Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Remarks:
Conducted according to guideline in effect at the time of study conduct.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 22.82% solids in water

Sampling and analysis

Analytical monitoring:
not specified

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Laboratory culture: A synthetic sewage feed was prepared by dissolving the following ingredients in dechlorinated water to a final volume of 500 mL. It had a pH of 7.21.

7.998 g Peptone
5.499 g Meat extract
1.504 g Urea
0.356 g NaCl
0.2007 g CaCl2.2H2O
0.1010 g MgSO4.7H2O
1.403 g K2HPO4

- Preparation of inoculum for exposure: At time 0 hour, 16 mL of synthetic sewage feed was diluted to 284 mL with dechlorinated tap water.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Hardness:
Not reported
Test temperature:
22.1-23.1°C
pH:
6.24 for the microbial inoculum
Dissolved oxygen:
Not reported
Nominal and measured concentrations:
1000 mg (solids)/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 mL glass flask; test solutions were transferred to 300-mL Biological Oxygen Demand (BOD) glass bottles for Dissolved Oxygen (DO) determinations
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass; 1000 mL headspace; 500 mL fill volume
- Aeration: Compressed air at a flow rate between 2-5 liters per minute at room temperature
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1 for abiotic control; 3 for reference substance

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water was prepared by aerating tap water for a minimum of 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: not reported
- Photoperiod: 3 h exposure
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption rates for 3 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable; the highest concentration required by the guideline was tested.
Reference substance (positive control):
yes
Remarks:
(3,5-Dichlorophenol (purity 99.5%; nominal concentrations 3.2, 10, and 32 mg/L)

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: solids
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
No significant difference (p = 0.05) in the microbial respiration of test systems with the test substance at 1000 mg solid/L compared to the positive controls.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: The effect concentration at which 50% inhibition occurred (EC50) was approximately 17.8 mg/L. The maximum difference between the respiration rates of the three positive controls was less than 10%.
Reported statistics and error estimates:
The 3-hour EC50 (Effective Concentration that inhibits respiration by 50%) and its 95% confidence limit were calculated by standard statistical methods.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Conclusions:
The 3-hour EC50 >1000 mg solids/L.
Executive summary:

The test substance was tested for respiration inhibition using the Activated Sludge, Respiration Inhibition test according to OECD Guideline 209 in the version dated July 22, 2010. The biological system was secondary activated sludge from the Elkton, Maryland (U.S.A) Publicly-Owned Treatment Works (POTW).
For the determination of the inhibitory behaviour of the test substance, activated sludge from the aeration tank of a municipal sewage treatment plant was exposed to the test substance at 1000 mg solids/L nominal concentration in triplicate. For the reference substance, 3,5-dichlorophenyl, activated sludge was exposed at 3.2, 10, and 32 mg/L nominal concentrations. After a 3-hour incubation period, the inhibition of the respiration rate of the activated sludge was determined in comparison to a test solution without any test or reference substance (positive control).
Under the conditions of the test, there was no significant difference in the microbial respiration of test systems with the test substance at 1000 mg solids/L compared to the positive controls. The Effective Concentration of the reference substance, 3,5-dichlorophenol, at which 50% inhibition occurred (EC50) was calculated as approximately 17.8 mg/L. The maximum difference between the respiration rates of the three positive controls was less than 10%. The test substance did not inhibit microbial respiration at a nominal concentration of 1000 mg solids/L. The test is valid.