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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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
Version / remarks:
22. Jul. 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
07. Dec. 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test Item
Designation in Test Facility: 17042002G
Date of Receipt: 20. Apr. 2017
Condition at Receipt: Room temperature, in proper conditions

Specification
Name sodium 3-sulfobenzoate
Batch no. 170103
Appearance White crystalline powder
Composition sodium 3-Sulfobenzoate
CAS No. 17625-03-5
EINECS-No. 241-602-5
Molecular formula C7H5SO5Na
Molecular weight 224.17 g/mol
Purity > 99.0% (Titration with NaOH, Glass indicating electrode, Calomel reference electrode)
Homogeneity totally homogeneous
Volatility not available
Stability H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: >1 g/l; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date 2017
Expiry date January 2019
Storage Room Temperature (20 ± 5°C), keep away from humidity

Safety Precautions
For handling, information given in the MSDS will be observed.

Storage
The test item was stored in a closed vessel at room temperature (20 ± 5°C), protected from humidity.
Analytical monitoring:
no
Details on sampling:
Activated sludge from a biologic sewage treatment plant was used. The chosen plant treats mostly domestic sewage.

Source and Pre-Treatment
The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf.
Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.

Specification
The specification of the test system is given below.
Data of collection 15 May 2017
Date of experiment 16 May 2017
pH 7.4
Dry matter of Sludge 2.46 g suspended solids/L
Dry matter in the Test 1.23 g suspended solids/L.
Vehicle:
yes
Details on test solutions:
On the day before the experiment, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution / L sludge.
One day before start of the test, the stock solution of the test item was prepared.
On the day of the experiment, the dry matter was determined once more. The stock solution of the positive control was prepared.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Origin sewage treatment plant Neustadt/W (if unavailable: Edenkoben. or Haßloch)
Date of collection 1 or 2 days before the start of the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
0.83 mmol/L.
Test temperature:
20.3 – 21.4 °C
pH:
pH 8.16.
pH has been always measured at the end of the test.
Dissolved oxygen:
Variation in O2-Consumption of the Blank Controls = 48.109 mg/(L*h) (standard deviation (1.788 mg/(L*h).
Salinity:
sodium 4.7 mg/L
potassium 2.6 mg/L
calcium 32 mg/L
magnesium 3.6 mg/L
aluminium 0.011 mg/L
iron <0.005 mg/L
manganese <0.005 mg/L
ammonium <0.05 mg/L
nitrate 5.5 mg/L
nitrite <0.005 mg/L
chloride 10 mg/L
sulphate 11 mg/L
total organic carbon (TOC) < 0.5 mg/L
antimony <0.001 mg/L
arsenic <0.001 mg/L
lead <0.001 mg/L
cadmium <0.0001 mg/L
chromium <0.001 mg/L
copper <0.001 mg/L
nickel <0.001 mg/L
mercury <0.0001 mg/L
selenium <0.001 mg/L
bor <0.01 mg/L
cyanide <0.005 mg/L
fluoride <0.1 mg/L
benzene <0.1 μg/L
polycyclic aromatic hydrocarbons <0.008 μg/L
chlorinated organic compounds <1 μg/L
pesticides and biocides < LOD μg/L
pH 8.16
Conductivity:
Conductivity at 25 °C 228 μS/cm
Nominal and measured concentrations:
Nominal concentration: 0, 1, 10, 100, 1000 mg/L
Details on test conditions:
Duration 3 hours
Replicates 1 replicate/concentration (positive control, test item lower concentrations)
5 replicates for test item treatment 1000 mg/L
Blank Control 2 replicates before and 2 after measuring positive control and test item, respectively
Water tap water
Aeration purified air, using Pasteur pipettes
Feeding nutrient solution, 16 mL/vessel
Temperature 20.3 – 21.4 °C
Reference substance (positive control):
yes
Remarks:
For the positive control 3,5-Dichlorophenol, the following treatments have been tested in each experiment using one replicate per concentration: 5 / 10 / 20 / 40 mg/L.
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Details on results:
One experiment was performed. The test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration.
For the treatment 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each.
Because no significant inhibition was observed, no additional experiment had to be performed. All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 8.9 mg/L mg/L (95 % confidence interval 5.6 – 12 mg/L) which lie within the recommended range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 hour.
No observations were made which might cause doubts concerning the validity of the study outcome.
The result of the test can be considered valid.
Results with reference substance (positive control):
The positive control gave an EC50 of 8.9 mg/L mg/L (95 % confidence interval 5.6 – 12 mg/L) which lie within the recommended range of 2 – 25 mg/L.
Reported statistics and error estimates:
For the treatments with the test item concentration 1000 mg/L, it was tested whether the differences between treatment and blank control were significant. For this determination, the values of the O2 consumption were used.
In order to select a suitable test for significance, it was checked whether equality of variance was given.
The calculated value F is compared with the F-test table (level of significance 95%). As equality of variance is given, the t-test is used. With the t-test, it was checked whether the differences are significant. Significance is given if the calculated t-value is bigger than the limit of significance (t-value taken from the table
with degree of freedom: n1 + n2 – 2, level of significance 95 %). The difference between treatment 1000 mg/L and the blank control can be considered as not significant as the calculated t-value lay below the significance limit and O2 consumption in the treatment was higher than in the blank control. Therefore, the concentration 1000 mg/L is stated as NOEC.
Validity criteria fulfilled:
yes
Conclusions:
During test with sodium hydrogen m-sulphonatobenzoate no significant inhibition was observed.
The following results for the test item sodium 3-sulfobenzoate were determined:
3h NOEC = 1000 mg/L
3h EC50 > 1000 mg/L
3h EC10 > 1000 mg/L.
Executive summary:

The test item was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.46 g suspended solids/L, giving a concentration of 1.23 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 8.9 mg/L (95% confidence interval: 5.6 – 12 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline.

Because no significant inhibition was observed, no additional experiment had to be performed. The following results for the test item sodium 3-sulfobenzoate were determined:

3h NOEC = 1000 mg/L

3h EC50 > 1000 mg/L

3h EC10 > 1000 mg/L

Description of key information

Sodium hydrogen m-sulphonatobenzoate was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. Duration of the test was 3 hours. Activated sludge was used as inoculum. It was taken from a domestic sewage treatment plant and washed before usage. The dry matter was determined as 2.46 g suspended solids/L, giving a concentration of 1.23 g suspended solids/L in the test.

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 8.9 mg/L (95% confidence interval: 5.6 – 12 mg/L) was determined, which lies within the recommended range of 2 – 25 mg/L stated in the OECD guideline.

Because no significant inhibition was observed, no additional experiment had to be performed. The following results for the test item sodium 3-sulfobenzoate were determined:

3h NOEC = 1000 mg/L

3h EC50 > 1000 mg/L

3h EC10 > 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information