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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 February 2004 to 19 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTIONS
The overnight sludge suspension was washed using the procedure described earlier except that the sludge solids were homogenised in a blender for 2 minutes after each wash. The final sludge solution was prepared in laboratory dilution water at a mixed-liquor suspended solid of approximately 4 g/L. The final sludge concentration in the test beakers was 1.7 g/L.
At time 0, 8 mL of each synthetic feed stock solution was placed in a 200 mL volumetric flask and brought to volume with laboratory water. This mixture was placed in the negative control test vessel (1 litre glass beaker). Next, 100 mL of laboratory water and 200 mL of sludge inoculum was added to the beaker to give a final volume of 500 mL. The test solution was gently stirred with a Teflon®coated stir bar and a stir plate and aerated at 0.5 to 1 L/minute using a pipette as an aeration device.
Exposure solutions were prepared at nominal concentrations of 25, 50, 100, 500 and 1000 mg/L by the addition of the appropriate amounts of the test substance to the test vessels. At time '12 min', the above was repeated for the second test vessel containing the lowest concentration of the test substance. The process was repeated at 12 minute intervals for the remaining test substance concentrations.
The positive control was tested on the microbial inoculum on the same way expect that 100 mL of the positive control stock solution was placed in the seventh beaker instead of 100 mL water. The process was repeated with different volumes of positive control stock solution (diluted with water to 100 mL) to provide a total of three positive control concentrations of 3.05, 9.77 and 31.25 mg/L.
Finally, a second negative control was prepared. Oxygen consumption was measured and recorded after an aeration time of three hours. Air temperature was recorded at test start. The pH of all test solutions were measured at test start and at test end.

1. Synthetic feed stock solutions were prepared as described below.
2. A positive control stock solution of 3,5-dichlorophenol was prepared according to the details below.
3. A test substance stock solution was not prepared. The test substance was added directly to the test beakers.

I. Synthetic Feed Stock Solutions
A. Organic Stock Solution: Peptone 16.0 g, Beef extract 11.0 g, Urea 3.0 g, Sodium chloride 0.7 g, Calcium chloride dihydrate 0.4 g and Magnesium sulfate 7-hydrate 0.2 g. Dissolve in and make up to 500 mL with distilled water

B. Phosphate Stock Solution
Potassium phosphate, dibasic 2.8 g. Dissolve in and make up to 500 mL with distilled water

Note: this synthetic feed is a 200-fold concentrate of that described in OECD Technical Report 'Proposed Method for the Determination of the Biodegradability of Surfactants used in Synthetic Detergents' (11 June 1976) with more dipotassium hydrogen phosphate added. This concentrate must be prepared in two separate stock solutions to prevent precipitation.

II. Positive Control Stock Solution
The solution of 3,5-dichlorophenol is prepared as follows:
1. Dissolve 0.5 g of 3,5-dichlorophenol in 10 mL of 1N NaOH.
2. Dilute to approximately 30 mL with 20 mL distilled water.
3. Add, while stirring, 1N H2SO4 to the point of incipient precipitation - approximately 8 mL of 1 N H2SO4 will be required.
4. Dilute the mixture to one litre with distilled water. The pH should then be in the range 7 to 8. Adjust with 1N H2SO4 if necessary.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
PREPARATION OF INOCULUM FOR EXPOSURE
On arrival at the laboratory, a small amount of fresh sludge was weighed, oven-dried and reweighed. A calculation was made from these results to determine the amount of wet sludge that must be suspended in laboratory dilution water (LDW) in order to obtain an activated sludge with a mixed-liquor suspended solids (MLSS) level of approximately 4 g/L. The sludge was washed three times with LDW using the following method: after centrifuging at approximately 7000 rpm for 15 minutes, the supernatant was decanted and the remaining sludge solids resuspended in LDW and mixed well. After the third spin, the wet solids were homogenised for 2 minutes in a blender. The wet solids were suspended in 3 L of LDW and 75 mL of each of the two synthetic feed stock solutions. The sludge suspension was aerated overnight (21 h) at 22 ºC. The pH of the overnight suspension was 7.199.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Air temperature at the test start was 22 ºC.
pH:
7.533 to 8.329
Dissolved oxygen:
0.30 mg/L
Nominal and measured concentrations:
25, 50, 100, 500 and 1000 mg/L (nominal)
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol; CAS No. 00591-35-5; Purity 99.4 %
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Details on results:
It was observed after the addition of the test substance to the test vessels at the start of the test that the test substance was not soluble in the test media for the period of the test.

