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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:
2011-11-10 till 2011-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
equivalent or similar 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:
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).
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, 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:
no
Total exposure duration:
3 h
Hardness:
No data
Test temperature:
20.3 °C (concerning the highest test concentration)
pH:
7.9 (concerning the highest test concentration)
Dissolved oxygen:
5.6 [mg O2/L] (O2-start)/ 3.3 [mg O2/L] (O2-end) (concerning the highest test concentration)
Nominal and measured concentrations:
Test item: 10, 100 and 1000 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 in physico-chemical oxygen consumption control : 1000 mg/L
- 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 (purity: 99.6 % , Batch number: A0298018) )
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:
10 mg/L: 0.0 % respiration inhibition
100 mg/L: 0.0 % respiration inhibition
1000 mg/L: 0.0 % respiration inhibition
Results with reference substance (positive control):
2.5 mg/L: 20.31 % respiration inhibition
5.0 mg/L: 20.31 % respiration inhibition
10 mg/L: 67.19 % respiration inhibition
20 mg/L: 85.94 % respiration inhibition
40 mg/L: 100.0 % respiration inhibition
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 substance showed 0.0 % respiration inhibition of activated sludge at a concentration of 1000 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed (Richter, 2011).
Executive summary:

In order to test the toxicity to microorganism of the test substance, conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984). The activated sludge from predominantly domestic sewage plant was exposed to the test solution of three nominal concentrations of the substance (10, 100, 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 claculated. 0.0 % respiration inhibition was observed at the highest concentration of test substance. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

Description of key information

The test substance showed 0.0 % respiration inhibition of activated sludge at a concentration of 1000 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed (Richter, 2011).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

In order to test the toxicity to microorganism of the test substance, conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984). The activated sludge from predominantly domestic sewage plant was exposed to the test solution of three nominal concentrations of the substance (10, 100, 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. 0.0 % respiration inhibition was observed at the highest concentration of test substance. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.