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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OCSPP 850.330 Modified Activated Sludge, Respiration Inhibition Test
Version / remarks:
2012
Deviations:
no
Principles of method if other than guideline:
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.
This preliminary test was used to estimate the range of concentrations of the test item needed in a possible definite test for determining the inhibition of oxygen consumption.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
At the start of the test defined amounts (1 x 3; 1 x 30; 3 x 300 mg test item that corresponded to the investigated 10, 100 and 1000 mg/L concentrations were directly weighed (administered) into each test flask and the subsequent calculations refer to the initial weighed nominal concentration.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
Species:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, 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, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. 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 fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.31), pH adjustment of the inoculum was considered not necessary.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
no
Hardness:
Not determined.
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: 20.3-21.6 °C.
The water temperature was recorded during the oxygen measurement in all test bottles.

pH:
The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all test concentrations, reference item concentrations and controls.
Dissolved oxygen:
The test flasks were aerated with compressed air (0.5 L/minute).
Salinity:
/
Conductivity:
/
Nominal and measured concentrations:
Nominal: 0 - 10 - 100 - 1000 mg/L
Details on test conditions:
The following test item properties were investigated in the pre-experiment: 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.
This preliminary test was designed according to the guideline requirements, triplicates were examined at the highest tested concentration of 1000 mg/L (as a limit concentration) and additionally two lower concentration levels of 10 and 100 mg/L were examined with one vessel each. Blank, abiotic (three abiotic controls with the highest concentration of the test item) and reference controls were included.
The nitrification potential of sludge was examined with additional control mixture (nitrification control in three parallels) that contained N-allylthiourea at 11.6 mg/L..
The composition of the test media is displayed under "Attached Document", below.

Controls
Blank Control (CB): In the test, eight replicates of blank control group (purified, deionized water, synthetic sewage and inoculum, but without addition of the test or reference item) were tested concurrently, four at the start and four at the end of the test series.
Abiotic Control (CA): Abiotic controls (purified, deionized water, synthetic sewage and the test item at the concentration of 1000 mg/L, but without inoculum) were prepared to measure the abiotic oxygen consumption. The abiotic control was tested concurrently at the highest test item concentration in triplicate.
Reference Control (R): Concurrent to the test item, the reference item 3,5-Dichlorophenol was tested at three concentrations each with three parallels (the nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions.
Nitrification Control (CN): In order to decide whether the sludge nitrifies and at what rate, mixtures in three parallels (same as the blank controls, however containing 11.6 mg/L N-allylthiourea) were included in the preliminary experiment.

Test solution with a final volume of 300 mL was tested per treatment in a glass flask.
The test flasks were incubated for 3 hours. The test vessels were aerated and shaken continuously such as to ensure an appropriate dissolved O2 level in the samples.
In this study analytical measurements were not performed.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol. Nitrification inhibitor: N-allythiourea (ATU).
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
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
Details on results:
In the experiment abiotic oxygen consumption of the test substance was not observed (in average -0.73 mg/Lh); therefore the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations. The measured nitrification potential of the applied activated sludge (see nitrification control values) did not necessitate further testing.

The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls, no inhibitory effect (the observed slight 0.65 % at 10 mg/L, 1.64 % at 100 mg/L and in average 1.05 % at 1000 mg/L inhibitions were within the biological variability of the applied test system) of the test item was observed.

The specific respiration rates of the highest dose, 1000 mg/L were compared with the blank control values using 2 Sample t-Test (α=0.05). No statistical significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/L.

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.



Results with reference substance (positive control):
Positive reference control: The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 13.87 mg/L, (95 % confidence limits: 11.23-17.87 mg/L).

Nitrification Controls: 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 50.53 mg/Lh, the heterotrophic respiration (RH) was 49.67 mg/Lh, the nitrification respiration (RN): 0.86 mg/Lh was calculated according to the equation: RN= RT-RH.
The obtained 0.86 mg/Lh was considered as not significant difference, being within a biological variability range of the applied test system, that lower than the 5 % of RT (2.53 mg/Lh) in blank controls
According to the above calculations and based on the OECD 209 guideline it was assumed that the heterotrophic oxygen uptake equals the total uptake.
Reported statistics and error estimates:
See above.

Validity of the Study:

The specific respiration rate of the blank controls (without the test substance or reference substance) was 33.69 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 2.87 %.

The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 13.87 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).

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Tests, the 3-hour EC50 and EC10 values of the test item were estimated to >1000 mg/L.
Executive summary:

The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions, according to the OECD guideline 209. 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.

Results: Under the conditions of the performed Activated Sludge Respiration Inhibition Tests, the 3-hour EC10 and EC50 values of the test item were estimated to >1000 mg/L.

The respiration due to nitrification was considered to be negligible.

A definite test was considered as unnecessary.

Description of key information

Under the conditions of the performed Activated Sludge Respiration Inhibition Tests, the 3-hour EC50 and EC10 values of the test item were estimated to >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The EC50 and EC10 are not 1000 mg/L, as one is forced to enter in the field above, but the EC50 and the EC10 are >1000 mg/L.