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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03 September 2018 to 13 August 2019
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 (Carbon and Ammonium Oxidation))
Version / remarks:
2010
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
There was no analytical monitoring of the test substance concentrations during the ASRI test; however, prior to this test, analytical investigations were conducted to assess the stability of the test substance at 10, 100 and 1000 mg/L over 3 hours.
Details on sampling:
- Concentrations: A preliminary test was run in order to assess the test substance stability over 3 hours. Three solutions with nominal concentrations of 10, 100 and 1000mg/L, each with three replicates, were prepared by diluting a stock solution (of known measured concentration). Each solution was added in 2L closed vessels, and then was incubated in the shaker at the speed of 150rpm.
- Sampling method: The test samples were taken after 0h, 30min and 3h from the 10, 100 and 1000 mg/L solutions prepared in the context of the preliminary assay intended to assess the test substance stability (i.e. there was no sample taken during the ASRI test for chemical analysis).
- Sample storage conditions before analysis: in refrigerator (2-8°C) after pretreatment if the samples were not analyzed directly within 24h.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 5 g/L test substance stock solution was prepared by dissolving 5.0002g test substance into 1L ultra-pure water. The pH of the solution was 7.51 and adjustment was not needed. The test substance stock solution was added into the test vessels of the test substance group as described below:
* Test substance group (FT): Definite volumes of test substance stock solution (concentration: 5 g/L) were mixed with 16mL synthetic sewage feed; ultra-pure water was then added till a volume of 250mL and then this was mixed with 250mL activated sludge suspended solution. Finally, concentrations of 10mg/L, 100mg/L and 1000mg/L were obtained.
- Controls: Different control groups were run in parallel:
* Blank control group (FB): 16mL synthetic sewage feed were mixed with ultra-pure water added up to a volume of 250mL, then this was mixed with 250mL activated sludge suspended solution.
* Abiotic control group (FA): Test substance stock solution (concentration: 5 g/L) was mixed with 16mL synthetic sewage feed; then ultra-pure water was added till a volume of 500mL. The final concentration was 1000mg/L.
* Reference substance group (FR): Definite volumes of reference substance stock solution (concentration: 1 g/L) were mixed with 16mL synthetic sewage feed; ultra-pure water was then added till a volume of 250mL and then this was mixed with 250mL activated sludge suspended solution. Finally, concentrations of 7.80mg/L, 12.60mg/L, 20.00 and 32.00mg/L were obtained.
- Test concentration separation factor: 10 for FT and ca. 1.6 for FR.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: No, sludge collected from a wastewater treatment plant.
- Name and location of sewage treatment plant where inoculum was collected: Aeration stage of Tianshan Wastewater Treatment Plant in Shanghai.
- Preparation of inoculum for exposure: The sludge was aerated till use. The sludge was washed with an isotonic solution containing 0.85% NaCl(w/v). After centrifuging the supernatant was decanted. This procedure was repeated three times (4000rpm, 4°C , centrifugation for 20 minutes).
- Initial biomass concentration: A small amount of the washed sludge was weighed and dried by moisture meter at 105°C for 1h to calculate the dry weight. The dry weight of sludge count up to 9.73%. The amount of wet sludge needed to be suspended in isotonic solution was calculated based on the dry weight. Then wet sludge was placed into a certain amount of solution to obtain the required sludge solids concentration of 3g/L. The obtained dispersion was suspended with a mixer and aerated till use. The final concentration of the activated sludge in the test medium was 1.5g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
No data available.
Test temperature:
Approximately 20.4°C.
pH:
FT: 7.59 to 7.89
FB: 7.52 to 7.89
FA: 7.42 to 7.50
FR: 7.46 to 7.58
Dissolved oxygen:
See in Table 1 in the field "Any other information on results incl. tables".
Salinity:
Not concerned.
Conductivity:
No data available.
Nominal and measured concentrations:
Nominal concentrations during the ASRI test: 10, 100 and 1000 mg/L.
Actual concentrations were not measured during the ASRI test but they were measured during a preliminary assay intended to assess the stability of 10-, 100- and 1000-mg/L test substance solutions over 3 hours. After 0h, 30min and 3h, all the measured concentrations were comprised between 97.6 and 104% of the nominal concentrations (see further details in the field "Any other information on materials and methods incl. tables").
Details on test conditions:
TEST SYSTEM
- Test vessel: 2L vessels.
- Type: closed.
- Fill volume: 500 mL.
- Aeration: Use of a shaker at the speed of 150rpm, keeping the dissolved oxygen concentration above 60-70% saturation to maintain the sludge flocs in suspension.
- No. of vessels per concentration in the test substance group (replicates): 1 for 10 and 100 mg/L test concentrations and 3 for the 1000 mg/L test concentration.
- No. of vessels for the blank control group (replicates): 2.
- No. of vessels for the abiotic control group (replicates): 1.
- No. of vessels per concentration in the reference substance group (replicates): 1.
- Sludge concentration: 1.5 g/L.
- Nutrients provided for bacteria: A synthetic sewage was made by dissolving the following ingredients in 1 liter of ultra pure water: 16.0 g peptone, 11.0 g meat extract, 3.0 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2H2O, 0.2 g MgSO4 x 7H2O, and 2.8 g K2HPO4.
- Nitrification inhibitor used: No.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra-pure water produced by Millipore MilliQ A10.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod and light intensity: no data available.
- Details on termination of incubation: After the exposure periods (3 hours), a sample was transferred from the aeration vessel to BOD flask and stirring was started immediately, then the concentration of dissolved oxygen was measured. The electrode was removed, the mixture was returned to the aeration vessel and aerating and stirring were continued.

