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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 10, 2003 to October 20, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
study conducted acc. to old OECD 209 version (1984) requiring less replicates and controls than updated guideline (2011)
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
The sample of 3-chloro-o-xylene (T.R. Wilbury Laboratories sample number 1763) used for the study was delivered on March 10, 2003. It was contained in four 50 mL amber bottles that were shipped in a plastic bucket at ambient temperature. The Label attached to the bottles included the following information: "3-Cl-1,2-dimethylbenzene, CAS # 608-23-1, Clariant Lot 26.11.02, 25 g, 98%, combustible". 3-chloro-o-xylene (a colorless liquid) was supplied by General Electric Corp., 1 Research Circle, Niskayuna, New York. Test concentrations were corrected for the purity of the test substance. Prior to use the test substance was stored at room temperature in the dark. The test substance was assumed to be stable under test conditions.
Analytical monitoring:
no
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
Five separate stock solution were prepared on June 4, 2003 for use in the definitive test. The first stock solution, with a nominal concentration of 70 000 mg/L, was prepared by bringing 3.5597 grams of test substance to a total volume of 50 mL with dimethylformamide (DMF) in a 50 mL class A volumetric flask. The second stock solution, with a nominal concentration of 22 000 mg/L, was prepared by bringing 1.6 mL of the 70 000 mg/L stock solution to a total volume of 5.0 mL with DMF in a 5.0 mL class A volumetric flask. The third stock solution, with a nominal concentration of 7 700 mg/L, was prepared by bringing 0.55 mL of the 70 000 mg/L stock solution to a total volume of 5.0 mL with DMF in a 5.0 mL class A volumetric flask. The fourth stock solution, with a nominal concentration of 2 600 mg/L, was prepared by bringing 0.185 mL of the 70 000 mg/L stock solution to a total volume of 5.0 mL with DMF in a 5.0 mL class A volumetric flask. The fifth stock solution, with a nominal concentration of 870 mg/L, was prepared by bringing 0.062 mL of the 70 000 mg/L stock solution to a total volume of 5.0 mL with DMF in a 5.0 mL class A volumetric flask. Nominal concentrations [0 (control and solvent control), 0.087, 0.26, 0.77, 2.3 and 7.0 mg/L] were formulated by the addition of 0.050 mL of the appropriate stock solution to 500 mL of test solution. The solvent control was prepared by the addition of 0.050 mL of DMF to 500 mL of solution.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge used for the test was obtained from the municipal wastewater treatment plant at Merrimac, Massachusetts on June 2, 2003. This treatment plant treats predominantly domestic waste. As collected, the suspended solids concentration of the sludge used in the definitive test was 6.9 g/L. The sludge was incubated at room temperature and aerated prior to use. It was adjusted to a suspended solids content of 4.4 g/L on the day of use for the definitive test. Because the sludge was not used the day it was collected, synthetic sewage, consisting of 16 g/L peptone, 11 g/L meat extract, 3 g/L urea, 0.7 g/L NaCl, 0.34 g/L CaCl2, 0.2 g/L MgSO4 .7H20, and 2.8 g/L K2HPO4 was combined with the sludge at the rate of approximately 50 mL/L. The pH of the sludge was 7.2 on the day of use and did not require adjusting.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
< 1 mg/L as CaCO3
Test temperature:
19.9 to 20.4°C
pH:
7.2
Dissolved oxygen:
initial: 5.1 - 7.7 mg oxygen/L
Nominal and measured concentrations:
Nominal concentration: 0.087, 0.26, 0.77, 2.3 and 7.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): open
- Size: 1.5 liter, fill volume: 500 mL (300 mL test solution and a 200 mL aliquot of microbial inoculum)
- Aeration: yes, aerated with 0.5 to 1.0 Liters of air per minute for 3 hour
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- No. of vessels per positive control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 4.4 g/L
- Nutrients provided for bacteria: synthetic sewage, consisting of 16 g/L peptone, 11 g/L meat extract, 3 g/L urea, 0.7 g/L NaCl, 0.34 g/L CaCl2, 0.2 g/L MgSO4 .7H20, and 2.8 g/L K2HPO4, approximately 50 mL/L (because the sludge was not used the day it was collected)
- Nitrification inhibitor used (delete if not applicable): none
- Biomass loading rate: 200 mL aliquot of microbial inoculum

