Registration Dossier

Ecotoxicological information

Toxicity to microorganisms

Currently viewing:

Administrative data

Link to relevant study record(s)

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:
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
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:
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. certificate)
Specific details on test material used for the study:
Batch No.: 31233510
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
At the start of the first preliminary 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, furthermore 3 x 300 mg for abiotic controls) of the test item were administered directly.
At the start of the complementary test defined amounts (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:
First test: Average of 20.4 °C. Second test: Average of 21.4 °C.
pH:
In each case the pH was between pH 7 and pH 8.
Dissolved oxygen:
Aeration with compressed air at 0.5 L/min
Salinity:
/
Conductivity:
/
Nominal and measured concentrations:
Nominal:
First test: 0 - 10 - 100 - 1000 mg/L
Complementary test: 0 - 1000 mg/L
Details on test conditions:
In the first test, 13.8 % inhibition was obtained at 10 mg/L, 7.2 % at 100 mg/L and in average 14.6 % at 1000 mg/L. Based on these results the performance of complementary preliminary experiment was considered to be necessary rather than a definitive test with a range of test item concentrations.
The test was designed according to the guideline requirements, triplicates in the first test and 5 parallels in the second test were examined at the highest tested concentration of 1000 mg/L and additionally two lower concentration levels of 10 and 100 mg/L were examined with one vessel each in the first experiment. 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 mixtures in two parallels) that contained N-allylthiourea at 11.6 mg/L. The test media were prepared freshly before the test.
Defined amounts of the test item were directly weighed (administered) into each test flask.
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.

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 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.
Abiotic controls were investigated in the preliminary test, only. The abiotic controls started after the test item treatments with the administration of the test item, without activated sludge inoculum.
In time intervals of approximately 12 minutes (an arbitrary but convenient interval) a maximum of four test vessels were started. The tests were performed using four oxygen electrodes that allow the simultaneous testing of four test vessels.

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-allylthiourea (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
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:
Nitrification Controls:
First test: The total respiration (RT) was 78.90 mg/Lh, the heterotrophic respiration (RH) was 79.80 mg/Lh, a nitrification respiration (RN) of -0.90 mg/Lh was calculated. According to the above calculations it was assumed that the heterotrophic oxygen uptake equals the total uptake.
Complementary test: The nitrification control was examined in the test with three parallels. 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.
It was assumed that the heterotrophic oxygen uptake equals the total uptake.
In this study the differentiation between heterotrophic respiration and nitrification was considered as not being necessary.

No abiotic oxygen consumption was noticed.

Controls:
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/gh1); in the complementary test it was 40.28 mg O2/gh [mg O2/gh: mg oxygen per gram of activated sludge (dry weight of suspended solids) per 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%.

Test substance:
In the first test equivocal results were obtained: 13.8 % inhibition was obtained at the lowest test item concentration of 10 mg/L, 7.2 % at 100 mg/L and in average 14.6 % 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 noticed in the comparison with the blank control values. Based on these results the performance of an additional test (complementary preliminary experiment) was considered as necessary. In this second test the observed oxygen consumption rates, and therefore the specific respiration rates, were in the range of the blank controls. No inhibitory effect of the test item was observed.
Results with reference substance (positive control):
Positive reference control:
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 63.66 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was 17.04 mg/L in the first test and 15.54 mg/L in the complementary test.
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.
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.
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

The EC10 and EC50 values of test item were determined to >1000 mg/L. Based on the statistical evaluation in this tests the NOEC was 1000 mg/L.

A value >1000 mg/L can not be entered in the key value below.

Key value for chemical safety assessment

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

Additional information