EFFECT PARAMETERS MEASURED
Dissolved oxygen contents were measured in order to calculate the oxygen uptake rate, the specific respiration rate and the percentage of respiration inhibition.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 for the test substance group and ca. 1.6 for the reference substance group.
- Range finding study:
* Test concentrations: The study was designed as a combined range-finding/limit test, three test substance concentrations being tested (10, 100 and 1000 mg/L), with only the highest one presenting three replicates.
* Results used to determine the conditions for the definitive study: Because no adverse effect was obtained at the highest concentration tested, no definitive test was performed.
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
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
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.
- Adsorption (e.g. of test material to the walls of the test container): no data available.
- Blank controls oxygen specific respiration rates: 29.41mg/(h*g SS) and 28.97mg/(h*g SS) for both replicates and thus in adequation with the validity criterion recommending a value superior to 20mg/(h*g SS).
- Coefficient of variation of oxygen uptake rate in control replicates: 1.08% and thus lower than 30% at the end of the test as required by the validity criterion.
Results with reference substance (positive control):
- Results with reference substance is valid.
- Relevant effect levels: the 3hours-EC50 of 3,5-dichlorophenol was 14.43 mg/L which lied in the acceptability range of 2mg/L to 25mg/L.
Reported statistics and error estimates:
t-Tests were used to indicate significant differences between test groups and blank controls.
Activated sludge respiration inhibition EC50 of Reference substance was calculated with Linear interpolation method.
The software was Toxcalc v5.0.32.

Results in the abiotic control group (FA)


The respiration rate of FA was 2.28mg/Lh, meaning that negligible abiotic oxygen consumption was observed.


 


Results in the test substance groups (FT)


Inhibition percentage of the test substance on the respiration of activated sewage sludge was 1.84% under the concentration of 10mg/L. Negative values were reported for inhibition percentages under the concentrations of 100mg/L (-5.15%) and 1000mg/L (-9.99%, -17.37% and -17.69%); indicating that the respiration rates were higher in the groups exposed to these concentrations as compared to the control groups. t-Test indicated these differences between test groups and blank controls were significant (p=0.0087, p<0.05); meaning that respiration rates of test groups were significantly higher as compared to blank control. Therefore, it is regarded that there was no inhibition effect of the test substance on the respiration of activated sewage sludge (see table 1).


 


Global results


Table 1: pH, oxygen consumption rates and percentage inhibition values of the test substance and control groups during the range-finding test.





























































































































































Test substance



pH



DO (mg/L)



T (min)



R (mg/Lh)



Rs (mg/gh)



IT (%)



0h



3h



Upper limit(Q1)



Lower limit(Q2)



FB (mg/L)



C1



7.53



7.89



6.88



2.10



6.50



44.12



29.41



-



C2



7.52



7.83



6.91



2.08



6.67



43.45



28.97



-



FT (mg/L)



10



7.78



7.89



5.98



2.08



5.17



45.26



30.17



1.84



100



7.69



7.81



5.96



2.07



4.83



48.32



32.21



-5.15



1000



7.61



7.73



5.35



2.13



3.83



50.44



33.63



-9.99



1000



7.63



7.69



5.76



2.03



4.17



53.67



35.78



-17.37



1000



7.59



7.71



5.82



2.08



4.17



53.81



35.87



-17.69



FA (mg/L)



