[No public or meaningful name is available]

Toxicity to microorganisms

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

I. Pre-treatment of test item and reference compound without ATU:
- Direct weighings were prepared to give the different test item concentrations
- 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 6.2–6.4
- The pH was adjusted to pH 7.4–7.5 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

II. Pre-treatment of test item with ATU:
- Direct weighings were prepared to give the different test item concentrations
- 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 6.3–6.4
- The pH was adjusted to pH 7.3–7.4 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 sewage treatment plant (Municipal STP Cologne-Stammheim)
The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 4000 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 volume with deionised water.
Storage of sludge : aeration of the activated sludge at 20 °C ± 2 °C, daily fed with synthetic medium
pH of the suspension before application : 7.7

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

No data

Test temperature:

20.5-21.4 °C

pH:

8.1-8.3

Salinity:

not applicable

Conductivity:

No data

Nominal and measured concentrations:

Test item: 100 mg/L (n)
Reference compound: 2.5, 5, 10, 20 and 40 mg/L (n)
The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.

Details on test conditions:

EXPOSURE 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
- Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge
- Oxygen consumption, temperature and pH were measured and recorded after an aeration time of 3 hours in all these vessels starting with control

* To determine the heterotrophic oxidation four additional controls and two replicates with the test item concentration 100 mg/L, all containing 1.25 mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L, were prepared.

Reference substance (positive control):

yes

Remarks:

(3,5-dichlorophenol (purity: 99.9 % , Batch number: A0357150))

Key result

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

Remarks:

respiration rate

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

Remarks:

respiration rate

Details on results:

100 mg/L: 0.0 % respiration inhibition

The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.

Results with reference substance (positive control):

The EC50 of the reference compound 3,5-Dichlorophenol should be in the range of 2–25 mg/L for total respiration. In the present test the EC50 of the reference compound was 14.677 mg/L. Thus the test is valid.

Validity criteria fulfilled:

yes

Remarks:

(respiratory rates of the 2 controls differ less than 15 % from each other; the EC50 of the reference compound 3,5-dichlorophenol is in the range 5–30 mg/L)

Conclusions:

The substance showed 0.0 % respiration inhibition of activated sludge at a concentration of 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed (Neuhahn, 2018).

Executive summary:

A study was performed to assess the toxicity 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 the substance at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours. The substance showed 0.0 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The EC50 is higher than 100 mg/L. The NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microorganism.

Description of key information

The substance showed 0.0 % respiration inhibition of activated sludge at a concentration of 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed (Neuhahn, 2018).

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

The EC50 was higher than 100 mg/L. The NOEC is equal or higher than 100 mg/L