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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:
2018
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 (Carbon and Ammonium Oxidation))
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 23ADB/50
- Expiration date of the lot/batch: 21/11/2023


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: 5 years
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water

Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Preparation of the test item
A stock solution of the test item was prepared at a concentration of 20500 mg/L (corresponding to 10024 mg/L of the active ingredient). Bottles were prepared with deionized water, synthetic sewage feed and the test were performed at 3 different concentrations of test item (1000 mg/L, 100 mg/L and 10 mg/L). pH of the solutions was checked. The mud was added before starting the test.

Preparation of the reference substance
A stock solution of reference substance 3,5 Dichlorophenol was prepared at a concentration of 1000 mg/I in deionized water. 3 different concentrations (50 mg/L, 10 mg/L and 1 mg/L) were prepared in the same way of the test item. pH of the stock solution was checked. The mud was added before starting the test.

Blank controls preparation
As blank was used deionized water containing an equal volume of activated sludge and synthetic medium without reference substance and test item.

Abiotic control
Abiotic control was prepared with the highest concentration amount of the test item, with the same amount of the synthetic sewage feed without the inoculum.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
For the preparation of the inocula a sample of aerobic mud was selected by the mixed treatment plant of urban (about 66%) and industrial (about 34%) liquid sewage situated in San Rocco near Monza (MB). The plant of treatment is managed by "BRIANZACQUE SRL (Monza- Italia)".
Mud preparation
Activated sludge was collected and used on day of collection.
No coarse particles were present so settling was not necessary. Then the mud samples, before their use, were washed and analyzed to prepare the inoculum. The sludge was continuously aerated at the test temperature.
The suspended solids in the mud solution was 3 g/I to have in the final mixture a concentration of 1.5g/L.
Test type:
not specified
Water media type:
other: deionised water
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
The bottles prepared were incubated at 20°C± 2°C for three hours.
Post exposure observation period:
After the exposure period each sample was transferred from the aeration vessel to a bottle fitted with a magnetic stirrer (see figure below). An oxygen probe was inserted into the neck of the bottle to measure the concentration of dissolved oxygen. The concentration of dissolved oxygen was recorded in turn continuously for a period of 6 minutes or until the oxygen concentration falls below 2 mg/I. The probe was washed with deionized water between measurements.
Test temperature:
The test was performed at 20°C ± 2°C.
pH:
-pH test item solutions 7.37-7.43
-pH synthetic sewage feed: 7.33
-pH reference substance stock solution: 7.61 after adjustment with NaOH (1 M)
Nominal and measured concentrations:
10-100-1000 mg/L
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
-the blank controls oxygen uptake rate: 27.49 mg oxygen per one gram of activated sludge in an hour.
-the coefficient of variation of oxygen uptake rate in control replicates: 13.07%
-oxygen consumption rate for abiotic control: 0.37 mg/Lh.

Mean percent inhibition total respiration rate of the test item "Trisodium (2S)-2,6-bis (3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" is detailed in the following table:
Concentration % of inhibition total respiration rate Test item "Trisodium (2S)-2,6-bis (3- carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt"
10 mg/L 17.38
100 mg/L 19.08
1000 mg/L 24.25

Results with reference substance (positive control):
Mean percent inhibition total respiration rate of the reference substance is detailed in the following table:

Concentration % of inhibition total respiration rate
Reference substance "3,5 - Dichlorophenol"
1 mg/L 23.12
10 mg/L 73.16
50 mg/L 86.66

Validity criteria fulfilled:
yes
Conclusions:
The obtained results, in compliance with assay validity criteria, interpreted in accordance to OECD 209: 2010, show that the EC50 value of the active ingredient of the test item "Trisodium (2S)-2,6-bis (3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" after 3 hours results greater than 1000 mg
Executive summary:

The toxicity to microrganisms from activated sludge has been tested for the test item "Trisodium (2S)-2,6-bis (3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" by measuring their respiration rate, according to OECD 209: 2010.

The respiration rates of activated sludge both in control, reference and treatment groups have been measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours at 20°C± 2°C.

This method can be applied to water soluble, poorly water soluble and volatile substances.

The results are referred to the active ingredient (48.9% of the test item).

The obtained results, in compliance with assay validity criteria, interpreted in accordance to OECD 209: 2010, show that the EC50value of the active ingredient of the test item "Trisodium (2S)-2,6-bis (3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" after 3 hours results greater than 1000 mg/L.

Description of key information

The EC50value of the active ingredient of the test item "Trisodium (2S)-2,6-bis (3-carboxylatopropanamido) hexanoate - Disuccinyl lysine sodium salt" after 3 hours results greater than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

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