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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November to 21 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss GLP Compliance Programme (inspected on 22-26 March and 12-16 April 2010 / signed on 11 January 2011)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: Chimex / 0141653- Expiration date of the lot/batch: 06 September 2012- Purity test date: 07 September 2011STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature at about 20 °C, in the dark
Analytical monitoring:
no
Vehicle:
yes
Remarks:
deionized water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTIONBased on the Sponsor’s information, the test item is soluble in water.- Method: For the definitive test, a stock solution with a nominal concentration of 2.5 g/L was prepared by dissolving 5.000 g of the test item in 2000 mL deionized water by intense stirring for 15 minutes. In this way a colorless solution was obtained. Adequate volumes of this stock solution were diluted with deionized water to prepare the test media with the lower test item concentrations. Then, 16 mL of synthetic sewage feed and 250 mL activated sludge inoculum were added.See table 6.1.7/1.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.- Justification: Activated sludge micro-organisms have been selected following the recommendations of OECD Guideline No 209.- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.Based on this ratio, a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed (see Section 3.4) per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and readjusted to the level of 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge inoculum used for the definitive test was adjusted from 6.9 to 7.1 with a diluted sodium hydroxide solution.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
None
Test temperature:
19 °C
pH:
At the start of the test, the pH measured in the treatments was between 7.1 to 7.5. At the end of the test, pH values of 8.1 to 8.3 were measured.
Dissolved oxygen:
At the start of the test, the oxygen concentration was between 7.4 to 7.8 mg O2/L. At the end of the test, the oxygen concentration was between 7.3 to 8.6 mg O2/L were measured.
Salinity:
None
Conductivity:
No data
Nominal and measured concentrations:
Nominal test item concentrations: 12.3, 37.0, 111, 333 and 1000 mg/L (corresponding to 3.7, 11.1, 33.3, 99.9 and 300 mg a.i./L, respectively)
Details on test conditions:
TEST SYSTEM- Test vessel: Test was performed in 2000-mL glass beakers containing 500 mL of incubation mixture- Synthetic Sewage Feed (The synthetic sewage feed was prepared to contain the following constituents per liter: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2H2O, 0.2 g MgSO4 × 7H2O, 2.8 g K2HPO4). The pH of this solution was measured to be pH 7.2.- At the start of the test, 16 mL synthetic sewage was made up to 250 mL with deionized water and 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) was added to this first control vessel. Thereafter, in time intervals of about fifteen minutes (an arbitrary but convenient interval) the procedure was repeated, except that the 16 mL synthetic sewage was added to the appropriate amounts of reference item or test item before adding deionized water to a volume of 250 mL, followed by 250 mL inoculum. The addition of the inoculum to the incubation mixture is considered as the start of the 3-hour incubation time period.- Aeration: During the incubation period of 3 hours all vessels were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.- No. of vessels per concentration (replicates): 3 - No. of vessels per control (replicates): Six blank controls- Sludge concentration (weight of dry solids per volume): 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixtureTEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Distilled or deionized water containing less than 1 mg DOC/L was used, except where chlorine free tap water is specified.OTHER TEST CONDITIONS- During the incubation period of 3 hours all vessels were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item. The concentration of dissolved oxygen did not drop below 60-70% saturation and the sludge flocs were maintained in suspension during the incubation period. Just before measurement of the respiration rates the dissolved oxygen concentrations were at least 7.3 mg/L.EFFECT PARAMETERS MEASURED- Measurement of the Respiration Rates: For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the linear part of the respiration curve, normally in the range 7-2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of typically 5 to 10 minutes.