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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 13th 2018 - June 20th 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch: 0400
Manufacturing date: 04/2018
Expiry date: 04/2019
Analytical monitoring:
no
Details on sampling:
After the exposure period each sample was transferred from the aeration vessel to a bottle fitted with a magnetic stirrer, An oxygen probe was inserted into the neck of the bottle to measure the concentration of dissolved oxygen.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of the test item was prepared after 1 hour of sonication and 24 hours of stirring at 35°C at a concentration of 2000 mg/l. Bottles were prepared with deionized water, synthetic sewage feed at 3 different concentrations of test item ( 1000 mg/L, 100 mg/L and 10 mg/L) using data of solubility in water. pH of the solutions was checked. The mud was added before starting the test.


PREAPRATION OF THE REFERENCE SUBSTANCE:
A stock solution of reference substance 3,5 Dichlorophenol was prepared at a concentration of 1000 mg/l 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.

PREPARATION OF CONTROLS:

Blank controls: A blank control was used consisting of deionised water and an equal volume of activated slide and synthetic medium without reference or test item.

Abiotic controls: Abiotic control was prepared with the highest concentration amount of test item with the same amount of synthetic sewage feed and without the inoculum.

Test organisms (species):
activated sludge
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: 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 treatmnet is managed by Brianzacque Srl (Monza-Italia)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20°C ± 2°C
pH:
7.47 - 7.68
Nominal and measured concentrations:
Nominal test concentrations of 1000 mg/L, 100 mg/L and 10 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass bottle
- Aeration: Vessels were aerated to keep the dissolved oxygen concentration above 60-70% saturation.
- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 3
- No. of vessels per test reference (replicates): 3
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): The suspended solids in the mud solution was 3 g/l to have in the final mixture a concentration of 1.5 g/l
- Weight of dry solids per volume of reaction mixture per unit of time:
-Synethtic sewage feed:
Peptone: 16 g/L
Meat extract: 11 g/L
Urea: 3 g/L
Sodium chloride: 0.7 g/L
Calcium chloride dihydrate: 0.4 g/L
Magneisum sulphate heptahydrate: 0.2 g/L
Anhydrous potassium monohydrogen phosphate: 2.8 g/L
Deionised water: To 1000 ml

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: pH was corrected with NaOH in 100 and 1000 mg/L vessels.


Reference substance (positive control):
yes
Remarks:
3,5 Dichlorophenol
Key result
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
Results with reference substance (positive control):
- Results with reference substance valid? Yes. Validity criteria was met as the EC50 o f 3,5 Dichlorophenol was between 2 mg/l and 25 mg/l
- Relevant effect levels: EC50 = 6.582 mg/L for total respiration

Table 1: Mean percent inhibition total respiration rate of the test item Balsalazide acid

Concentration

% of inhibition total respiration rate of Balsalazide acid

10 mg/L

11.68

100 mg/L

21.26

1000 mg/L

21.92

Table 2: Mean percent inhibition total respiration rate of the reference substance

Concentration

% of inhibition total respiration rate of Balsalazide acid

1mg/L

21.00

10 mg/L

54.07

50 mg/L

83.86

Validity criteria fulfilled:
yes
Remarks:
Blank controls oxygen uptake rate: 25.40 mg O2/g activated sludge in 1h; coefficient of variation of O2 uptake rate in controls: 5.33%.; EC50 of 3,5-Dichlorophenol: 6.582 mg/L; O2 consumption rate for abiotic control: 0.20 mg/L h
Conclusions:
The EC50 of Balsalazide acid after 3 hours was > 1000 mg/L.

Description of key information

The effect of Balsalazide acid on micro-organisms from activated sludge was determined in accordance with OECD Guideline 209. The 3 hour EC50 of Balsalazide acid was found to be >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information