The respiration rates for the test oxygen uptake values, starting from the lowest concentration to the highest were 55.02, 51.77, 54.28, 52.00 and 55.75 mg O2L^-1h^-1 resulting in inhibition values of -9.2, -2.8, -7.8, -3.3 and -10.7 %, respectively (negative inhibition values represent enhanced respiration rates). These values are not considered significant due to the inherent experimental error of the test. Respiration rates for the first and last negative controls were 51.32 and 49.40 mg O2L^-1h^-1, respectively, resulting in a mean value of 50.4 mg O2L^-1h^-1.

Test validity
A test is considered valid if:
1. The two negative control respiration rates are within 15 % of each other.
2. The 3 hour EC50 value of 3,5-dichlorophenol is in the range 5-30 mL.

These conditions were fulfilled during the course of the study.
Results with reference substance (positive control):
Oxygen uptake values for the positive control (concentrations 3.05, 9.77 and 31.25 mg/L) resulted in respiration rates of 52.80, 36.11 and 15.00 mg O2L-1h-1 respectively resulting in inhibition values of -4.8, 28.3 and 70.2 %, respectively. An EC50 value of 15.8 mg/L was calculated by regression analysis.

Table 1: Dissolved oxygen measurements

Beaker No.

Conc. (mg/L)

O2 uptake

Time (sec.)

Time (min.)**

Resp. Rate***

% Inhibition

Upper Value*

Lower Value*

1 Neg Control

0

4.77

1.99

195

3.25

51.32

2 Test

25

5.15

2.17

195

3.25

55.02

-9.2

3 Test

50

5.03

2.01

210

3.5

51.77

-2.8

4 Test

100

4.93

1.99

195

3.25

54.28

-7.8

5 Test

500

5.22

1.97

225

3.75

52

-3.3

6 Test

1000

5.01

1.99

195

3.25

55.75

-10.7

7 Pos. Control

3.05

4.73

2.09

180

3

52.8

-4.8

8 Pos. Control

9.77

5.63

2.47

315

5.25

36.11

28.3

9 Pos. Control

31.25

7.52

6.27

300

5

15

70.2

10 Neg. Control

0

4.51

2.04

180

3

49.4

*mg of oxygen from dissolved oxygen meter test readings (as mg/L)

**Time (seconds) converted to minutes for Resp. Rates calculations

***Respiration rate as mg of oxygen per liter per hour

Note: the negative controls should be within 15 % = 50.4 ± 3.8

(Relative Percent Difference between reps/MEAN x 100)

Validity criteria fulfilled:
yes
Conclusions:
The 3 h respiration inhibition test resulted in an EC50 value of >1000 mg/L. A NOEC of 100 mg/L, the highest concentration tested, was obtained for this study. The test substance is not expected to inhibit respiration of secondary waste water treatment microorganisms at a nominal concentration of 1000 mg/L
Executive summary:

In a GLP compliant respiration inhibition study conducted in line with OECD Guideline 209, the respiration inhibition of secondary waste water treatment microorganisms of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo [1,5-b][1,2,4]triazol-2-yl] phenyl]amino]-4-oxo-, tetradecyl ester was investigated. The EC50 of the substance was determined to be >1000 mg/L and the NOEC to be 1000 mg/L.

Description of key information

Key study:- Moulton (2004) 'Activated sludge respiration inhibition' conducted in line with OECD Guideline 209 and EU Method C.11. The EC50 has been determined to be >1000 mg/L and the NOEC to be 1000 mg/L.

Key value for chemical safety assessment

Additional information

The key study (Moulton, 2004) was performed in compliance with GLP and to OECD Guideline 209 and EU Method C.11 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with accepted, standardised guidelines and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The substance is not expected to inhibit respiration of secondary waste water treatment microorganisms at a nominal concentration of 1000 mg/L. The EC50 has been determined to be >1000 mg/L and the NOEC to be 1000 mg/L.