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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-06 to 2017-09-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 2018-12-30
- Purity: 98.2%
- Purity test date: 2017-01-04 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: not specified

OTHER SPECIFICS:
Physical state/Appearance: clear colorless liquid

Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2500 mg (nominal) of test item was dissolved in water and the volume adjusted to 1L to obtain a stock solution from which dilutions of 250 and 25 mg/L were made. Subsequently, a solution of 10 mg/L was made with an 200 mL aliquot from the 25 mg/L stock solution which was dispersed with 16mL of synthetic sewage, 250 mL of activated sewage sludge and water, to a final volume of 500 mL. Test concentrations of 100 and 1000 mg/L (in triplicates) were also prepared similarly. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.
- Differential loading: 10, 100 and 1000 mg/L (nominal concentrations)
- Controls: 4 vessels of negative control (not exposed to test material) and 3 vessels of positive control (with reference item) in concentrations of 3.2, 10 and 32 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: A mixed population of activated sewage sludge micro-organisms from the aeration stage
- Name and location of sewage treatment plant where inoculum was collected: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test.
- Method of cultivation: activated sewage sludge (6 liters) was fed synthetic sewage (300 mL)
- Initial biomass concentration: 3 g SS/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
no data; however it is being described in detail in the study report that the sythetic sewage feed was prepared according to OECD Guideline 209
Test temperature:
19-20 °C
pH:
Control: 0 hr: 7.2-7.4; 3 hrs: 7.2 - 7.3
Test item: 0 hr: 7.3 - 7.8; 3 hrs: 7.4 - 7.6
3,5- dichlorophenol: 0 hr: 7.2 - 7.4; 3 hrs: 7.6 - 7.7
Dissolved oxygen:
Control: 8.01 - 8.48 mg O2/L
Test item: 7.66 - 8.42 mg O2/L
3,5- dichlorophenol: 8.56 - 8.81 mg O2/L

In some instances, the initial and final DO concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
Nominal and measured concentrations:
Loading rate for the test substance:
10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flask
- Material, size, headspace, fill volume: 500 mL
- Aeration: yes, with clean, oil-free compressed air
- No. of vessels per concentration (replicates): 1 (3 replicates of the 1000 mg/L test concentration)
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 3 g SS/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A synthetic sewage feed was made by dissolving the following amounts of substances in 1 litre of deionized water:
16.0 g peptone,
11.0 g meat extrakt,
3.0 g urea,
0.7 g NaCl,
0.4 g CaCl2 x 2 H2O,
0.2 g MgSO4 x 7 H2O,
2.8 g K2HPO4

OTHER TEST CONDITIONS
- Adjustment of pH: pH of stock solution was 6.4 and adjusted to pH 7.4 using 1.0 M NaOH
- Light intensity: laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed was measured after a contact time of 3 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 10, 100, 1000
- Results used to determine the conditions for the definitive study: Yes. No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Key result
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
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge
- Observations on Test Preparations:
* Control: after 3hrs: Dark brown dispersion (all replicates)
* Test item: Dark brown dispersion, no undissolved test item visible (all test concentrations)
* 3,5-dichlorophenol: Dark brown dispersion, no undissolved reference item visible (all test concentrations)
- Blank controls oxygen uptake rate: not less than 20 mg O2/g.h
- Coefficient of variation of oxygen uptake rate in control replicates: the coefficient of variation of oxygen uptake rate in control replicates is not more than 30% at the end of the test.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3-h EC50 value of 5.4 mg/L, 95% C.L. 4.0 to 7.3 mg/L.
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC10, EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal
Concentration
(mg/L)

Dissolved Oxygen Concentration
(mg O2/L)

Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L

Control

R1

8.48

95

R2

8.38

94

R3

8.12

91

R4

8.01

90

Test Item

10

7.82

88

100

8.42

95

1000 R1

7.66

86

1000 R2

7.79

88

1000 R3

7.89

89

3,5-dichlorophenol

3.2

8.81

99

10

8.56

96

32

8.75

98

R1– R4= Replicates 1 to 4

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

Nominal
Concentration
(mg/L)

Initial O2
Reading
(mg O2/L)

Measurement Period
(minutes)

Final O2Reading
(mg O2/L)

O2Consumption Rates
(mg O2/L/hour)

%
Inhibition

Control

R1

5.8

6

2.0

38.00

-

R2

6.5

8

1.7

36.00

-

R3

5.5

6

1.9

36.00

-

R4

5.4

6

1.7

37.00

-

Test Item

10

6.2

6

2.4

38.00

[3]

100

6.0

6

2.2

38.00

[3]

1000 R1

5.4

5

2.3

37.20

[1]

1000 R2

5.6

6

2.0

36.00

2

1000 R3

6.0

6

2.3

37.00

[1]

3,5-dichlorophenol

3.2

7.5

10

3.8

22.20

40

10

6.8

5

5.6

14.40

61

32

8.4

10

7.8

3.60

90

R1– R4= Replicates 1 to 4

[Increase in respiration rate as compared to controls]

Validity criteria fulfilled:
yes
Conclusions:
An activated sludge respiration inhibition test (according to OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plat resulted in a 3-h EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The results of the study can be considered reliable without restriction.

Description of key information

An activated sludge respiration inhibition test (according to OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information