Registration Dossier

Administrative data

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:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

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 time of study conduct
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 28 wt%

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
no
Details on test solutions:
No test vehicle was used, the test substance was added directly to the test vessels.

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
Secondary activated sludge from Elkton, Maryland, USA Publically Owned Treatment Works (POTW) was used as the microbial inoculum. The effluent was kept in glass containers under aerobic conditions in the period between sampling and application on return to the laboratory.

Study design

Water media type:
freshwater
Total exposure duration:
3 h

Test conditions

Test temperature:
The water temperature was measured in the positive control medium at the start and the end of the incubation period.
pH:
6.2
Dissolved oxygen:
A well-mixed sample of each treatment was transferred into a BOD bottle after 3 hours ± 5 min incubation. The dissolved oxygen (DO) concentration was measured with an oxygen electrode and recorded over a period of about 10 minutes. During measurement, the samples were continuously stirred with a stirrer built into the oxygen electrode. The rate of oxygen consumption (mg O2/L/min) was determined from the most linear part of the respiration curve, usually between approximately 6.5 to 2.5 mg O2/L, or, in case of low oxygen consumption, over a period of about 10 minutes.
Nominal and measured concentrations:
100 mg a.i. /L
Details on test conditions:
At time 0 h, 16 mL of synthetic sewage feed was diluted to 300 mL with dechlorinated tap water. The test was initiated with the first positive control by adding an appropriate amount of microbial inoculum (sludge), the amount of which was determined in a pre-experiment and by determining the sludge dry weight. Aeration was started. After approximately 15 minutes the above procedure was repeated, except that a known amount of the reference substance was added to the dechlorinated tap water and synthetic sewage feed and the test initiated with the addition of the microbial inoculum to a final volume of 500 mL. The procedure was repeated at approximately 15-minute intervals with the remaining two reference concentrations. Then the second positive control and then three replicates of the test substance added at a nominal concentration of 100 mg a.i./L. The final flask should be the third positive control, prepared exactly as the first two.
During pretesting, the test substance in the vessels foamed excessively. The foaming could be controlled by performing the test at an air flow rate of 2 L/min. across the surface and a test substance concentration of 100 mg a.i./L. An antifoaming agent was added to all test vessels, including the positive control, reference substance, and test substance vessels. The antifoam agent used was Antifoam A Concentrate, a silicon-based agent purchased from Sigma-Aldrich. It was added at a rate of 0.05mL/500mL.

An abiotic control was not performed.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
100 other: mg a.i./L
Nominal / measured:
nominal
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: highest concentration tested. Testing at higher concentrations was not possible due to excessive foaming.
Details on results:
Under the conditions of the test, there was no significant activated sludge respiration inhibition (p = 0.05) at 100 mg a.i./L, the highest concentration tested compared to the positive controls to which the test substance was not added.

Solids content of the microbial inoculum averaged 0.44g solids/L, which resulted in a concentration of 0.88g/L in the test vessels when 100 mL inoculum was added to a final volume of 500 mL. The pH of the microbial inoculum was 6.2.

The measured TOC of the reference substance stock solution averaged 16.9 mg C/L. (n = x). Based on the structure and purity of the reference substance, this corresponds to a stock solution concentration of 500 mg/L.
Results with reference substance (positive control):
The Effective Concentration of the reference substance 3, 5- Dichlorophenol at which 50% inhibition occurred (EC50) was approximately 11 mg/L.
The maximum difference between the respiration rates of the three positive controls was less than 10%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). The Effective Concentrations of the test substance at which 20, 50, and 80% inhibition occurred (EC20, EC50, EC80, respectively) could not be determined because there was no inhibition at the highest test concentration of 100 mg/L. Testing at higher concentrations was not possible due to excessive foaming.
Executive summary:

The test substance was tested for respiration inhibition using the Activated Sludge, Respiration Inhibition test according to OECD Guideline 209. The biological system used was secondary activated sludge from the Elkton, Maryland (U.S.A.) Publicly-Owned Treatment Works. 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 100 mg a.i./L/ nominal concentration in triplicate. Testing at higher concentrations was not possible due to excessive foaming. For the reference substance 3,5-dichlorophenol, activated sludge was exposed at 3.2, 10, and 32 mg a.i./L nominal concentrations. After a three 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 (p = 0.05) in the microbial respiration of test systems with the test substance at 100 mg a.i./L (100 a.i. ppm) compared to the positive controls. The Effective Concentration of the reference substance 3,5- Dichlorophenol at which 50% inhibition occurred (EC50) was approximately 10 mg/L. The maximum difference between the respiration rates of the three positive controls was less than 10%.