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water used for dilution of the sludge and for all toxicity testing was deionized water collected at Marblehead, Massachusetts with a hardness less than 1 mg/L as CaCO3. A representative sample of water was analyzed to verify that it was free of measurable quantities of pesticides. Intervals of analytical water quality measurement are 2 times a year.
- Particulate matter: As collected, the suspended solids concentration of the sludge used in the definitive test was 6.9 g/L. It was adjusted to a suspended solids content of 4.4 g/L on the day of use for the definitive test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: ambient laboratory lighting
- Dissolved oxygen: measured with a YSI Model 57 meter (instrument number 18)

TOXICITY TEST / EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The test was conducted under static conditions at 20 + 2°C. At time 0 for each test vessel, 16 mL of synthetic sewage and an appropriate amount of the test substance stock solution were brought up to 300 mL in dilution water in a 1.5 liter glass beaker. A 200 mL aliquot of microbial inoculum was added to this mixture within 5 minutes of its preparation. Test vessels were established at 15 minute intervals and vessels were aerated with 0.5 to 1.0 liters of air per minute for 3 hours. The first and last vessels established were controls. After the 3 hour incubation period, a portion of the contents of each test vessel were transferred to a BOD bottle equipped with a magnetic stirrer and the dissolved oxygen concentration in this sealed bottle was monitored for 10 minutes. Test vessels were maintained under ambient laboratory lighting during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3
- Range finding study: yes
- Test concentrations: not given
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Details on results:
The test substance did not inhibit respiration of the activated sludge in the tested range of concentrations. The 3 hour EC50 for activated sludge exposed to 3-chloro-oxylene was greater than 7.0 mg/L and the 3h-NOEC (activated sludge, total respiration inhibition) >= 7 mg/L, the highest tested concentration. Insoluble material was not observed at any tested concentration during the test.

Validity criteria acc. to OECD 209 (1984)
The two control respiration rates were within 15% of each other (12%).
The positive control 3,5-dichlorophenol was 9.4 mg/L and within the acceptable range of 5 to 30 mg/L.

Validity criteria acc. to OECD 209 (2010)
The respiration rate was > 20 mg O2/L (should not be less than 20 mg O2/L).
The coefficient of variation of four control respiration rate replicates (2 control replicates and 2 solvent control replicates) were 15% of each other (should be not more than 30% within control replicates: note, this validity criteria could not be fully assessed, because data requirements regarding replicates were less in the old version of OECD 209; therefore, control and solvent control replicates were pooled in this study to calculate the coefficient of variation).
The EC50 of 3,5-DCP for total respiration was within the range of 2 mg/L to 25 mg/L, i.e. 9.4 mg/L.

Note: Data generated by the respiration inhibition test are presented in any other information on results.
Results with reference substance (positive control):
The EC50 determined during the reference toxicant test with the batch of activated sludge used for the definitive test and 3,5-dichlorophenol was 9.4 mg/L and within the acceptable range of 5 to 30 mg/L.
Reported statistics and error estimates:
The respiration rate was calculated as the mg oxygen/liter/hour consumed between 6.5 (or the initial dissolved oxygen concentration if it was less than 6.5 mg/L) and 2.5 mg/L or over a 10 minute period if the respiration rate was low. The percent inhibition, which was used to calculate EC50 values, was calculated as (1 minus [2 times the respiration rate of the test substance concentration] divided by the sum of the 2 control respiration rates) times 100. The 3 hour EC50 value for the definitive activated sludge respiration inhibition test could not be calculated for the test substance by standard statistical techniques (Stephan, 1978), because there was less than 50% inhibition at all tested concentrations. The 3 hour EC50 value for the reference substance was calculated by the probit method.

