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
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))
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Pre-treatment of test item and reference compound without ATU
Direct weighings were prepared to give the specific test item concentration. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 3.5 – 3.6. The pH was adjusted to pH 7.1 – 7.4 with NaOH.
For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 - 8 with HCl.

Pre-treatment of test item with ATU
Direct weighings were prepared to give the specific test item concentration. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 3.4 – 3.5. The pH was adjusted to pH 7.2 – 7.3 with NaOH.
For the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Date of collection: 2019-09-02
- Microbial inoculum:The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Storage of sludge: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.6
- Synthetic sewage feed: A synthetic waste water feed was prepared.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
18.6 - 19.9 °C
pH:
8.4 - 8.5; 7.4 (physico-chemical oxygen consumption control)
Nominal and measured concentrations:
100 mg/L (nominal)
Concentrations are given as nominal concentrations and were not confirmed by analytical methods.
Details on test conditions:
- Test item concentration/s: 100 mg/L, 3 replicates; 100 mg/L with ATU, 2 replicates
- Control: 6 replicates; 4 replicates with ATU
- Test item concentration in physico-chemical oxygen consumption control: 100 mg/L
- Concentration of reference compound 3,5-Dichlorophenol: 2.5, 5, 10, 20 and 40 mg/L
The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.
- Test vessels: 300 mL glass Erlenmeyer flasks
- Method of application: direct weighing
- Test concentration of the activated sludge: 800 mg/L suspended solids
- Test temperature: 20 ± 2°C
- Stirring period of the test item before start of incubation time: 17 hours
- Incubation time: 3 hours with permanent aeration
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (Acros Organics, purity: 99.9%, Batch no. A0357150)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 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.:
> 100 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.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
The effect value relates to a nominal concentration, since no analytical monitoring was performed.
The oxygen consumption in the presence of N-allylthiourea was determined. As no inhibition was observed for the total oxygen consumption at 100 mg/L no differences between the heterotrophic and the nitrification oxygen uptake rates have to be calculated.
Results with reference substance (positive control):
The EC50 of the reference compound 3,5-Dichlorophenol was 15.3 mg/L.
Reported statistics and error estimates:
As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50.
The No Observed Effect Concentration was calculated according to STUDENT-t test for Homogeneous Variances using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).
The EC50 value for the reference substance was calculated from the respiration rates at different test item concentrations using the same statistics programme mentioned above.
Validity criteria fulfilled:
yes
Remarks:
oxygen uptake rate of blank controls should not be < 20 mg O2/g of activated sludge per h; coefficient of variation of O2 uptake in control replicates should not be > 30 % at test end; EC50 of reference compound should be 2 – 25 mg/L for total respiration
Conclusions:
The activated sludge was exposed to Adipic acid at a limit test item concentration of 100 mg/L for 3 h. An EC50 of higher than 100 mg/L, an EC10 of higher than 100 mg/L and a NOEC equal or higher than 100 mg/L were determined.
Executive summary:

A study was performed to assess the toxicity of Adipic acid to bacteria in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03 -02). This test method is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008).


The activated sludge was exposed to Adipic acid at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours.


To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode).


Six controls without the test item were included in the test design, three at the start and the others at the end of the test series.


A limit test was performed with 3 replicates with a test item concentration of 100 mg/L. Each batch of activated sludge was checked using 5 concentrations in the range of 2.5 – 40 mg/L of 3,5-Dichlorophenol as a reference compound.


The respiration rate is classified into two processes of oxidation. The oxidation of organic carbon and the ammonium oxidation (nitrification). The use of the specific nitrification inhibitor, ATU (N-allylthiourea), enables the direct assessment of the inhibitory effects of test substances on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification may be calculated. The oxygen uptake rates of four additional controls and two replicates of the test item concentration 100 mg/L, all in the presence of N-allylthiourea, were prepared and measured after the exposure period. These values represent the heterotrophic respiration.


Since some substances may consume oxygen by chemical reactivity, a physico-chemical oxygen consumption control was carried out additionally. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.


An EC50 of higher than 100 mg/L, an EC10 of higher than 100 mg/L and a NOEC equal or higher than 100 mg/L were determined.


The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Description of key information

A study was performed to assess the toxicity of Adipic acid to bacteria in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03 -02). This test method is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008).

The activated sludge was exposed to Adipic acid at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours.

To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode).

Six controls without the test item were included in the test design, three at the start and the others at the end of the test series.

A limit test was performed with 3 replicates with a test item concentration of 100 mg/L. Each batch of activated sludge was checked using 5 concentrations in the range of 2.5 – 40 mg/L of 3,5-Dichlorophenol as a reference compound.

The respiration rate is classified into two processes of oxidation. The oxidation of organic carbon and the ammonium oxidation (nitrification). The use of the specific nitrification inhibitor, ATU (N-allylthiourea), enables the direct assessment of the inhibitory effects of test substances on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification may be calculated. The oxygen uptake rates of four additional controls and two replicates of the test item concentration 100 mg/L, all in the presence of N-allylthiourea, were prepared and measured after the exposure period. These values represent the heterotrophic respiration.

Since some substances may consume oxygen by chemical reactivity, a physico-chemical oxygen consumption control was carried out additionally. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.

An EC50 of higher than 100 mg/L, an EC10 of higher than 100 mg/L and a NOEC equal or higher than 100 mg/L were determined.

The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L
EC10 or NOEC for microorganisms:
100 mg/L

Additional information

"Should read as EC50 >100mg/L, EC10 >100mg/L, NOEC >= 100 mg/L"