- The pH values and dissolved oxygen concentrations were determined after the addition of synthetic sewage feed and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The temperature of the incubation mixture was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic sewage feed, the appearance of the test media was recorded.TEST CONCENTRATIONS- Range finding study: Based on the test guideline, a range-finding test using the following three nominal test item concentrations of 10 mg/L, 100 mg/L and 1000 mg/L (corresponding to 3 mg a.i./L, 30 mg a.i./L and 300 mg a.i./L, respectively) was performed. Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration five replicates were prepared. Five blank controls, containing an equal volume of activated sludge and synthetic medium, but no test or reference item, were included in the range-finding test.- Results used to determine the conditions for the definitive study: The results of the preliminary test indicated a clear concentration effect relationship and no atypical respiration curves were observed. Because the concentration related respiration curves were not bi-phasic, dose-response plots were not distorted and EC50 value was not unexpectedly low, respiration of the test item by nitrification was not assessed. In conformity with the guideline, since in the range-finding test the test item showed inhibitions of the respiration rate of 97, 27 and 3% for the test item concentrations of 1000, 100 and 10 mg/L, respectively, a definitive test was performed. Based on the results of the range-finding test and in agreement with the Sponsor a definitive test with the following nominal test item concentrations was performed: 12.3, 37.0, 111, 333 and 1000 mg/L (corresponding to 3.7, 11.1, 33.3, 99.9 and 300 mg a.i./L, respectively) with three replicates per concentration to primarily permit the determination of the EC50.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
70.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% Cl: 65.0 – 76.2 mg/L
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
99.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% Cl: 93.7 - 106 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
194 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% Cl: 186 - 202 mg/L
Key result
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
375 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% Cl: 354 - 400 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The inhibition of the respiration rates for all tested concentrations was in the range -5.9 to 98% compared to the control. Up to and including the concentration of 37.0 mg/L the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours (result of a Welch t-test with Bonferroni-Holm adjustment, one-sided smaller, α = 0.05). At the higher test item concentrations of 111 to 1000 mg/L the inhibitory effect increased and reached 24% at 111 mg/L, 75% at 333 mg/L and 98% at 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested. A clear concentration-effect relationship on the inhibition of the respiration rate was determined.The 3-hour NOEC, derived by per-vessel statistics using the Welch t-test with Bonferroni-Holm adjustment (one-sided smaller, α = 0.05), was: NOEC: 37.0 mg/LRespiration rates of the Blank Controls- The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 41 and 43 mg O2 per dry weight of activated sludge per hour, for the two blank controls prepared after the reference item group were 42 and 41 mg O2/gh and for the two replicates prepared at the end of the test were 41 and 42 mg O2/gh. All six values are higher than the 20 mg O2/gh stipulated in the guideline.- The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 2% and is therefore, as stipulated in the guideline, not more than 30%.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 9.6 mg/L with 95% confidence limits from 4.3 to 16 mg/L. The 3-hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming suitability of the activated sludge used.
Reported statistics and error estimates:
The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration using suitable software (ToxRat Professional).The NOEC was derived by per-vessel statistics using the Welch t-test with Bonferroni-Holm adjustment (one-sided smaller, α = 0.05).The 3-hour EC10, EC20, EC50 and EC80 values and their 95% confidence limits were derived by Probit Analysis. Adverse effects of the test item compared to the control were investigated using one-tailed (smaller) hypothesis testing at p ≤0.05 and per treatment means.The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Weibull Analysis.

