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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:
April from 18th to 19th, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to guideline
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
GLP compliance:
yes (incl. QA statement)
Vehicle:
no
Details on test solutions:
TEST SUBSTANCE
2000.0 mg of the test substance were added to about 800 ml of demineralized water and stirred at room temperature for about 30 min. The test substance was completely dissolved, verified by visual inspection. The stock solution was made up to 1l with demineralized water. The stock solution appeared blue and clear.

REFERENCE SUBSTANCE
500.0 mg of the reference substance was added to about 800 ml of demineralized water and stirred at room temperature until the reference substance was completely dissolved. The pH value of the stock solution was measured and adjusted to 7.5 ± 0.5 with 1 M sodium hydroxide solution. Following this, the stock solution was made up to 1l with demineralized water. The stock solution appeared colourless-clear.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: activated sludge from the municipal wastewater treatment plant of Mannheim/Baden Württemberg, Germany, collected on 18 April 2007 from the aeration tank of the plant.
- Pretreatment: the activated sludge suspension was washed 3 times with tap water and sieved with a fine woven mesh (mesh size about 1 mm).
- Preparation of inoculum for exposure: was adjusted to a concentration of 5.0 g/l dry weight and pre-aerated over night hours. After the pre-aeration, the lost water of the suspension was replaced.
- Initial biomass concentration: an aliquot of this suspension was added to the test vessels to obtain a sludge concentration of 1.0 g/l dry substance.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
22 ± 2 °C
pH:
7.7 - 8
Dissolved oxygen:
Oxygen concentration during aeration was > 2.5 mg/l.
Oxygen concentration immediately before measurement was > 6.5 mg/l.
Nominal and measured concentrations:
1000, 500, 250, 125 and 62.4 mg/l, nominal.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer-vessel (nominal volume 250 ml).
- Fill volume: after addition of demineralized water to adjust a volume of 150 ml, 8 ml nutrient medium was filled in each test vessel. Volumes of the test assays were made up to 200 ml with demineralized water.
- Aeration: incubation was started by aeration of the test vessels with pressure air.
- No. of organisms per vessel: 50 ml of the inoculum suspension (dry weight 5.0 g/l).
- Sludge concentration in the test vessel: 1.0 g/l.
- No. of vessels per concentration: 1 replicate.
- No. of vessels per control: 2 replicates of the blank controls.
- No. of vessels per blank control: 1 replicate.
- No. of vessels per positive control: 3, one for each test concentration (1, 10 and 100 mg/l).

TEST MEDIUM
Concentrated OECD medium.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was in the required range of 7.5 ± 0.5, thus a correction was not necessary.

EFFECT PARAMETERS MEASURED
After 180 minutes incubation, the mixture in the test vessels were placed one after the other in a oxygen measuring cell which was able to prevent the inflow of ambient oxygen in sequence of measurement. A chart recorder recorded the oxygen respiration rates.
Duration of the measurement of oxygen consumption: 8 - 10 min.
Reference substance (positive control):
yes
Remarks:
3,5 -dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
580 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.:
> 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 values were given with an accuracy of 2 significant digits. The degree of inhibition was evaluated by Probit analysis according to Finney.
EC10 (3h): 580 mg/l (nominal)
EC20 (3h): 830 mg/l (nominal)
EC50 (3h) > 1000 mg/l (nominal)
EC80 (3h) > 1000 mg/l (nominal)
Curve slop: 1.237
Results with reference substance (positive control):
EC20 (3h): 1.1 mg/lEC50 (3h): 6.8 mg/lEC80 (3h): 42.5 mg/l

Oxygen consumption measurement

Assay identification BC1 BC2 BC3 BC mv TS11000 mg/l TS2500 mg/l TS3250 mg/l TS4125 mg/l TS562.4 mg/l RS11 mg/l RS210 mg/l RS3100 mg/l
O2concentration start (mg O2/l) 8.1 8.0 6.9 - 7.0 7.8 7.1 7.1 7.7 8.1 8.4 8.7
O2concentration after 6 min (mg O2/l) 6.0 6.0 5.0 - 5.5 6.0 5.0 5.0 5.5 6.5 7.5 8.5
O2consumption rate (mg O2/l * 6 min) 2.1 2.0 1.9 2.0 1.5 1.8 2.1 2.1 2.2 1.6 0.9 0.2
O2consumption rate (mg O2/l*h) 21 20 19 20 15 18 21 21 22 16 9 2
Specific O2consumption rate (mg O2/g*h) 21 20 19 20 15 18 21 21 22 16 9 2
Inhibition of respiration (%) ** - - - - 25 10 -5 -5 -10 20 55 90
Conclusions:
EC50 (3h) > 1000 mg/l (nominal)
Executive summary:

The toxic potential of the test substance to microorganisms was tested according to OECD guideline 209. Activated sludge from a municipal waste water treatment plant was used. The test duration was 3 hours and the nominal concentrations of the test substance used were 1000, 500, 250, 125 and 62.4 mg/l. 3,5 - dichlorophenol was tested as reference substance at concentrations of 1, 10 and 100 mg/l.

The values were given with an accuracy of 2 significant digits. The degree of inhibition was evaluated by Probit analysis according to Finney.

EC10 (3h): 583.8 mg/l (nominal; graphically determined)

EC20 (3h): 833.1 mg/l (nominal; graphically determined)

EC50 (3h): 1645.0 mg/l (nominal; graphically determined)

EC80 (3h): 3247.9 mg/l (nominal; graphically determined)

Curve slop: 1.237

Conclusion

EC50 (3h) > 1000 mg/l (nominal)

Description of key information

EC50 (3h) > 1000 mg/l (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
580 mg/L

Additional information

The toxicity to microorganisms of the Basic Blue 140 (BBl140) was assessed performing a test in accordance with the OECD Test Guideline N. 209.

Activated sludge from a municipal waste water treatment plant was used collected for the experiment and the following concentration were assayed: 1000, 500, 250, 125 and 62.4 mg/l. 3,5 - dichlorophenol was used as reference substance at concentrations of 1, 10 and 100 mg/l.

The Effect Concentration (EC50) after 3 hours was identified as greater than 1000 mg/l (nominal).