Activated sludge respiration data from the test with 3-chloro-o-xylene

Nominal
concentration of
test item

(mg/L)

 

Dissolved oxygen
concentration

(mg/L)

Time to
6.5 mg/L
oxygen

Time to 2.5 mg/L oxygen (minutes)

Respiration
rate
(mg/L/hr)

Inhibition

(%)

Rep.

Initial

Final

(minutes)

0 (Control)

1

5.7

0.2

 -

2.5

77

 -

 

2

6.0

0.2

 -

3.0

70

 -

0 (Solvent control)

1

5.9

0.2

 -

2.5

82

-12

 

2

5.1

0.2

 -

1.5

104

-42

0.087

1

6.7

0.2

<0.5

3.5

69

6

 

2

6.7

0.1

<0.5

3.5

69

6

0.26

1

6.6

0.2

<0.5

3.0

80

-9

 

2

6.4

0.2

 -

3.5

67

9

0.77

1

6.9

0.2

0.5

3.5

80

-9

 

2

7.1

0.2

0.5

4.0

69

6

2.3

1

6.8

0.2

<0.5

3.5

69

6

 

2

7.1

0.2

0.5

4.5

60

18

7.0

1

7.7

0.2

1.0

4.5

69

6

 

2

7.5

0.2

1.0

4.5

69

6

Validity criteria fulfilled:
yes
Remarks:
(OECD 209 adopted 1984, 2010 as far as applicable)
Conclusions:
3h-EC50 (activated sludge, total respiration inhibition) > 7 mg/L (nominal), 3h-NOEC (activated sludge, total respiration inhibition) >= 7 mg/L
Executive summary:

The inhibition of activated sludge respiration by 3-chloro-o-xylene was investigated according to OECD 209 and in compliance with GLP. The test was performed at 20 ± 2°C under static conditions with a control, a solvent control (0.1 mL/L dimethylformamide) and test concentrations of test substance (0.087, 0.26, 0.77, 2.3 and 7.0 mg/L). Nominal concentrations of 3-chloro-o-xylene were used for all calculations.

Activated sludge was obtained from the Merrimac Wastewater Treatment Facility, Merrimac, Massachusetts. The total suspended solids content of the activated sludge was 6.9 g/L on the day of collection and 4.4 g/L on the day of the respiration inhibition test. During exposure, the test substance did not inhibit respiration of the activated sludge in the tested range of concentrations. The 3 hour EC50 for activated sludge exposed to 3-chloro-oxylene was therefore greater than 7.0 mg/L and the 3h-NOEC (activated sludge, total respiration inhibition) >= 7 mg/L. The EC50 determined during a reference toxicant test with this batch of activated sludge and 3,5-dichlorophenol was 9.4 mg/L, which is within the acceptable range.

Deviation from the new test guideline (adopted 2011) compared to the old test guideline (adopted 1984; e.g. fewer replicates and controls were required, i.e. 2 replicates instead of 5 for the test solutions and an additional abiotic and/or nitrification control) were considered to not affect the outcome of the toxicity test. The validity criteria of OECD 209 (1984, or 2011 as far as applicable) were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment.

Description of key information

3h-EC50 (activated sludge, total respiration inhibition) > 7 mg/L; 3h-NOEC (activated sludge, total respiration inhibition) >= 7 mg/L (OECD 209, 2003)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
7 mg/L

Additional information

A reliable key study was performed according to OECD 209 and in compliance with GLP (RL2, 2003). The inhibition of activated sludge respiration by 3-chloro-o-xylene was investigated according to OECD 209 (adopted 1984) and in compliance with GLP. The test was performed under static conditions with 0.087, 0.26, 0.77, 2.3 and 7.0 mg/L test item solution. During exposure, the test substance did not inhibit respiration of the activated sludge in the tested range of concentrations. The 3h-EC50 for activated sludge exposed to 3-chloro-oxylene was therefore > 7.0 mg/L and the 3h-NOEC (activated sludge, total respiration inhibition) >= 7 mg/L. Deviation from the new test guideline (adopted 2011) compared to the old test guideline (adopted 1984) were considered to not affect the outcome of the toxicity test. The validity criteria of OECD 209 (1984, or 2011 as far as applicable) were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment.