Preparations of the test flasks
One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask.
A volume of 12.8 mL synthetic sewage and an adequate amount of the test item were placed into the test flask by direct addition and filled up with deionised water to 200 mL before the start of the incubation. At the start of the test, 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids was added to all flasks, with the exception of the abiotic control flasks.
Abiotic controls in 5 parallels (A1, A2, A3, A4 and A5), and one untreated blank (B1) control was started as the first step of the test. Then at appropriate time intervals of approximately 30 minutes the further test groups were started. The second series were 4 different concentrations (without replicates) of the reference item (REF1, REF2, REF3, REF4) and to the second blank control (B2). The third series was the 5 replicates of the test item concentration of 10 mg/L nominal concentration (TI11, TI12, TI13, TI14 and TI15) and the third blank control (B3). The fourth series was the five replicates of test item concentration 100 mg/L nominal concentration (TI21, TI22, TI23, TI24 and TI25) and the fourth blank control (B4). The final series was the five replicates of test item concentration 1000 mg/L nominal concentration (TI31, TI32, TI33, TI34 and TI35) and the fifth blank control (B5).
In summary, 3 test item concentration series and one abiotic control series were prepared in 5 parallels with one blank control series by series. In parallel with the above test mixtures four reference item concentrations without replicates were also prepared with one blank control.

Measurement of Respiration Rate (Dissolved Oxygen)
For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after about 3 hours incubation time, and was not further aerated. The oxygen concentration was measured for about fifteen minutes, with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

Measurement of pH and temperature
The pH of the test mixtures was determined at the start and at the end of the incubation period in all test vessels. The temperature was measured in the laboratory with a min/max thermometer during the experiment.
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
Details on results:
In comparison to the inoculum controls the inhibition of the respiration rate in the case of the activated sludge was between -0.13 % and -6.24 % in the examined range of
10 – 1000 mg/L of test item.
The average specific respiration rate (RS) of the untreated (Blank) controls (B1-B5) was 21.23 mg O2/L/h/g solid.
The variation coefficient of oxygen uptake rate in above untreated replicates was 2.09 %.
Results with reference substance (positive control):
The following nominal concentrations of the reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as in the case of the test item: 1.0, 3.2, 10.0 and 32.0 mg/L.
In comparison to the controls the inhibition of the respiration rate of the activated sludge was 73.82 % at the highest nominal concentration of 32.0 mg/L.
At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L 18.68 %, 36.39 % and 48.07 % inhibition of the respiration rate was calculated respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 8.37 mg/L with 95% confidence limits of 6.09 – 11.51 mg/L.
Reported statistics and error estimates:
Not specified

Influence of test item on oxygen consumption of activated sludge

Test group

Conc. of test item in test mix (mg/L)

Total oxygen consumption rate (RT) (mg O2/L/h)

Specific respiration rate (RS) (mg O2/L/h/g solid)

Inhib. (%)

pH values#

Temperature (°C) (measured in the Laboratory)*

Variation coefficient of O2consumption rate (%)

ID

Name

Start

End

 

A1

Abiotic

1000

-0.17

-

-

7.08

7.58

19.8/20.7

-

A2

1000

-0.50

-

-

7.05

7.59

-

A3

1000

-0.54

-

-

7.14

7.59

-

A4

1000

-0.21

-

-

7.08

7.60

-

A5

1000

-0.29

-

-

7.15

7.59

-

 

B1

Blank

0.00

31.76

21.17

-

7.05

7.41

19.8/20.7

2.09

B2

0.00

31.44

20.96

-

7.25

7.81

B3

0.00

32.40

21.60

-

7.35

7.89

B4

0.00

31.02

20.68

-

7.41

7.91

B5

0.00

32.63

21.75

-

7.44

7.91

 

REF1

Ref. item

1.00

25.56

17.04

18.68

7.23

7.78

19.8/20.7

-

REF2

3.20

19.92

13.28

36.39

7.24

7.79

-

REF3

10.00

16.20

10.80

48.07

7.24

7.82

-

REF4

32.00

8.00

5.33

73.82

7.24

7.83

-

 

TI1

Test item

10

31.89

21.26

-1.21

7.33

7.88

19.8/20.7

-

TI2

100

31.55

21.04

-0.13

7.39

7.89

-

TI3

1000

33.50

22.33

-6.24

7.49

7.97

-

#: Individual pH values of the BOD flasks, except TI1-3, where the average values are reported

*: Minimum/Maximum temperature (°C) measured in the Laboratory, during the experiment

Validity criteria fulfilled:
yes
Conclusions:
Activated sludge respiration inhibition test (carbon and ammonium oxidation) was carried out with NAUGARD® XL-1 to evaluate the influence of the test item on the activity of activated sludge microorganisms by measuring the respiration rate under defined conditions.

Under the conditions of the Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of NAUGARD® XL-1 was the following:
The 3-hour EC50: > 1000 mg/L

Based on the results of this study, the test item NAUGARD® XL-1 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.
Executive summary:

A laboratory test was carried out with NAUGARD® XL-1 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

 

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

 

Under the conditions of the Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of NAUGARD® XL-1 was the following:

The 3-hour EC50: > 1000 mg/L

 

Based on the results of this study, the test item NAUGARD® XL-1 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.

Description of key information

The 3-hour EC50: > 1000 mg/L

 

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

A laboratory test was carried out with NAUGARD® XL-1 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

Under the conditions of the Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of NAUGARD® XL-1 was the following:

The 3-hour EC50: > 1000 mg/L

Based on the results of this study, the test item NAUGARD® XL-1 had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.