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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
2016
Qualifier:
according to guideline
Guideline:
other: OCSPP 850.330 Modified Activated Sludge, Respiration Inhibition Test
Version / remarks:
2012
Deviations:
no
Principles of method if other than guideline:
Two preliminary tests were performed. The second one (called complementary test), was started because of an inhibitory tendency of the results of the first test. According to the results of both experiments, no further tests with a range of test substance concentrations were performed.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
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, furthermore 3 x 300 mg for abiotic controls) of the test item were administered directly.
At the start of the complementary test defined amounts (2 x 5 x 300 mg test item, that corresponded to the investigated 1000 mg/L concentration of the test item) were administered directly.
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 preliminary experiment and one day before the complementary experiment.
Preparation:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge 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.
Preparation of Activated Sludge Inoculum:
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 activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 48 and 24 hours, respectively and fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.36 and 7.25, respectively) and before use (pH: 7.88 and 7.69, respectively). pH adjustment of the inoculum was considered not necessary in any case.
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:
In the preliminary test the temperature in the test mixtures during the measurements was in average: 20.4 °C, the measured minimum: 19.6 °C, maximum: 21.0 °C.
In the complementary preliminary test the temperature in the test mixtures during the measurements was in average: 21.4 °C, the measured minimum: 19.2 °C, maximum: 22.0 °C.
pH:
The preliminary test was performed without pH adjustment, but in the complementary preliminary test in one series of the test vessels additional pH adjustment was performed with 1N NaOH to reach the acceptable pH range (pH 7-8).
The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum, prepared test mixtures (except the test mixtures without pH setting) were in the acceptable pH 7-8 range at the start of the tests.
Dissolved oxygen:
The mixtures were aerated at 0.5 L/min in the temperature range of 20 ± 2 °C.
Salinity:
/
Conductivity:
/
Nominal and measured concentrations:
Nominal:
First test: 0 - 10 - 100 - 1000 mg/L
Complemenrtary test: 0 - 1000 mg/L
Details on test conditions:
First test:
The study design of the present pre-test (test item concentrations, controls) fulfilled the guideline criteria; however regarding the inhibitory tendencies of the test item in this preliminary test equivocal results were obtained: 11.60 % inhibition was obtained at the lowest test item concentration of 10 mg/L, -1.52 % (considered as zero inhibition) at 100 mg/L and in average 5.77 % at the highest examined concentration of 1000 mg/L. Furthermore, the test item had significant effect on the pH: at the highest examined concentration of 1000 mg/L. The pH of the test mixture was in the range of 4.19-4.25, without inoculum addition. To investigate the real test item effect within this test system and the possible effect of the pH an additional complementary experiment (complementary preliminary experiment) was considered as necessary.
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 two parallels) that contained N-allylthiourea at 11.6 mg/L.
Second test:
The complementary preliminary test was designed according to the guideline requirements, and based on the experiences obtained in the preliminary test. Five parallels were examined at the tested concentration of 1000 mg/L (as a limit concentration).
One series of test item treatments was investigated with pH adjustment, one series without pH adjustment (the test item mixture was neutralized with addition of 1N NaOH prior to inoculum addition).
In parallel, blank (in eight parallels), nitrification (in three parallels) and reference controls (in three concentrations in three parallels) were included.
Controls:
Blank Control (CB):
In the preliminary test six replicates of blank control group (purified, deionized water, synthetic sewage and inoculum, but without addition of the test or reference item) were tested concurrently, three at the start and three at the end of the test series. In the complementary preliminary test eight replicates of blank control group were tested concurrently, four at the start and four at the end of the test series.

Abiotic Control (CA):
In the preliminary test 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. In the complementary test abiotic control was not investigated.

Reference Control (R):
Concurrent to the test item, the reference item 3,5-Dichlorophenol was tested at three concentrations (the proposed nominal test concentrations of 2, 7 and 24.5 mg/L) under otherwise identical test conditions. The reference item was investigated in one vessel per concentration in the preliminary test and in three parallels per concentration in the complementary test.

Nitrification Control (CN):
In the preliminary test in order to decide whether the sludge nitrifies and at what rate, two concurrent nitrification controls (same as the blank controls, however containing 11.6 mg/L N-allylthiourea) were included.
For the same reason nitrification control in three parallels was included in the complementary test.

Course of the Experiments
Test solution with a final volume of 300 mL was tested per treatment in a glass flask. 9.6 mL synthetic sewage and an adequate volume of the stock solution of the N-allylthiourea adequate volumes of the stock solution of reference item were filled up with water (with purified, deionized water) to 150 mL before the start of the test. At the test item treatments the appropriate amounts of the test item were measured directly into the empty test containers. At the start of the test 150 mL activated sludge inoculum with a sludge concentration of 3 g/L (on dry weight basis) was added to the test containers, first to the controls (blank, nitrification, reference) thereafter to the test item (containing the adequate amount of test item, the 150 mL synthetic sewage-water mixture that was filled into the test containers just before the inoculation, and in one test series the pH was additionally set up with 1N NaOH) and finally to the “end” controls. The Tables 1 and 2 contain exact order and number of examined parallels of the test vessels regarding the controls and test item treatments.