Table 6.1.7/2: Influence of CHIMEXANE CL (Test Item) and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

 

Vessel no.

Nominal concentration of test chemical (mg/L)

Oxygen consumption rate

Inhibition (%)

Respiration rate (R) [mg O2 /Lh]

Specific respiration rate (RS) [mg O2 /gh]

 

Mean

Control

1

0

61.87

41.24

NA

2

0

64.11

42.74

6

0

63.30

42.20

7

0

62.20

41.47

23

0

61.07

40.71

24

0

62.40

41.60

Mean

62.49

41.66

SD

1.08

0.72

Coefficient of variation

1.72%

1.72%

Test item

8

12.3

64.00

42.67

-2.4

-5.9

9

12.3

64.35

42.90

-3.0

 

10

12.3

70.20

46.80

-12.3

 

11

37

62.28

41.52

0.3

0.6

12

37

62.91

41.94

-0.7

 

13

37

61.08

40.72

2.3

 

14

111

48.72

32.48

22.0

24.2

15

111

46.50

31.00

25.6

 

16

111

46.89

31.26

25.0

 

17

333

15.60

10.40

75.0

75.5

18

333

14.28

9.52

77.1

 

19

333

16.13

10.76

74.2

 

20

1000

1.58

1.05

97.5

97.7

21

1000

1.47

0.98

97.7

 

22

1000

1.27

0.85

98.0

 

Reference item

3

3.2

46.08

30.72

26.3

NA

4

10

31.20

20.80

50.1

5

32

10.04

6.69

83.9

- % inhibition: Increased oxygen consumption rate relative to control.

 

Validity of the Test

The test is regarded as valid since the validity criteria are fulfilled.

- The specific respiration rate of the blank controls was in the range 41 to 43 mg oxygen per gram dry weight of sludge per hour and therefore, higher than 20 mg O2/gh.

- the coefficient of variation of oxygen uptake rate in control replicates was 2% and therefore, less than 30%.

- the EC50 (3-hour) of the reference item 3,5-dichlorophenol was 9.6 mg/L and therefore, in the range 2 to 25 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The test item CHIMEXANE CL had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to and including the test item concentration of 37.0 mg/L (corresponding to 11.1 mg a.i./L). Therefore, the 3-hour NOEC of CHIMEXANE CL was 37.0 mg/L. The 3-hour EC50 of CHIMEXANE CL was found to be 194 mg/L (corresponding to 58.2 mg a.i./L) with 95% confidence limits from 186 – 202 mg/L.
Executive summary:

The inhibitory effect of the test item CHIMEXANE CL on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline 209in compliance with GLP.

Nominal test item concentrations: 12.3, 37.0, 111, 333 and 1000 mg/L (corresponding to 3.7, 11.1, 33.3, 99.9 and 300 mg a.i./L, respectively) with three replicates per concentration to primarily permit the determination of the EC50.

As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions at concentrations of 3.2, 10 and 32 mg/L (all single replicates).

Six blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item were included in the definitive test.

The inhibition of the respiration rates for all tested concentrations was in the range -5.9 to 98% compared to the control. Up to and including the concentration of 37.0 mg/L the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours. At the higher test item concentrations of 111 to 1000 mg/L the inhibitory effect increased and reached 24% at 111 mg/L, 75% at 333 mg/L and 98% at 1000 mg/L. Concentrations exceeding 1000 mg/L were not tested. A clear concentration-effect relationship on the inhibition of the respiration rate was determined.

The test item CHIMEXANE CL had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to and including the test item concentration of 37.0 mg/L (corresponding to 11.1 mg a.i./L). Therefore, the 3-hour NOEC of CHIMEXANE CL was 37.0 mg/L. The 3-hour EC50 of CHIMEXANE CL was found to be 194 mg/L (corresponding to 58.2 mg a.i./L) with 95% confidence limits from 186 – 202 mg/L.

Description of key information

The inhibitory effect of the test item CHIMEXANE CL on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline 209 in compliance with GLP.

The test item CHIMEXANE CL had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours up to and including the test item concentration of 37.0 mg/L (corresponding to 11.1 mg a.i./L). Therefore, the 3-hour NOEC of CHIMEXANE CL was 37.0 mg/L. The 3-hour EC50 of CHIMEXANE CL was found to be 194 mg/L (corresponding to 58.2 mg a.i./L) with 95% confidence limits from 186 – 202 mg/L.

This study was considered reliable for that endpoint.

Key value for chemical safety assessment

EC50 for microorganisms:
58.2 mg/L
EC10 or NOEC for microorganisms:
11.1 mg/L

Additional information