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:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
yes
Remarks:
Only 92% of stock solution of test substance achieved in first Range-finding study (later addition of sludge solution and synthetic sewage feed). A limit test with modification performed; test substance concentration of 100% established.
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
Only 92% of stock solution of test substance achieved in first Range-finding study (later addition of sludge solution and synthetic sewage feed). A limit test with modification performed; test substance concentration of 100% established.
Qualifier:
according to
Guideline:
other: EPA OCSPP 850.3300 (Modified Activated Sludge, Respiration Inhibition Test)
Deviations:
yes
Remarks:
Only 92% of stock solution of test substance achieved in first Range-finding study (later addition of sludge solution and synthetic sewage feed). A limit test with modification performed; test substance concentration of 100% established.
Qualifier:
according to
Guideline:
other: HJ/T 153-2004, The guidelines for the testing of chemicals[S]. Beijing: SEPA, 2004
Deviations:
yes
Remarks:
Only 92% of stock solution of test substance achieved in first Range-finding study (later addition of sludge solution and synthetic sewage feed). A limit test with modification performed; test substance concentration of 100% established.
Qualifier:
according to
Guideline:
other: CRC-MEP. The Guidelines for the Testing of Chemicals, Effects on Biotic Sysmtem[M]. The 2nd edition. Beijing: China Enviroment Press. 2013:94-112
Deviations:
yes
Remarks:
Only 92% of stock solution of test substance achieved in first Range-finding study (later addition of sludge solution and synthetic sewage feed). A limit test with modification performed; test substance concentration of 100% established.
Qualifier:
according to
Guideline:
other: GB/T 21976-2008, Chemicals - Activated sludge, respiration inhibition test[S]. Beijing: SAC, 2008
Deviations:
yes
Remarks:
Only 92% of stock solution of test substance achieved in first Range-finding study (later addition of sludge solution and synthetic sewage feed). A limit test with modification performed; test substance concentration of 100% established.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Details on test material:
- Purity: 99.9945%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations Sampled: Test substance in 100 mg/L of Tt3, Tt4, Tt5 was analysed.
-Sampling method: 1 mL upper air and 1 mL water in test bottles were drawn and both injected into headspace bottle which was pre-filled with 29 mL of deionized water. The headspace bottles were placed in a water bath at 80±2°C and heated for 30 minutes. 0.6 mL upper air and 0.6 ml water in headspace bottles was drawn and both injected into another headspace bottle which was pre-filled with 9.4 mL of deionized water. The headspace bottles were placed in a water bath at 80±2°C and heated for 30 minutes. 1 mL upper gas was drawn out from the headspace bottle and was manually injected into the gas chromatograph to determine the concentration.

Test solutions

Vehicle:
yes
Remarks:
Water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: 16 mL of the synthetic sewage feed and 8.36 g wet sludge (dry weight content wsa 8.97%) was added into a test bottle, and brought to a final volume of 500 mL with deionized water. Then the bottle was sealed. The test substance was then injected into the test bottles. Using the ideal gas law, 5, 50, and 500 mg test substance has a volume of 0.73 mL, 7.3 mL, and 73 mL (when T is 20°C) were used to prepare the test concentrations of 10, 100, and 1000 mg/L, respectively.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Activated sludge microorganisms
- Name and location of sewage treatment plant where inoculum was collected: Chendong waste water treatment plant receiving predominantly domestic sewage
- Method of cultivation: The sludge suspension was aerated overnight at 20±2°C.
- Preparation of inoculum for exposure: Before use, the sludge was washed 3 times with deionized water using the following method: after centrifuging at approximately 2500 r/m for 10 minutes, the supernatant was decanted and the remaining sludge solids re-suspended in deionized water and mixed well with a mechanical stirrer. After the third spin, a small amount of the fresh sludge was weighed, oven dried, and reweighed. The dry weight content of this wet sludge was calculated to be 8.97%. The wet sludge was added into the test bottles directly.

