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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
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
Deviations:
yes
Remarks:
The air temperatures in the test room was recorded as being above the acceptable range of 18-22°C. However, the measured temperature of the control samples at the start and end of the test was within the required range. No adverse effects on the study.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch: CDD-005
Analytical monitoring:
not required
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Test item preparation: Nominal amounts of test item were seperately dispersed in ca. 200 mL of water and subjected to ultrasonnication for ca. 15 minutes, followed by magnetic stirring for 24 hours in order to maximise the dissolved test item concentration (all vessels were shielded from light during this process).

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: The activated sludge sample was maintained on continuous aeration in the laboratory at a temperature of approx. 21°C, and was used on the day of collection. The pH of the samples was measured as 7.1.
- Pretreatment: None
- Initial biomass concentration: 3.0 g/L suspended solids.
- Source: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Hardness:
Not applicable
Test temperature:
A temperature controlled room at measured air temperatures of between 18 and 23°C. Although the range of air temperatures recorded in the room was above the acceptable range of 18-22°C, the measured temperature of the controls at the start and end of the test were within the required range. no temperature measurements were made of the reference or test item preparations, however given that the temperature of the control samples which spanned the whole test period were within the required temperature range, it was considered that the reference and test item preparations would have also been within the required temperature range. As such, this deviation was considered not to have adversely affected the study.
pH:
The pH values were maintained within acceptable ranges (start and end of exposure in test vessels 7.6).
Dissolved oxygen:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60-70% of the dissolved oxygen saturation level of 8.9 mgO2/L.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations of 10, 100 and 1000 mg/L. The test items were dispersed directly in water.
Details on test conditions:
TEST SYSTEM
- Test vessel: > 500 mL glass vessels
- Type: open
- Material, size, headspace, fill volume: Filled to 500 mL (16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL flask).
- Aeration: Continuously aerated by oil free compressed air (0.5 to 1.0 L/min).
- No. of vessels per concentration (replicates): Triplicate test vessels at 1000 mg/L, single test vessels at 10 or 100 mg/L.
- No. of vessels per control (replicates): 4 replicates
- Biomass loading rate: 3.0 g/L suspended solids (inoculum).

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

OTHER TEST CONDITIONS
- Adjustment of pH: Not required for test vessels.
- Photoperiod: Continuous

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rates used to calculate oxygen uptake rates and percentage of inhibition.

TEST CONCENTRATIONS
- Range finding study/limit test
- Test concentrations: 10, 100 and 1000 mg/L

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
Remarks:
respiration rate
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
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid: The EC50 for the positive substance was within the range 2 to 25 mg/L (7.4 mg/L), indicating the test is valid

 

Test substance

3,5-dichlorophenol

ECx(3 hours) (mg/L)

95% confidence limits (mg/L)

ECx(3 hours) (mg/L)

95% confidence limits (mg/L)

EC10

>1000

-

1.6

-

EC20

>1000

-

2.3

-

EC50

>1000

-

7.4

5.8 - 9.5

EC80

>1000

-

23

-

NOEC

1000

-

-

-

Validity criteria fulfilled:
yes
Remarks:
All validity criteria as per the test guideline met.
Conclusions:
The effect of the test item on the respiration of activated sludge micro-organisms gave a 3 hour EC50 value of >1000 mg/L. The NOEC value after 3 hours exposure was 1000 mg/L.

Description of key information

In a 3-hour activated sludge respiration inhibition test (ASRIT), the toxicity of the test substance to sewage treatment plant micro-organisms was assessed.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The effect of the test item on the respiration of activated sludge micro-organisms gave a 3 hour EC50 value of >1000 mg/L. The NOEC value after 3 hours exposure was 1000 mg/L.