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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-02-19 to 2018-07-05
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:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
May 30, 2008 (updated on 1st March 2016)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In the main test the test item concentration (as a limit concentration) of 1000 mg/L was
examined with five replicates. The defined amounts (5 x 300 mg) of the test item were
(administered) measured directly into empty containers that were filled up with water and
synthetic sewage, just before the inoculation. Concentrations in excess of nominal
1000 mg test item/L were not tested.
- Controls: eight replicates of control group (deionized water, synthetic sewage and inoculum, but without addition of the test or reference item) were tested in parallel, four at the start and four at the end of the test series.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): NA
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): NA
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 19 February 2018 (two days before the test).
- Preparation of Activated Sludge Inoculum: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (7.379 g wet weight), dried and the
ratio of wet sludge to dry weight (0.6669 g dry weight) determined. Based on this ratio,
calculated amount of wet sludge (27 g dry weight that was equivalent to 299 g wet sludge)
was suspended in isotonic saline solution (ad. 9 L) to yield a concentration equivalent to
about 3 g per litre (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids,
containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The activated sludge was not used on the day of the collection but continuously aerated (2
L/minute) at the test temperature for about 48 hours (2 days) and was fed daily with 50 mL
synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.38),
additional pH adjustment of the inoculum was considered not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
The test was carried out in a controlled environment room (during the incubation, during the formulation and oxygen concentration measuring) at a temperature of 20 ± 2 °C according to the guideline. The recorded temperatures in the environmental room varied between: 20.5-21.8 °C.
Nominal and measured concentrations:
Nominal: 1000 mG/L
Details on test conditions:
TEST SYSTEM
- Test vessel/Material, size, headspace, fill volume: Erlenmeyer glass bottles of approximately 300 mL volume.
- Type (delete if not applicable): open
- Aeration: The test vessels were aerated and shaken continuously such as to ensure an appropriate dissolved O2 level in the samples.
- No. of vessels per concentration (replicates): 5 replicates
- No. of vessels per control (replicates): Blank Control (CB): eight controls (four at the start and four at the end of the test series)
- No. of vessels per vehicle control (replicates): NA
- No. of vessels per abiotic control (replicates): In the preliminary range-finding the abiotic control (deionized water, synthetic sewage and the test item in the corresponding concentration, but without inoculum) was tested at the highest test item concentration in three parallels. In the main test the abiotic control was not repeated because oxygen uptake was not observed in the abiotic control group in the preliminary experiment.
- Reference Control (R): in parallel to the preliminary test with the test item, the reference item 3,5-Dichlorophenol was tested at three concentrations, each with one test vessel (the proposed nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions. In the main test the reference item 3,5-dichlorophenol was tested at three concentrations with three parallels (at the nominal test concentrations of 2, 7 and 24.5 mg/L).
- Sludge concentration (weight of dry solids per volume): In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria: Synthetic Sewage Feed: 9.6 mL for 300 mL (final test volume)
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea : In order to decide whether the sludge nitrifies and at what rate, mixtures in two parallels
(same as the blank controls, however containing 11.6 mg/L N-allylthiourea) were included in the preliminary experiment. The nitrification respiration was not significant and it can be assumed that the heterotrophic oxygen uptake equals the total uptake and no significant nitrification occurred.
The main test was performed in one set of test vessels, without ATU addition, because of a differentiation between heterotrophic respiration and nitrification was considered as not necessary. In the main test (for informative reason) a nitrification control group (containing N-allylthiourea) was investigated with three parallels.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not specified
- Light intensity: not specified
- Details on termination of incubation: The measurement of the respiration rate in a well-mixed aerated sample of each treatment was performed after exactly 3 hours incubation time. The treatment sample was not further aerated. The oxygen concentration was measured with O2 electrode (working based on LDO
method) under stirred conditions and was recorded for about 4-10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason four O2 electrodes were used. Simultaneous (a maximum of four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in 7 cycles with the available four O2 electrodes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : See above. The oxygen consumption rate (in mg O2 L-1 hour-1) was determined from the linear part of the respiration curve (in the range between approximately 2 -7 mg O2/L).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: NA, only one limit concentration was tested.
- Justification for using fewer concentrations than requested by guideline: based on the results of the preliminary tests
- Range finding study : Yes
- Test concentrations: the test item effect on the pH within the test system and the test item effect on the oxygen consumption of the activated sludge inoculum at the concentrations of 10 mg/L, 100 mg/L and 1000 mg/L. According to the guideline requirement triplicates at the highest tested concentration of the
preliminary test (1000 mg/L), blank, abiotic (three abiotic controls with the highest concentration of the test substance), reference and nitrification controls were included in the preliminary test. Test media were prepared freshly before the test. The pH of the solutions of the item in the test was in the acceptable range (pH 7-8) prior to and after inoculum addition. In the preliminary experiments the test item did not show an inhibitory effect (inhibition was not detected at the nominal concentration of 1000 mg/L) on the applied test system.
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:
EC10
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: NA, no analytical monitoring of the tests substance was performed.
- Effect concentrations exceeding solubility of substance in test medium: NA
- Adsorption (e.g. of test material to the walls of the test container): not specified
- Blank controls oxygen uptake rate and Coefficient of variation of oxygen uptake rate in control replicates: The specific respiration rate of the blank controls (without the test substance or reference substance) was 45.93 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 5.74 %.
Additionally, the difference between “start” and “end” blank control respiration rates (CBA, CBB, CBC and CBD beginning and CBE, CBF, CBG, and CBH end of the exposure period, respectively) were minimal (~6 %), within the biological variability of the applied system.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 17.66 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).
Reported statistics and error estimates:
The EC50 value of the reference item was calculated using Probit analysis using the IBM® SPSS® Statistics, Version 24 (2016) statistical software program.
The specific respiration rates of the highest test item concentration level were compared to the blank control values by 2 Sample t-Test (α=0.05) by IBM® SPSS® Statistics, Version 24 (2016) software.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect (the observed slight in average 2.10% inhibition was within the biological variability of the applied test system) of the test item was observed.

