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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 August- 25 September, 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:
July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1st March 2016
Deviations:
no
Guideline:
other: Ecological Effects Test Guidelines of the United States Environmental Protection Agency (EPA 712-C-014): OCSPP 850.3300, "Modified Activated Sludge, Respiration Inhibition Test", January 2012.
Version / remarks:
January 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Date of production: 28 July 2015
Expiration date: 28 July 2020
Analytical monitoring:
no
Details on sampling:
The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen concentration measuring) at a temperature of 20 +/- 2 °C according to the guideline. The recorded temperatures in the environmental room varied between: 20.3-21.6 °C.
The test flasks were aerated with compressed air (0.5 L/minute).
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations, reference item concentrations and controls. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.
The test conditions were measured with suitable instruments and documented in the raw data.


Vehicle:
no
Details on test solutions:
For correct administration, ~3 g test item was ground with a pestle and mortar (as fine as possible). Thereafter defined amounts of the ground test item (1 x 3; 1 x 30; 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly weighed (administered) into each
test flask. The subsequent calculations refer to the initial weighed nominal concentration.
Concentrations in excess of nominal 1000 mg test item/L were not tested.



Test organisms (species):
activated sludge, domestic
Details on inoculum:
Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 07 August 2017 (two days before the test).
Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study
was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (6.613 g wet weight), dried and the ratio of wet sludge to dry weight (0.5668 g dry weight)
determined. Based on this ratio, calculated amount of wet sludge (60 g dry weight that was equivalent to 700 g wet sludge) was suspended in isotonic saline solution (ad. 20 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 48 hours (2 days) and fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.31), pH adjustment of the inoculum was considered not necessary.

Test type:
static
Water media type:
other: deionized
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen concentration measuring) at a temperature of 20 +/- 2 °C according to the guideline. The recorded temperatures in the environmental room varied between: 20.3-21.6 oC.

The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.
pH:
The test was performed without pH adjustment.

The pH was determined in all test vessels before the inoculum addition, at the start (just after the inoculation) and at the end of the incubation period in all test concentrations, reference item concentrations and controls.
Dissolved oxygen:
The oxygen concentrations was determined in all test vessels before the inoculum addition, at the start (just after the inoculation) and at the end of the incubation period in all test concentrations, reference item concentrations and controls.
Nominal and measured concentrations:
Nominal concentration: 10, 100 and 1000 mg/L
Details on test conditions:
Equipment

Normal laboratory equipment were used in the study,
Complex measuring system with O2 electrode and pH electrode
and built in temperature probe
Aeration system,
Orbital shaker
Min/Max. Thermometer,
Balance,
Centrifuge,
Moisture content analyzer

The Preliminary Experiment

The following test item properties were investigated in the pre-experiment: the test item effect on the pH within the test system and the test item effect on the oxygen consumption of the activated sludge inoculum.
This preliminary test was designed according to the guideline requirements, triplicates were examined at the highest tested concentration of 1000 mg/L (as a limit concentration) and additionally two lower concentration levels of 10 and 100 mg/L were examined with one vessel each. Blank, abiotic (three abiotic controls with the highest concentration of the test item) and reference controls were included.
The nitrification potential of sludge was examined with additional control mixture (nitrification control in three parallels) that contained N-allylthiourea at 11.6 mg/L.
The test media were prepared freshly before the test.

Test Mixtures

For correct administration, ~3 g test item was ground with a pestle and mortar (as fine as possible). Thereafter defined amounts of the ground test item (1 x 3; 1 x 30; 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations) were directly weighed (administered) into each test flask. The subsequent calculations refer to the initial weighed nominal concentration.
Concentrations in excess of nominal 1000 mg test item/L were not tested.


