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Ecotoxicological information

Toxicity to microorganisms

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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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Pre-treatment of test item and reference compound
Direct weighings were prepared to give the 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 16 hours. The pH was measured and ranged between pH 9.3-9.4. The pH was adjusted to pH 7.0-7.4 with HCl.
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 7.1 with HCl.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Type: mixed population of aquatic microorganisms (activated sludge)
- Origin: aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim)
- Date of collection: 2014-03-10
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500/4500 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
Test type:
static
Water media type:
freshwater
Limit test:
yes
Test temperature:
19.2 - 21.4
pH:
8.0 - 8.3
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
Exposure conditions
- Test item concentration/s: 100 mg/L, 3 replicates
- Control: 6 replicates
- 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: 16 hours
- Incubation time: 3 hours with permanent aeration
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (Acros Organics, purity: 99.9 %, batch no. A0330535)
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:
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.
Results with reference substance (positive control):
In the present test the EC50 of the reference compound was 11.77 mg/L; thus the test is valid.
Validity criteria fulfilled:
yes
Remarks:
oxygen uptake rate of blank controls >= 20 mg O2 / g activated sludge (dw) in an hour; coefficient of variation of oxygen uptake in control replicates <= 30 % at end of test; EC50 of reference should be in the range of 2 – 25 mg/L for total respiration
Conclusions:
The test item showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L.
Executive summary:

A study was conducted in accordance with OECD Guideline 209 (Carbon and Ammonium Oxidation) (2010) which is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008) to assess the toxicity of the test substance to bacteria.

The activated sludge was exposed to the test substance at a limit test item concentration of 100 mg/L. 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 physico-chemical oxygen consumption control was carried out additionally. A limit test was performed with 3 replicates with a test item concentration of 100 mg/L.

The respiration rate of each mixture was determined after aeration periods of 3 hours.

The test substance showed 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

Description of key information

A study was conducted in accordance with OECD Guideline 209 (Carbon and Ammonium Oxidation) (2010) which is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008) to assess the toxicity of the test substance to bacteria.

The activated sludge was exposed to the test substance at a limit test item concentration of 100 mg/L. 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 physico-chemical oxygen consumption control was carried out additionally. A limit test was performed with 3 replicates with a test item concentration of 100 mg/L.

The respiration rate of each mixture was determined after aeration periods of 3 hours.

The test substance showed 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

Key value for chemical safety assessment

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

Additional information

Should read EC50 > 100 mg/L, NOEC >= 100 mg/L.