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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May, 10 2017 to 28, July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The purpose of the test was only to assess the effects on total oxygen uptake and inhibition effect in respect of nitrification process was not determined.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: The product properties test guideline OCSPP 850.3300 of the EPA
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test concentrations in the preliminary test: 10, 100, 1000 mg/L (five replicates per concentration).
Test concentrations in the definitive test: the test was performed at five nominal concentrations of the test substance and 5 replicates per concentration: 62.5 mg/L, 125 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L were used.

Definitive Test
Preparations of the test flasks
One test solution with a final volume of 400 mL (ratio of composition of each test mixture referring to 500 mL according to the guideline) was prepared per treatment in a glass flask. A volume of 12.8 mL synthetic sewage and an adequate amount of the test substance given into the test flask by direct addition was filled up with deionized water to 200 mL before the start of the incubation (using ultrasonic bath for 30 minutes). At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3 g per litre of suspended solids was added.

Test organisms (species):
activated sludge, domestic
Details on inoculum:
Source: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary

Conditioning: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use. The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged. 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 amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.20. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
other: deionized water
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.0 – 21.9 °C
pH:
7.20
Nominal and measured concentrations:
Nominal test concentrations in the preliminary test: 10, 100, 1000 mg/L
Nominal test concentration in the definitive test: 62.5 mg/L, 125 mg/L, 250 mg/L, 500 mg/L and 1000 mg/L
Details on test conditions:
Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in the preliminary test further testing was required.
5 concentration series were prepared with 5 replicates and with one blank control series by series. In parallel with the above test mixtures four reference substance concentrations without replicates, were also prepared with one blank control.

Four reference controls with different concentrations and one untreated blank control were started as the first step of the test. Then at appropriate time intervals of approximately 30 minutes the further test groups were started. The second series were the five replicates of the lowest test substance concentration of 62.5 mg/L and the second blank control. The third series was the five replicates of the next test substance concentration of 125 mg/L and the third blank control. This procedure was repeated up to the highest test substance concentration of 1000 mg/L and the last blank control. The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test.

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
Details on results:
Test substance
In comparison to the inoculum controls the inhibition of the total respiration rate in the case of the activated sludge was between -2.15 % and 39.71 % in the examined concentration range of 62.5 – 1000 mg/L of the test substance.

The average specific total respiration rate (RTS) of the untreated (Blank) controls was 26.30 mg O2/L/h/g solid. The variation coefficient of oxygen uptake rate in above untreated replicates was 3.13 %.
Results with reference substance (positive control):
Reference substance
The following nominal concentrations of the reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions: 1.0, 3.2, 10.0 and 32.0 mg/L. In comparison to the controls the inhibition of the total respiration rate of the activated sludge was 69.58 % at the highest nominal concentration of 32.0 mg/L.
At the nominal concentrations of 1.0, 3.2 and 10.0 mg/L, values of 16.56 %, 21.32% and 28.43 % inhibition of the respiration rate respectively were calculated.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 17.17 mg/L with 95%
confidence limits of 11.57 – 25.49 mg/L.
The EC50 values of the positive control substance 3,5-Dichlorophenol (3,5-DCP) were in the recommended range for total respiration using activated sludge derived from domestic sewage, demonstrating the adequacy of the testing design.
Reported statistics and error estimates:
- The 3-hour EC20, EC50 and EC80 values of the test substance and the 3-hour EC50 values of the reference substance with their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.
- The oxygen uptake rates (R) was calculated from the measured values, based on the linear part of the graphs of oxygen concentration (values taken in consideration between 2.0 mg/L and 7.0 mg/L only) versus time.
- The oxygen uptake rates, percentage of inhibition and specific respiration rates were calculated by using Excel for Windows Software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, with respect to the inhibitory effect of the test substance on the total respiration rate, the 3 h EC50 value of the test substance was determined to be higher than 1000 mg/L.
Executive summary:

A study was conducted to determine the effect of the test substance on the microorganisms by measuring their respiration rate according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)), EU Method C.11 and OCSPP 850.3300, in compliance with GLP. The purpose of the test was to assess the effects on total oxygen uptake only. Inhibition effects in respect of nitrification process were not determined. The test substance concentrations inthe preliminary test were of 10, 100, 1000 mg/L. Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in this test, a further definitive test was conducted with the following concentrations: 62.5, 125, 250, 500 and 1000 mg/L. The concentration series were prepared with 5 replicates and with one blank control series by series. In parallel with the above test mixtures four reference substance concentrations without replicates, were also prepared with one blank control. The results of this experiment were inconclusive regarding the toxicity of the test substance, since large differences in respiration rates were observed between the preliminary study and this first main study. In order to confirm the toxicity of the test substance, the experiment was repeated using the same experimental concentrations as in the first definitive test. The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test. The reference control results showed that in the second main experiment, the inoculum and methodology provided a good measure of inhibition of activated sludge respiration rate. Under the study conditions, with respect to the inhibitory effect of the test substance on the total respiration rate, the 3 h EC50 value of the test substance was determined to be higher than 1000 mg/L (Sipos, 2017).

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

A study was conducted to determine the effect of the test substance on the microorganisms by measuring their respiration rate according to OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)), EU Method C.11 and OCSPP 850.3300, in compliance with GLP. The purpose of the test was to assess the effects on total oxygen uptake only. Inhibition effects in respect of nitrification process were not determined. The test substance concentrations inthe preliminary test were of 10, 100, 1000 mg/L. Since significant inhibition effect (on the total respiration rate of the microorganisms of the activated sludge) was detected in this test, a further definitive test was conducted with the following concentrations: 62.5, 125, 250, 500 and 1000 mg/L. The concentration series were prepared with 5 replicates and with one blank control series by series. In parallel with the above test mixtures four reference substance concentrations without replicates, were also prepared with one blank control. The results of this experiment were inconclusive regarding the toxicity of the test substance, since large differences in respiration rates were observed between the preliminary study and this first main study. In order to confirm the toxicity of the test substance, the experiment was repeated using the same experimental concentrations as in the first definitive test. The abiotic control did not need to be tested as there was no oxygen uptake in the preliminary test. The reference control results showed that in the second main experiment, the inoculum and methodology provided a good measure of inhibition of activated sludge respiration rate. Under the study conditions, with respect to the inhibitory effect of the test substance on the total respiration rate, the 3 h EC50 value of the test substance was determined to be higher than 1000 mg/L (Sipos, 2017).