Controls

Blank Control (CB):
In this preliminary range-finding test eight replicates of blank control group (water, synthetic sewage and inoculum, but without addition of the test or reference item) were tested concurrently, four at the start and four at the end of the test series.
Abiotic Control (CA):
Abiotic controls (water, synthetic sewage and the test item at the concentration of 1000 mg/L, but without inoculum) were prepared to measure the abiotic oxygen consumption. In this preliminary range-finding the abiotic control was tested at the highest test item concentration (for that reason 3 x 300 mg ground test item was directly weighed into assigned containers) in three parallels.
Reference Control (R):
Concurrent to the test item, the reference item 3,5-Dichlorophenol was tested at three concentrations each with three parallels (the nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions.
Nitrification Control (CN):
In order to decide whether the sludge nitrifies and at what rate, mixtures in three parallels (same as the blank controls, however containing 11.6 mg/L N-allylthiourea) were included in the preliminary experiment.

Test solution with a final volume of 300 mL was tested per treatment in a glass flask. 9.6 mL synthetic sewage and an adequate volume of the stock solution of the N-allylthiourea adequate volumes of the stock solution of reference item were filled up with water (with purified, deionized water) to 150 mL before the start of the test. The composition and order of the test media are summarized in the Table 2. At the test item treatments the 3, 30 and 3 x 300 mg (respectively) test item was measured directly into the empty test containers. At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added to the test containers, first to the blank controls (CBA, thereafter CBB, CBC and CBD the “start” CB group), then to the nitrification controls (CNA, CNB, CNC), to the test solutions of the reference item (R1(A-C), R2(A-C), R3(A-C)) and to the test item (containing the adequate amount of test item, the 150 mL synthetic sewage-water mixture that was filled into the test containers just before the inoculation); and finally to the “end” CB group (CBE, CBF, CBG, and CBH).

The abiotic controls were started after the test item treatments with the administration of the test item (3 x 300 mg) only, without activated sludge inoculum.
In time intervals of approximately 12 minutes (an arbitrary but convenient interval) a maximum of four test vessels were started.
Due studies run in parallel and common controls between the common “start” and “end” controls and test item groups of the present study (T1 T3 and abiotic controls, Table 2), test item containers from other study (not further detailed in this report) were started and examined.
The test was performed using four oxygen electrodes that allow the simultaneous testing of four test vessels.
The difference between “start” and “end” blank control respiration rates (CBA, CBB, CBC and CBD beginning and CBE, CBF, CBG, and CBH end of the exposure period, respectively) were minimal, within the biological variability of the applied system.
The test flasks were incubated for 3 hours. The test vessels were aerated and shaken continuously such as to ensure an appropriate dissolved O2 level in the samples.
In this study analytical measurements were not performed.

Measurement of Respiration Rates
The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time. The treatment sample was not further aerated. The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 5.5-10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason four O2 electrode were used. Simultaneous (a maximum of four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in 7 cycles with the available four O2 electrodes. The oxygen consumption rate (in mg O2 L-1 hour-1) was determined from the linear part of the respiration curve (in the range between approximately 2 7 mg O2/L).

Statistical Analysis

The EC50 value of the reference item was calculated using Probit analysis.
The analysis was done using the IBM® SPSS® Statistics, Version 24 (2016) statistical software program.
The specific respiration rates of the highest test item concentration level were compared to the blank control values by 2 Sample t-Test (2-sided, α=0.05) by IBM® SPSS® Statistics, Version 24 (2016) software.



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:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
Validity of the Study

The specific respiration rate of the blank controls (without the test substance or reference substance) was 33.69 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 2.87 %.
The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 13.87 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

10.2 Specific Respiration Rates in the Test Item Concentrations

Defined amounts of the test item (1 x 3, 1 x 30, 3 x 300 mg in the test item treatments and 3 x 300 mg in the abiotic controls) were directly added in each test container, and after a pH measurement inoculated. The test item was investigated up to and including the concentration of 1000 mg/L without any pH adjustment before the inoculum addition. Analytical verification of test concentrations was not carried out.

