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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 - 16 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland
Specific details on test material used for the study:
Reference Item:
The reference item and the following information were supplied by Acros Organics BVBA/Belgium:

Identity: 3,5-Dichlorophenol
Product No.: 11361
Batch No.: A0323035
Purity: 99.6% (GC)
Expiration Date: 30 July 2017 (retest date)
Storage Conditions (as provided by the supplier): Store in a cool, dry place, in a tightly closed container.
Storage Conditions (as handled at Harlan Laboratories): At room temperature at 20 ± 5°C, in the dark.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Procedure: At the start of the test, 16 mL synthetic sewage were made up to 250 mL with water and then 250 mL activated sludge inoculum with a sludge concentration of nominally 3 g/L (dry weight) were 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 were added to the appropriate amounts of reference item or test item in water to obtain a volume of 250 mL, followed by the addition of 250 mL inoculum. The addition of the inoculum to the incubation mixture is considered as the start of the 3-hour incubation time period.

Dosage: Based on the results of the range-finding test, the test item was soluble at a concentration of at least 5 g/L in tap water. A stock solution with a nominal concentration of 5 g/L was prepared by dissolving 5009 mg of the test item in 1000 mL water and thoroughly mixed by intense stirring for 15 minutes. In this way a clear solution was obtained. The pH of the stock solution was adjusted from pH 4.0 to pH 7.4 with a 1M NaOH solution. Adequate volumes of this stock solution were diluted with water to prepare the test media with the lower test item concentrations. Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- 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 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 was 7.1.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Nominal and measured concentrations:
Control, 62.5, 125, 250, 500 and 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM-

Test vessel:
- Material, size, headspace, fill volume: 2000-mL glass beakers containing 500 mL of incubation mixture
- Aeration: continuous- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 6
- No. of vessels per positive control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Synthetic seage feed: The synthetic sewage feedwas prepared to contain the following constituents per liter filled up to the final volume with deionized water: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 K2HPO4The pH of this solution was pH 7.3.

- Test water: Distilled or deionized water, containing less than 1 mg/L dissolved organic carbon was used except where chlorine free tap water is specified.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): respiration rate, pH values and dissolved oxygen concentrations were determined in all test media and the controls at the start and at the end of the 3-hour incubation period

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Results used to determine the conditions for the definitive study: The test item was soluble at a concentration of at least 5 g/L in tap water.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
555 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 289 - >1000 mg/L
Key result
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 782 - >1000 mg/L
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
Remarks:
respiration rate
Remarks on result:
other: 95% CL: >1000 - >1000 mg/L
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The 3 hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 9 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3 hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming the 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 Dunnett t-test (Dunnett, 1955; Dunnett, 1964) (one sided smaller, α= 0.05). The 3-hour EC10, EC20, EC50 and EC80 values and their 95% confidence limits were, as far as possible, derived by Probit Analysis (Davies, 1971; Finney, 1971).The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Probit Analysis(Davies, 1971; Finney, 1971).DAVIES, R.G. (1971): Computer programming in quantitative biology. Academic Press, London. FINNEY, D.J. (1971): Probit analysis,3rd edition. University Press, London. DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. Journal of the American Statistical Association, 50, pp. 1096–1121.DUNNETT, C.W. (1964): New tables for multiple comparisons with a control. Biometrics, 20, pp. 482–491.

Respiration Rates of the Blank Controls:

The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 47 and 44 mg O2 per dry weight of activated sludge per hour, for the two blank controls prepared after the reference item group were 50 and 45 mg O2/gh and for the two replicates prepared at the end of the test were 45 and 44 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 5% and is therefore, as stipulated in the guideline, not more than 30%.

Table: Influence of Cyrene™ (Test Item) and 3,5-Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

Vessel No.

[mg/L] Nominal concentration of test chemical

Oxygen consumption rate

Inhibition %

pH values

Oxygen

Concentration [mg O2/L]

Respiration rate ( R) [mg O2/Lh]

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

Start*

End*

Start*

End*

Control

1

0

70.53

47.02

 

7.1

7.8

 8.1

 8.4

2

0

65.47

43.64

7.1

7.8

 8.1

 8.6

6

0

74.53

49.69

7.0

7.9

 8.6

 9.6

7

0

66.84

44.56

7.0

7.9

 8.5

 8.4

23

0

66.93

44.62

7.1

7.8

 8.1

 8.3

24

0

66.00

44.00

7.2

7.8

 8.3

 8.6

Mean

68.38

45.59

 

 

Standard Deviation

3.50

2.33

Coefficient of Variation

5.11%

5.11%

Test Item

Mean

 

8

62.5

72.67

48.44

 -6.3

-3.4

7.1

7.9

 8.0

8.5

9

62.5

67.05

44.70

 2.0

7.2

7.8

 8.4

8.5

10

62.5

72.48

48.32

 -6.0

7.1

7.9

 8.0

8.9

11

125

70.40

46.93

 -2.9

-5.4

7.1

7.8

 7.9

9.0

12

125

74.67

49.78

 -9.2

7.1

7.9

 8.1

8.6

13

125

71.07

47.38

 -3.9

7.2

7.9

 8.0

8.7

14

250

76.80

51.20

 -12.3

-10.3

7.1

7.8

 8.0

8.2

15

250

72.67

48.44

 -6.3

7.0

7.9

 8.2

8.5

16

250

76.80

51.20

 -12.3

7.1

7.9

 7.9

8.5

17

500

65.40

43.60

 4.4

6.2

7.1

7.9

 7.9

8.1

18

500

68.04

45.36

 0.5

7.2

8.0

 8.0

8.6

19

500

59.00

39.33

 13.7

7.2

8.0

 8.0

9.0

20

1000

56.40

37.60

 17.5

17.1

7.2

8.0

 8.4

8.6

21

1000

56.22

37.48

 17.8

7.1

8.0

 8.1

8.8

22

1000

57.43

38.29

 16.0

7.1

8.0

 7.9

9.0

Reference Item

3

3.2

52.11

34.74

23.8

 

7.0

7.9

8.0

9.1

4

10

34.07

22.71

50.2

 

7.0

7.8

8.0

8.9

5

32

9.60

6.40

86.0

 

7.0

7.7

8.8

9.5

* Start and end of the 3-hour incubation period.

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

Validity criteria fulfilled:
yes
Conclusions:
A 3 hour EC50 of >1000 mg/L and NOEC of 500 mg/L were determined for the substance using a relevant test method and in compliance with GLP. The result is considered to be reliable.

Description of key information

Toxicity to microorganisms: ASRI 3 hour EC50 value of >1000 mg/l and NOEC of 500 mg/l (nominal concentrations).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
500 mg/L

Additional information

A 3 hour EC50 value of >1000 mg/l (highest concentration tested) and a NOEC of 500 mg/l (nominal concentrations) have been determined for the effects of the test substance, Cyrene™ on respiration inhibition of activated sludge, in accordance with OECD Test Guideline 209. This indicates that the test substance was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/l.