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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-27 to 2017-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D26-3-3-1
- Expiration date of the lot/batch: December 2018
- Purity test date: April 11, 2017 (certificate of analysis release date)
- Purity: 47.4 wt%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

OTHER SPECIFICS:
- Physical state/Appearance: Brown viscous liquid
Analytical monitoring:
no
Remarks:
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and is reflected in the GLP compliance statement.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
* Test Item Preparation: Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. 2500 mg of test item was dissolved in water and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity. The pH was then adjusted to 7.0 - 8.0.
* Reference Item Preparation: A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 25 minutes. The pH of this stock solution was measured to be pH 6.2 and was adjusted to pH 7.1 using 1.0 M NaOH. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.
- Controls: test medium without test item and treated in the same way as the test item solutions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- 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 21ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (6.5 liters) was fed synthetic sewage (325 mL). The pH of the sample on the day of the test was 7.5. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.
- Initial biomass concentration: 3.0 g/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
Not reported
Test temperature:
21”C
pH:
prior to adjustment: 7.8 - 12.0
after adjustment: 7.5 - 7.7
Dissolved oxygen:
5.7 - 8.2 mg O2/L or 64 - 92% of Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Loading rate for the test substance:
- Range-Finding Test: 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks of 500 mL
- Type (delete if not applicable): closed (see below for aeration)
- Material, size, headspace, fill volume: 500 mL
- Aeration: aerated with clean, oil-free compressed air via narrow bore glass tubes
- No. of vessels per concentration (replicates): one flask each for 10 and 100 mg/L and three for 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per abiotic control (replicates): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
* 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.  
* The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.
* The oxygen concentrations in all vessels were measured after 30 minutes contact time.
* The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Range finding study : yes
- Test concentrations: 10, 100 and 1000 mg/L
- 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:
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: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
- Blank controls oxygen uptake rate: ~40 mg O2/L/hour
- Coefficient of variation of oxygen uptake rate in control replicates: not more than 30% at the end of the test.
- The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
- It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
- 3-h EC50 = 6.6 mg/L, 95% confidence limits 4.9 to 9.0 mg/L which lies within the range 2 to 25 mg/L for total respiration suggesting the suitability of the test conditions and the sludge.
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.
- 95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
- For 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).

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

6.2

70

R2

6.6

74

R3

7.2

81

R4

6.0

67

Test Item

10

6.2

70

100

6.1

69

1000 R1

5.9

66

1000 R2

5.7

64

1000 R3

6.0

67

3,5-dichlorophenol

3.2

6.5

73

10

7.2

81

32

8.2

92

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

5.7

6

1.7

40.0

-

R2

6.0

6

2.0

40.0

-

R3

5.9

6

1.9

40.0

-

R4

5.8

5

2.3

42.0

-

Test Item

10

5.5

5

2.0

42.0

[4]

100

5.7

6

1.8

39.0

4

1000 R1

5.9

6

1.8

41.0

[1]

1000 R2

5.9

6

1.8

41.0

[1]

1000 R3

6.1

6

2.0

41.0

[1]

3,5-dichlorophenol

3.2

6.5

10

2.0

27.0

33

10

7.3

10

4.6

16.2

60

32

8.3

10

7.4

5.4

87

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 plant resulted in a 3-h EC50 > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. The results of the test can be considered reliable without restriction.

Description of key information

The study of Bayliss (2016), investigating the toxicity of the test item to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage. The 3-h EC50 was > 1000 mg/L. The 3-h EC10 and the 3-h NOEC were >1000 and 1000 mg/L, respectively.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information