In the experiment abiotic oxygen consumption of the test substance was not observed (in average 0.05 mg/Lh, see Table 4); therefore the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations. The measured nitrification potential of the applied activated sludge (see nitrification control values) did not necessitate further testing. The influence of Blue Sema on the oxygen consumption and specific respiration rate of activated sludge is presented in Tables 4-5.
The observed oxygen consumption rates and consequently the specific respiration rates were in the range of the blank controls. No inhibitory effect of the test item was observed. The observed 0.06 % at 10 mg/L, -0.61 % (stimulation) at 100 mg/L and in average 2.43 % at 1000 mg/L inhibitions were within the biological variability of the applied test system.
The specific respiration rates of the highest dose, 1000 mg/L were compared with the blank control values using 2 Sample t-Test (2-sided, α=0.05). No statistical significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/L.
Based on measured oxygen consumption values and calculated specific respiration rates it is concluded that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L.
The EC50 value was determined as: EC50 > 1000 mg/L.
Results with reference substance (positive control):
The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 7.13 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 27.09 % and 68.57 %, respectively. The results are summarized in Appendix I, Tables 4 and 5.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 13.87 mg/L, (95 % confidence limits: 11.23-17.87 mg/L).

The Oxygen Concentrations and pH Values in the Test Item Groups and Controls

Identifi-cation

Concentration
(mg/L)

Oxygen concentration
(mg O2/L)

pH

Start of the 3-hour aeration

End of the 3-hour aeration

Before the addition of inoculum 

Start of the 3-hour aeration

End of the 3-hour aeration

CBA

0.00

7.95

7.62

7.60

7.79

8.13

CBB

7.72

7.52

7.60

7.79

8.22

CBC

7.62

7.55

7.59

7.81

8.18

CBD

7.62

7.57

7.59

7.82

8.14

CBE

7.86

7.76

7.55

7.87

8.22

CBF

7.68

7.31

7.56

7.88

8.30

CBG

7.73

7.57

7.56

7.91

8.20

CBH

7.81

7.50

7.57

7.90

8.03

CNA

11.6 mg
ATU/L

7.87

7.55

7.60

7.86

8.17

CNB

7.70

7.77

7.60

7.87

8.10

CNC

7.72

7.28

7.59

7.86

8.05

R1A

2 mg
3,5-DCP/L

7.69

7.33

7.59

7.87

8.12

R1B

7.87

7.10

7.62

7.87

8.21

R1C

7.74

7.77

7.60

7.83

8.24

R2A

7 mg
3,5-DCP/L

7.82

7.37

7.61

7.86

8.25

R2B

7.78

7.37

7.60

7.88

8.11

R2C

7.63

7.19

7.58

7.81

8.18

R3A

24.5 mg
3,5-DCP/L

7.80

7.57

7.63

7.86

8.10

R3B

7.91

7.15

7.63

7.86

8.22

R3C

7.70

7.17

7.63

7.87

8.23

T1/A

10 mg
Test Item/L

8.24

7.33

7.56

7.93

8.12

T2/A

100 mg
Test Item/L

7.89

7.91

7.41

7.76

8.03

T3/A

1000 mg
Test Item/L

7.92

7.43

4.93

7.49

7.95

T3/B

7.92

7.44

4.91

7.47

7.96

T3/C

7.92

7.72

4.93

7.87

8.04

CA1

1000 mg
Test Item/L

8.04

7.57

‑ *

5.16

4.92

CA2

8.03

7.35

‑ *

4.96

4.88

CA3

8.04

7.87

‑ *

4.95

4.79

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

Remarks:         *     At the Abiotic controls no inoculum was added; therefore one pH measurement was carried out at the start of the test.

The Q1, Q2 and the applied Δt valuesin the Test Item and Control Groups; theOxygen Consumption Rate (R), and % Inhibition of R

Identifi-cation

Concentration
(mg/L)

Oxygen concentration
(mg O2/L)

Δt (min)

Oxygen Consumption Rate (R)
(mg O2/Lh)

Average R
(mg O2/Lh)

Inhibition of R (%)

