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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
Study period:
2012-08-06 to 2012-08-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to relevant OECD TG 209
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: appropriate amounts of test item were weighed onto glass slides using an analytical balance and transferred into the designated test vessels, then 234 mL water was added and the mixture was intensely stirred at room temperature; nominal test concentrations were 10, 100 and 1000 mg/L
- Controls: four blank controls containing an equal volume of activated sludge and synthetic medium were tested; additionally, a positive control with the reference substance 3,5-dichlorophenol was tested as single replicates at 3.2, 10 and 32 mg/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no emulsifiers or solvents were used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all loading rates were clearly above the water solubility limit of the test substance
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: municipal sewage treatment plant ARA Ergolz II, Füllinsdorf, Switzerland treating predominantly domestic sewage
- Preparation of inoculum for exposure: sludge was washed twice with chlorine free tap water by centrifugation. The supernatant liquid phase was decanted. A homogenised aliquot of the sludge suspension was weighed and then dried and the ratio of wet to dry weight was calculated. An appropriate amount of wet sludge was then resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During a holding period of two days before use, the sludge was fed with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration. Immediately before use the dry weight of the sludge was measured again and adjusted to 3 g dry weight per liter. The pH of the activated sludge used in the test was 7.2.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
not applicable
Hardness:
not reported
Test temperature:
20 °C
pH:
The pH value of the dispersions of test material ranged from 6.4 to 6.8 and was adjusted to 7.5 to 7.7 before the test.
Dissolved oxygen:
The dissolved oxygen concentration did not drop below 60 to 70% of saturation; the dissolved oxygen concentration was at least 8.0 mg/L at the end of the incubation period.
Salinity:
not applicable
Nominal and measured concentrations:
10, 100 and 1000 mg/L nominal concentrations
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 2000 mL glass flasks
- Aeration: continuous during the incubation
- No. of vessels per concentration (replicates): one for the low and medium concentration, three for the high concentration; one for each concentration of positive control substance
- No. of vessels per control (replicates): four for inoculum control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: chlorine free tap water

OTHER TEST CONDITIONS
- Adjustment of pH: dispersions of test substance were adjusted to pH 7.5-7.7

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : continuously after 3 hours of incubation
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
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 test substance had no inhibitory effect to the respiration of microorganisms in the tested activated sludge up to the highest nominal test concentration of 1000 mg/L.
Results with reference substance (positive control):
The 3-hour EC50 value obtained with the reference substance 3,5-dichlorophenol was 10 mg/L, which is within the range from 2 to 25 mg/L given in the guideline. This confirms the suitability of the tested activated sludge.
Validity criteria fulfilled:
yes
Conclusions:
The substance had no inhibitory effect on the respiration of microorganisms in activated sludge when tested up to a nominal concentration of 1000 mg/L over a period of 3 hours.
Executive summary:

The toxicity of the test substance to microorganisms was studied under GLP conditions in a valid test in accordance with OECD TG 209. The tests were performed in 2-L flasks with a final volume of 500 mL (consisting of 234 mL water, 16 mL synthetic sewage and 250 mL suspended activated sludge with a sludge concentration of 3 g dry weight per liter). Four inoculum controls were conducted in parallel to the tests with the substance and with the positive control substance 3,5-dichlorophenol. The nominal concentrations of the test substance were 10 and 100 mg/L (single replicates) and 1000 mg/L (triplicate). The positive control substance was tested at 3.2, 10 and 32 mg/L (single replicates). Dispersions of the test substance were prepared by weighing appropriate amounts of test substance onto glass slides, adding these amounts to water without solvent or emulsifier and stirring vigorously at room temperature. There was evidence of non-dissolved substance at all three nominal concentrations. The pH value of the dispersions was adjusted to the range of pH values from 7.5 to 7.7 and sludge was then added. Incubation for three hours was performed at 20 °C under continuous aeration ensurring dissolved oxygen concentrations of 60 to 70% of saturation. The dissolved oxygen concentration was at least 8 mg/L at the end of the incubation period when aeration was stopped. After three hours of incubation, the respiration rate of well-mixed samples of each test medium was measured by determining the dissolved oxygen concentrations continuously using an oxygen meter WTW Oxi 539. The oxygen consumption rate was determined from the linear part of the respiration curve. The test substance had no inhibitory effect on the respiration of the activated sludge up to the nominal test concentration of 1000 mg/L. The 3-hour NOEC was equal to or greater than 1000 mg/L. The EC50 obtained with the positive control substance was 10 mg/L, which is in the recommended range from 2 to 25 mg/L showing the suitability of the tested sludge. Additionally, the other validity criteria were fulfilled: the specific respiration rate of the blank controls was higher than 20 mg oxygen/g per hour and the coefficient of variation of oxygen uptake rate between control replicates was less than 30%.

Description of key information

The substance did not inhibit the microbial activity in activated sludge up to a nominal concentration of 1000 mg/L over a period of 3 hours resulting in a NOEC value of equal to or greater than 1000 mg/L.

Key value for chemical safety assessment

Additional information

The available respiration inhibition test with activated sludge from a municipal STP processing mainly domestic sewage was performed in accordance with OECD TG 209. All validity criteria were fulfilled. The substance was tested at nominal concentrations of 10, 100 and 1000 mg/L. There was evidence of non-dissolved substance at all three concentration levels. The substance did not inhibit the microbial activity of the activated sewage sludge after three hours of incubation with continuous aeration. The 3-hour NOEC for inhibition of respiration was equal to or greater than 1000 mg/L.