1000



7.50



7.42



8.78



8.40



10.00



2.28



1.52



-



 


 


FR (mg/L)


 


 


 



7.80



7.46



7.58



6.52



2.02



7.33



36.83



24.55



15.88



12.6



7.54



7.54



6.98



2.74



10.67



23.84



15.89



45.55



20.0



7.47



7.51



7.73



5.07



10.00



15.96



10.64



63.55



32.0



7.54



7.57



7.95



6.32



10.00



9.78



6.52



77.66



FB: Blank control groups (C1 and C2: 2 replicates)


FT: Test substance groups


FA: Abiotic control group


FR: Reference substance groups


Q1: Oxygen concentration at the beginning of the selected section of the linear phase (mg/L)


Q2: Oxygen concentration at the end of the selected section of the linear phase (mg/L)


T: Time interval between these two measurements (min)


R: Oxygen uptake rate (mg/Lh)


Rs: Specific respiration rate (mg/gh)


IT: Percentage inhibition of total oxygen uptake relative to the blank controls


 


 


Validation Criteria
• The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 14.43 mg/L mg/L and therefore within the acceptance range of 2 to 25 mg/L for total respiration.
• The specific respiration rate of the controls was 29.41mg/(h*g SS) and 28.97mg/(h*g SS) for both replicates and therefore greater than the threshold of 20 mg oxygen per gram dry weight of sludge per hour.
• The coefficient of variation of oxygen uptake in the control vessels was 1.08% and therefore less than the threshold of 30%.


=> All validation criteria for the study were therefore satisfied.

Validity criteria fulfilled:
yes
Remarks:
(see above).
Conclusions:
The 3-hour EC50 and 3-hour NOEC of 2,2-Difluoroethyl acetate to activated sludge based on respiration inhibition were determined to be > 1000 mg/L and ≥ 1000 mg/L, respectively.
Executive summary:

The toxicity of 2,2-Difluoroethyl acetate to the respiration of activated sludge was investigated in a 3-hour test according to the OECD guideline 209 under GLP compliance.


Four different treatments were carried out for 3 hours:



  • Blank control group: containing synthetic sewage feed and activated sludge.

  • Test substance group: containing test substance (at 10, 100 or 1000 mg/L), synthetic sewage feed and activated sludge.

  • Abiotic control group: containing test substance (at 1000 mg/L) and synthetic sewage feed.

  • Reference substance group: containing reference substance (at 7.8 mg/L, 12.6 mg/L, 20 and 32 mg/L), synthetic sewage feed and activated sludge.


After 3 hours, dissolved oxygen contents were measured in order to calculate the oxygen uptake rate and the percentage of respiration inhibition.


 


In the abiotic control group, negligible abiotic oxygen consumption was observed. In the test substance group, respiration inhibition percentage was 1.84% under the concentration of 10 mg/L. Negative values were reported for inhibition percentages under the concentrations of 100 mg/L (-5.15%) and 1000 mg/L (-9.99%, -17.37% and -17.69%); indicating that the respiration rates were higher in the groups exposed to these concentrations as compared to the control groups. Therefore, it is regarded that there was no inhibition effect of the test substance on the respiration of activated sewage sludge. The 3-hour EC50 value for the reference item, 3,5-dichlorophenol, was 14.43 mg/L mg/L and therefore within the acceptance range of 2 to 25 mg/L for total respiration, confirming the suitability of the test system.


Therefore, the 3-hour EC50 and 3-hour NOEC of 2,2-Difluoroethyl acetate to activated sludge based on respiration inhibition were determined to be > 1000 mg/L and ≥ 1000 mg/L, respectively.

Description of key information

The 3-hour EC50 and 3-hour NOEC of 2,2-Difluoroethyl acetate to activated sludge based on respiration inhibition were determined to be > 1000 mg/L and ≥ 1000 mg/L, respectively.

Key value for chemical safety assessment

Additional information

An experimental study performed under GLP compliance and in accordance with OECD test guideline 209 was flagged as a key study and assigned a Klimisch score of 1. It revealed no adverse effects of 2,2-Difluoroethyl acetate on the respiration of activated sewage sludge. Therefore, the 3-hour EC50 and 3-hour NOEC of 2,2-Difluoroethyl acetate to activated sludge based on respiration inhibition were determined to be > 1000 mg/L and ≥ 1000 mg/L, respectively.