Q1

Q2

CBA

0.00

7.20

2.04

6

51.60

50.53

0.00

CBB

7.08

2.40

5.5

51.05

CBC

7.12

2.38

5.5

51.71

CBD

7.18

2.24

6

49.40

CBE

7.36

2.19

6.5

47.72

CBF

7.31

2.29

6

50.20

CBG

7.13

2.35

5.5

52.15

CBH

7.08

2.04

6

50.40

CNA

11.6 mg
ATU/L

7.14

2.19

6.0

49.50

49.67

1.71

CNB

7.35

2.34

6

50.10

CNC

7.28

2.34

6

49.40

R1A

2 mg
3,5-DCP/L

7.33

2.37

6.5

45.78

46.93

7.13

R1B

7.10

2.28

6

48.20

R1C

7.00

2.32

6

46.80

R2A

7 mg
3,5-DCP/L

7.06

2.11

8

37.13

36.84

27.09

R2B

7.06

2.14

8

36.90

R2C

7.19

2.02

8.5

36.49

R3A

24.5 mg
3,5-DCP/L

7.57

4.83

10

16.44

15.88

68.57

R3B

7.15

4.19

10

17.76

R3C

7.17

4.93

10

13.44

T1/A

10 mg
Test Item/L

7.33

2.28

6

50.50

50.50

0.06

T2/A

100 mg
Test Item/L

7.06

2.40

5.5

50.84

50.84

-0.61

T3/A

1000 mg
Test Item/L

7.02

2.05

6

49.70

49.30

2.43

T3/B

7.03

2.16

6

48.70

T3/C

7.30

2.35

6

49.50

CA1

1000 mg
Test Item/L

7.57

7.61

8.5

-0.28

0.05

99.91

CA2

7.35

7.37

8.5

-0.14

CA3

7.87

7.79

8.5

0.56

Q1:             the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);

Q2:             the oxygen concentration at the end of the selected section of the linear phase (mg/L);

Δt:              the time interval between these two measurements (min.).

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

The Specific Respiration Rate (RS) in the Test Item and ControlGroups

Identification

Concentration
(mg/L)

Specific Respiration Rate
(mg O2/gh)

Average RS
(mg O2/gh)

CBA

0.00

34.40

33.69

CV(%)= 2.87

CBB

34.04

CBC

34.47

CBD

32.93

CBE

31.82

CBF

33.47

CBG

34.76

CBH

33.60

CNA

11.6 mg
ATU/L

33.00

33.11
CV(%)= 0.82

CNB

33.40

CNC

32.93

R1A

2 mg
3,5-DCP/L

30.52

31.29
CV(%)= 2.58

R1B

32.13

R1C

31.20

R2A

7 mg
3,5-DCP/L

24.75

24.56
CV(%)= 0.87

R2B

24.60

R2C

24.33

R3A

24.5 mg
3,5-DCP/L

10.96

10.59
CV(%)= 13.94

R3B

11.84

R3C

8.96

T1/A

10 mg
Test Item/L

33.67

33.67

T2/A

100 mg
Test Item/L

33.89

33.89

T3/A

1000 mg
Test Item/L

33.13

32.87NS
CV(%)= 1.07

T3/B

32.47

T3/C

33.00

CA1

1000 mg
Test Item/L

-0.19

0.03
CV(%)= 964.37#

CA2

-0.09

CA3

0.38

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

CV:            Coefficient of variation

NS:   There is no statisticallysignificant difference when compared to the control (2-Sample t-Test; 2-sided; α = 0.05)

#:     The high CV value was caused by the low calculated mean value and a standard deviation of 0.30.

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.
In conclusion this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L. Therefore, in line with OECD 209, a definite test is not required.
Executive summary:

The toxicity of the test item to microorganisms was assessed in a GLP-compliant Activated Sludge Respiration Inhibition Test according to the guidelines OECD No. 209, EU Method C.11 and US EPA OCSPP 850.3300. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the ground test item were added directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification controls and abiotic controls were investigated. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 32.87 mg O2/gh). No inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-h EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.


Description of key information

In the study according to OECD 209 with the test item no influence on the specific respiration rates up to 1000 mg/L were shown. The 3-h EC50 and NOEC was determined to be > 1000 mg/L and 1000 mg/L, respectively.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The toxicity of the test item to microorganisms was assessed in a GLP-compliant Activated Sludge Respiration Inhibition Test according to the guidelines OECD No. 209, EU Method C.11 and US EPA OCSPP 850.3300. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The test item was investigated at the nominal concentrations of 10, 100 and 1000 mg/L. Defined amounts of the ground test item were added directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore, blank (inoculum) control, nitrification controls and abiotic controls were investigated. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L: 32.87 mg O2/gh). No inhibitory effect of the test item was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-h EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.