Study design

Test type:
not specified
Water media type:
other: Deionized water
Limit test:
yes
Total exposure duration:
30 min

Test conditions

Test temperature:
20±2°C
pH:
7.04~7.74
Dissolved oxygen:
8.50 mg O2/L (beginning of test)
8.51 mg O2/L (end of test)
Nominal and measured concentrations:
10 mg/L (nominal)
100 mg/L (nominal)
1000 mg/L (nominal) and 862 mg/L (Measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): closed
- No. of vessels per concentration (replicates):1 at 10 and 100 mg/L; 3 at 1000 mg/L
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration: dry weight content was calculated to be 8.97%

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : After 30 minutes, the respiraton rate was measured directly in the vessel until the oxygen concentration fell from about 7 to 2 mg/L, or over a period of up to 10 minutes. This determination was repeated on the contents of each vessel at 15 minute intervals so that the exposure time in each beaker was 3 hours.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.92, 9.2, 92% stock solution
- Results used to determine the conditions for the definitive study: A range-finding test was preformed where the sludge microorganisms were exposed to the test substance at concentrations of 0.92, 9.2, and 92% of the stock solution. The results indicated that the 30-minute EC50 was >92% stock concentration in water. A limit test was then performed where the sludge microorganisms were exposed to the test substance at a concentration of 100% of the stock solution with 3 parallels. The result indicated that the 30-minute EC50 was >100% stock concentration in water (measured concentration of 4.11 mg/). Stock solution was prepared and added into the test system both in the first and second test. However, measured concentration of stock solution was much lower than water solubility provided by the sponsor because the test substance is easily evaporated, causing the preparation and transformation of saturated solution to be very difficult. In order to achieve a higher test concentration, a third test was carried out. The test substance was directly injected into the test bottles to prepare the nominal concentration of 10, 100, and 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol

Results and discussion

Effect concentrations
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The % inhibition was -28.7 and -7.83% at 10 and 100 mg/L, respectively. The % inhibition in the 2 replicates at 1000 mg/L was -14.2, -6.33, and -11.7%.
The results show that there was no obvious oxygen uptake of abiotic blank of test substance for dissolved oxygen was 8.50 and 8.51 mg O2/L at the beginning and end of test. The oxygen uptake rates of negative controls were 37.9 and 44.7 mg O2/(L•h), which were 25.3 and 29.8 mg O2/(g•h) after considering the concentration of sludge of 1.5 g/L, fulfilling the validity criterion of not less than 20 mg O2/(g•h). Additionally, the coefficient of variation of the two negative control respiration rates was 17%, which was within 30% of each other.
Results with reference substance (positive control):
The 30-min EC50 value of the positive control (3,5-dichlorophenol) was 13.9 mg/L, and its 95% confidence limits ranged from 11.1 mg/L to 17.4 mg/L, which was within the range of 2 mg/L to 25 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
30-minute EC50: >1000 mg/L (nominal); >862 mg/L (analytical)
Executive summary:

The study was conducted according to the guidelines of HJ/T 153-2004, "The Guidelines for the Testing of Chemicals, Effects on Biotic Systems" (2nd edition) (Beijing: China Environment Press. 2013), and with the reference to OECD 209 "Activated Sludge, Respiration Inhibition Test" (Paris, 2010). Under valid conditions, a 30-min activated sludge respiration inhibition test was performed using activated sludge from a domestic waste water treatment plant. The results show that there was no obvious oxygen uptake of abiotic blank of test substance for dissolved oxygen was 8.50 and 8.51 mg O2/L at the beginning and end of test. The 30-min EC50 value of the positive control (3,5-dichlorophenol) was 13.9 mg/L, and its 95% confidence limits ranged from 11.1 mg/L to 17.4 mg/L, which was within the range of 2 mg/L to 25 mg/L. The oxygen uptake rates of negative controls were 37.9 and 44.7 mg O2/(L•h), which were 25.3 and 29.8 mg O2/(g•h) after considering the concentration of sludge of 1.5 g/L, fulfilling the validity criterion of not less than 20 mg O2/(g•h). Additionally, the coefficient of variation of the two negative control respiration rates was 17%, which was within 30% of each other. The validity criteria were fulfilled. Under the valid test conditions, the 30-minute EC50 of the test substance was >1000 mg/L in water (measured concentration 862 mg/L).