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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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
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 16 hours.
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 ± 0.5 with HCl.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
-mixed population of aquatic microorganisms (activated sludge), origin of an aeration tank of a domestic sewage treatment plant (Municipal STP Cologne-Stammheim, Germany)
- pretreatment: aeration, daily feed with synthetic medium
-pH 7.5 +/- 0.5
-test concentration of the activated sludge: 800 mg/L suspended solids

Preparation of inoculum for exposure:
- first, the sludge was settled and the supernatant was decanted
- after centrifuging (15 min at 4000 rpm and 20 °C) the supernatant was decanted again
- approx. 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 first dissolved in synthetic medium and than filled up to a defined end volume with deionised water
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
No data
Test temperature:
20.6-21.2 (including all test concentrations, controls and reference substance)
pH:
7.4-8.0 (including all test concentrations, controls and reference substance
Salinity:
No data
Nominal and measured concentrations:
Nominal: 10, 100, and 1000 mg/L
Details on test conditions:
- Test vessel: 300 mL glass Erlenmeyer flasks
- type of application: direct weighing
- temperature: 20 +/- 2 °C
- stirring period of the test item before start of the incubation time: 16h
- test parameter: respiration rate
- Two controls without test item
- One physico-chemical oxygen consumption control (without sludge) with the highest concentration of test substance was carried out as well
- no replicates was set up

Test system
Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8.
8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C.
Control vessels (inoculated sample without test item) were prepared the same way.
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 was measured and recorded after an aeration time of 3 hours in all these vessels starting with control 1. Thereafter, temperature and pH were measured as well. Then the other test vessels were measured. Control 2 terminated the measurements.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol, concentration (2.5, 5, 10, 20, 40 mg/L))
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
Remarks:
respiration rate
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
Remarks:
respiration rate
Details on results:
The substance showed 0% respiration inhibition of activated sludge within the test.
Results with reference substance (positive control):
The EC50 of the reference compound 3,5-Dichlorophenol is in the range 5 – 10 mg/L.
Inhibition: at 2.5 mg/L 15.84 %, at 5 mg/L 17.09 %, at 10 mg/L 64.55 %, at 20 mg/L 87.53 % and at 40 mg/L 94.55 %
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 test item showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L. An EC10 and an EC50 of >1000 mg/L were obtained in this test. The effect values relate to nominal concentrations, since no analytical monitoring was performed.
Executive summary:

In order to test the toxicity to microorganism of the substance, activated sludge from predominantly domestic sewage plant was exposed to the test solution of three nominal concentrations of the substance (10, 100, and 1000 mg/L), blank control solution and solution with positive reference substance for a period of 3 hours. Dissolved oxygen was recorded and respiration rates were calculated. No physico-chemical oxygen consumption was observed at the highest concentration of test substance. No respiration inhibition was observed at 10, 100 and 1000 mg/L (EC10 and EC50 > 1000 mg/L). This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

Description of key information

The test item showed 0.0 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L. An 3h-EC10 and an 3h-EC50 of >1000 mg/l were obtained in this test.
The effect values relate to nominal concentration, since no analytical monitoring was performed.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

"should read: EC10 >1000 mg/L"