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.
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
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:
First test:
In this experiment the abiotic oxygen consumption of the test substance was not noticed, see Table 6); 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 (nitrification control) did not necessitate further testing.
Equivocal results were obtained: 11.6 % inhibition was obtained at the lowest test item concentration of 10 mg/L, -1.52 % (considered as zero inhibition) at 100 mg/L and in average 5.77 % at the highest examined concentration of 1000 mg/L. 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) by TOXSTAT software. Statistical significant difference was not noticed in the comparison with the blank control values; however the inhibitory tendencies were equivocal. Based on these results the performance of an additional complementary test (complementary preliminary experiment) was considered as necessary.
Second test:
The observed oxygen consumption rates consequently the specific respiration rates were in the range of the blank controls in both series (without and with pH adjustment), no inhibitory effect of the test item was observed.
There was only negligible difference between the oxygen consumption rates (and the specific respiration rates) between the test groups without and with pH adjustment.
The specific respiration rates of the 1000 mg/L dose (without and with pH adjustment) were compared with the blank control values using 2 Sample t-Test (α=0.05) by TOXSTAT software. No statistical significant differences were observed in the comparison with the blank control values.
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 greater than 1000 mg/L. The EC50 value was determined as: EC50 > 1000 mg/L.

These tests demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L. Therefore, a definite test was considered as unnecessary.
Results with reference substance (positive control):
Positive reference control:
First test:
In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 9.43% at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 21.22 % and 63.90 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 17.04 mg/L, (95 % confidence limits: 12.93-22.45 mg/L).
Second test:
In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 8.17 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 23.42 % and 80.04 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 15.54 mg/L, (95 % confidence limits: 13.66-17.42 mg/L).

Nitrification Controls:
First test:
The total respiration (RT) was 78.90 mg/Lh, the heterotrophic respiration (RH) was 79.80 mg/Lh, the nitrification respiration (RN): -0.90 mg/Lh was calculated according to the following equation: RN= RT-RH.
The obtained -0.90 mg/Lh was considered as not significant difference within a biological variability range of the applied test system, that lower than the 5 % of RT (3.95 mg/Lh) in blank controls.
According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.
Second test:
The total respiration (RT) was 60.42 mg/Lh, the heterotrophic respiration (RH) was 60.16 mg/Lh, the nitrification respiration (RN): 0.26 mg/Lh.
The obtained 0.26 mg/Lh was considered as not significant difference within a biological variability range of the applied test system, that lower than the 5 % of RT (3.02 mg/Lh) in blank controls.
According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Validity of the Study

The specific respiration rate of the blank controls (without the test substance or reference substance) was higher than 20 mg/gh in both performed experiments. In the preliminary test it was 52.60 mg O2/gh *; in the complementary test it was 40.28 mg O2/gh.

* mg O2/gh: mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.

The coefficient of variation of oxygen uptake rate in control replicates was lower than 30% in both tests. In the preliminary test it was 5.43 %; in the complementary test it was 4.80%.

The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was in the range of 2 mg/L to 25 mg/L for total respiration (in these experiments the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

In the preliminary test the 3-hour EC50 of the reference item 3,5-Dichlorophenol was 17.04 mg/L; in the complementary test it was 15.54 mg/L.

Environmental Conditions (pH, Temperature):

The preliminary test was performed without pH adjustment, but in the complementary preliminary test in one series of the test vessels additional pH adjustment was performed with 1N NaOH to reach the acceptable pH range (pH 7-8). The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum, prepared test mixtures (except the test mixtures without pH setting) were in the acceptable pH 7-8 range at the start of the tests.

The mixtures were aerated at 0.5 L/min in the temperature range of 20 ± 2oC. In the preliminary test the temperature in the test mixtures during the measurements was in average: 20.4oC, the measured minimum: 19.6 oC, maximum: 21.0 oC.

In the complementary preliminary test the temperature in the test mixtures during the measurements was in average: 21.4 oC, the measured minimum: 19.2 oC, maximum: 22.0 oC.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the performed Activated Sludge Respiration Inhibition Tests, the EC10 and EC50 values of test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this tests the NOEC was 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.

Under the conditions of the performed Activated Sludge Respiration Inhibition Tests, the EC10 and EC50 values of test item were determined as greater than 1000 mg/L. Based on the statistical evaluation in this tests the NOEC was 1000 mg/L.

In conclusion these tests demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L. Therefore, a definite test was considered as unnecessary.

Description of key information

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