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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 April 2012 to 23 April 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Obtained on 23rd April 2012, from Seven Trent Water Plc at Loughborough, Leicestershire, UK
- Taken from the aeration stage
- Preparation of inoculum: The sample was maintained on continuous aeration at 21 ºC and pH 7.1
- The suspended solids concentration was determined prior to use and was equal to 3.0 g/L
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
- Oxygen concentration was measured up to 30 minutes after the exposure time
Test temperature:
20 ± 2 ºC
pH:
6.4 - 7.6 (test item)
Dissolved oxygen:
1.8 - 6.0 mg O2/L (test vessels)
Nominal and measured concentrations:
Nominal concentrations of 10, 100 and 1000 mg/L
Details on test conditions:
PRELIMINARY RANGE-FINDING TEST
Concentrations and Replications
- Activated sludge was exposed to the following nominal concentrations of the test material 10, 100 and 1000 mg/L
- The 1000 mg/l concentration was performed in triplicate, all others test concentrations were performed once
- The reference substance was included in the preliminary test at 3.2, 10 and 32 mg/L
- The reference substance was performed once for each concentration
- The control was performed 4 times

Preparation of the Test Material and Inoculum
- Method: 16 mL of synthetic sewage, 250 mL of activated sewage sludge and reverse osmosis water were added to create a final volume of 500 mL, to give the required concentrations. The mixture was subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours to maximise the amount of dissolved test material
- Test solutions were prepared in triplicate

Application of the Reference Substance/Positive Control
- Method: A stock solution of 0.5 g/L was prepared in water with the aid of untrasonication for 20 minutes. The pH was adjusted to 7.0 using 0.1 M NaOH. Aliquots of the stock solution were removed and dispersed with 250 mL of activated sewage sludge, 16 mL of synthetic sewage and water to give the final concentrations
- The exposure conditions were the same as the for the test group

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water was used
- Total dissolved organic carbon: < 1mg/L

RESPIRATION SUBSTRATE
- The following was added as a synthetic sewage substrate:
16 g Peptone, 11 g Meat Extract, 3g Urea, 0.7g NaCl, 0.4g CaCl2.2H2O, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4, dissolved in 1 litre of water with the acid of ultrasonication

CONTROL
- The control group was maintained under identical conditions although the test material was omitted

MEASUREMENT OF OXYGEN CONSUMPTION
- Apparatus: A Yellow Springs dissolved Oxygen meter fitted with a BOD prode
- Method: An aliquot was removed from the conical flask and poured into the measureing vessel, a 250 mL darkened glass BOD, and the rate of respirsation was measured. The rate of respiration was measured over the linear portion of the oxygen consumption trace, where in the case of low oxygen consuption the rate was determined over an approximate 10 minute period.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- As no toxic effects or inhibition were observed during the range-finding study, further testing was deemed unnecessary and unrealistic
- No statistically significant toxic effects were shown at any of the test concentrations
- The coefficient of variation of the oxygen uptake rate in control vessel was 4.2%
- The specific respiration rate of the control was 26.89 mg oxygen per gram dry weight of sludge per hour
Results with reference substance (positive control):
- 3 hour EC50 = 9.1 mg/L with the 95% confidence limits 7.3-11
- The result for the reference substance was valid

Table 1. Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Substance

Nominal Concentration (mg/l)

Initial O2Reading (mg/O2/l)

Measurement Period (min)

Final O2 Reading (mg/O2/l)

O2Consumption Rates (mg O2/l/hour)

% Inhibition

Control

R1

5.3

5

1.9

40.80

-

R2

5.7

6

1.9

38.00

-

R3

4.6

4

1.9

40.50

-

R4

5.2

4

2.4

42.00

-

Test Material

10

4.6

4

1.9

40.50

0

100

4.2

3

2.0

44.00

[9]

1000 R1

4.2

3

1.9

46.00

[14]

1000 R2

3.7

2

2.2

45.00

[12]

1000 R3

3.4

2

1.8

48.00

[19]

Positive Control

3.2

6.0

7

2.1

33.43

17

10.0

7.4

10

4.5

17.40

57

32.0

8.2

10

7.2

6.00

85

[ ] = an increase in respiration rate.

R1 - R4 = replicates 1 to 4.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, the EC50 of the test material was determined to be > 1000 mg/L and the NOEL was shown to be 1000 mg/L. The data from the range-finding study were considered sufficient for classification as the toxicity of the test material is beyond the limits of classification according to Regulation (EC) 1272/2008 and thus it was deemed unnecessary to test beyond 1000 mg/L.
Executive summary:

In a GLP compliant study performed according to the standardised guideline OECD 209, the toxicity of the test material was determined in an activated sludge respiration inhibition test. Activated sludge was exposed to the test material for 3 hours at the following concentrations; 10, 100 and 1000 mg/L, after which the rate of respiration was calculated from the oxygen consumption. Under the conditions of the test the EC50was determined to be > 1000 mg/L and the NOEL was shown to be 1000 mg/L. This data was considered sufficient for classification purposes as the toxicity of the test material is beyond the limits of classification according to Regulation (EC) 1272/2008.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
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
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Description of key information

3 hour EC50 = > 1000 mg/L for a structural analogue (EC 265-205-1), 3 hour NOEC = 1000 mg/L (OECD 209).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The effect of an analogue test material (EC 265-205-1) on the respiration rate of activated sludge was investigated in accordance with the standardised guideline OECD 209. Activated sludge was exposed to the test material for 3 hours at the following concentrations; 10, 100 and 1000 mg/L, after which the rate of respiration was calculated from the oxygen consumption. Under the conditions of the test the EC50was determined to be > 1000 mg/L and the NOEL was shown to be 1000 mg/L.

The study was performed in line with GLP and accepted standardised guidelines with a high standard of reporting. The study was conducted with a structural analogue and was therefore assigned a reliability score of 2 and considered suitable for assessment as an accurate reflection of the test material. Read across is justified on the basis of similar chemical structures and analogous results from a batch of physico-chemical tests, including water solubility.