Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 August, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
White solid

Sampling and analysis

Analytical monitoring:
not specified
Details on sampling:
In a non-GLP preliminary test (Study No. Gl 729A002A), the test concentrations included 100, 300, 1000 and 3000 mg/L and an abiotic control at concentration of
1000 mg/L was performed, the contact time was 3 hours. According to the results, there was no oxygen consumption at the abiotic control and EC50 of test item may locate in the range of 100 - 3000 mg/L at contact times.

Based on the results of the preliminary test, the definitive test was conducted with 5 concentrations of 100, 234, 548, 1782 and 3000 mg/L with 5 replicates, contact
time for exposure was 3 hours. Two sets of test item mixtures at each concentration were prepared, 5.0 ml of ATU stock solution (1.16 g/L) was added to each mixture of one set. The test mixture without ATU was used to determine inhibition effects of total oxygen uptake and the determined values of test mixture containing ATU represented heterotrophic respiration only, and the differences between these and the corresponding total respiration rates represented nitrification. Because there was no oxygen uptake at the abiotic control in the preliminary test, the abiotic control was not repeated in the definitive test.

Test solutions

Vehicle:
yes
Details on test solutions:
Based on the results of preliminary test, the definitive test was conducted with 5 concentrations of 100, 234, 548, 1282 and 3000 mg/L (5 replicates for each concentration). Two sets of test item mixtures at each concentration were prepared, one set of test mixture was used to determine inhibition effects of total oxygen uptake, another set contained 11.6 mg ATU/L was used to determine inhibition effects of heterotrophic respiration only, the differences between heterotrophic and the corresponding total respiration rates represent nitrification. Inoculums blank controls without ATU and containing ATU were performed with 6 replicatesat the beginning, medium and end of the exposure period. The rate of respiration of each incubation mixture was determined after 3 hours contact time.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sludge was derived from a treatment plant receiving predominantly domestic sewage. A fresh sample of activated sludge was collected from the aeration tank of Shenyang North Sewage Treatment Plant 2 day before the test was started and the sludge was fed daily with synthetic sewage feed.

The activated sludge was fed with the synthetic sewage feed (50 mL synthetic sewage feed/L activated sludge) and continuously aerated at the test temperature (19.8-21.3°C) 2 days prior to use in the test. Before the activated sludge was used as the inoculums, the concentration of the sludge was determined again by drying method, and the concentration was adjusted to be 3.0 g/L.

The prepared medium was stored in the dark at 0 to 4 °C until used for the test and for no longer than 2 days.

Study design

Test type:
not specified
Water media type:
other: distilled
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
The contact time for exposure was 3 hours.
Post exposure observation period:
After the exposure period of 3 hours, a sample from the first aeration vessel was transferred to the measuring cell, an oxygen probe with a sleeve adaptor was then inserted into the neck of the flask and the magnetic stirrer was started, and the concentration of dissolved oxygen was immediately measured. The concentration of dissolved oxygen was continuously measured and recorded for 10 minutes. The cells were rinsed with water between measurements

Test conditions

Hardness:
Not specified
Test temperature:
20±2°C in a temperature controlled room, actual recorded temperature was 19.4-21.3°C.
pH:
pH of this solution was 6.93, adjust pH to 7 .03 with NaOH.
Dissolved oxygen:
Dissolved oxygen concentration above 60 - 70% saturation
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Not specified
Details on test conditions:
Contact time for exposure: 3 hours
Test water: distilled water
Air for aeration: clean oil-free air, flow rate 0.5-1 L/min
Test temperature: 20±2°C in a temperature controlled room, actual recorded temperature was 19.4-21.3°C.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
410.2 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
820.4 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of heterotrophic respiration
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
625.2 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
inhibition of respiration due to nitrification
Details on results:
The specific oxygen uptake rate of the blank controls (without the test item or reference item) was 32.8 mg/(g·h) oxygen. The coefficient of variation of oxygen uptake rate in control replicates was 5.9% at the end of definitive test. The validity criteria were satisfied.
Results with reference substance (positive control):
Based on the results of reference item test, the EC (3 hours) of 3,5-dichlorophenol for three different respirations are as follows:

Inhibition of respiration EC50(mg/L) 95% confidence limits (mg/L)
Total 4.0 2.4-6.6
Heterotrophic 5.3 3.7-7.7
Nitrification 1.6 0.5-4.8

The validity criteria for the reference item EC50 values were also satisfied.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of test item, the EC10 and EC50 (3 hours) of the test item for three different respirations are as follows:

Inhibition of respiration EC10 and 95% confidence limits (mg/L) EC50 and 95% confidence limits (mg/L)
Total 275.3 (128.6-589.2) 410.2 (191.7-877.9)
Heterotrophic 591.0 (257.7-1355.5) 820.4 (357.7-1881.5)
Nitrification 129.9 (82.4-204.6) 625.2 (396.8-984.9)