An additional nitrification control was examined in the test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 68.89 mg/Lh, the heterotrophic

respiration (RH) was 68.89 mg/Lh, the nitrification respiration (RN): 0.01 mg/Lh was calculated according to the equation: RN= RT-RH.

The obtained low value of 0.01 mg/Lh (calculated by Excel software) was considered as not significant difference, being within a biological variability range of the applied test system, that lower than the 5 % of RT (3.44 mg/Lh) in blank controls.

Validity criteria fulfilled:
yes
Remarks:
Average specific respiration rate of the blank was 45.93 mg O2/g activated sludge (on dry weight basis) in an hour (criteria>20) with a CV of 5.74 %. The 3H-EC50 of 3,5-Dichlorophenol was 17.66 mg/L within the range of 2 mg/L to 25 mg/L.
Conclusions:
Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.
Executive summary:

The purpose of the test was to evaluate the influence of the test item 2,5 -Dimethoxytetrahydrofuran on the activity of activated sludge by measuring the respiration rate under defined conditions, according to OECD 209 guideline and following GLP. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The sensitivity of the used batch of activated sludge was checked with a suitable reference substance on the day of exposure. The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. All validity criteria of the study were met. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

Description of key information

Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.

Key value for chemical safety assessment

Additional information

The purpose of the test was to evaluate the influence of the test item 2,5 -Dimethoxytetrahydrofuran on the activity of activated sludge by measuring the respiration rate under defined conditions, according to OECD 209 guideline and following GLP. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. The sensitivity of the used batch of activated sludge was checked with a suitable reference substance on the day of exposure. The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. All validity criteria of the study were met. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L. The NOEC was determined to be 1000 mg/L.