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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-02-21 - 2017-03-23
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to
other: ISO International Standard 10634. "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium", (1995)
GLP compliance:

Test material

Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Storage condition of test material: At room temperature

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Storage length: The freshly obtained sludge was used immediately
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (33 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: The concentration of suspended solids was determined to be 3.0 g/L in the concentrated sludge.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
28 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium:
Stock solutions of mineral components
A) 8.50 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.50 g NH4Cl dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
Mineral medium
1 litre mineral medium contains: 10 mL of solution (A), 1 mL of solutions (B) to (D) and Milli-RO water.
- Test temperature: 22 - 24 °C (recorded in a vessel with water in the same room)
- pH: 7.6 at test start, 7.4 - 7.6 on day 28
- pH adjusted: not specified
- Aeration of dilution water: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air (CO2 < 1 ppm) overnight to purge the system of CO2.
- Suspended solids concentration: 3.0 g/L
- Continuous darkness: yes

- Culturing apparatus: 2 litre brown coloured glass bottles
- Number of culture flasks/concentration:
Test suspension: containing test item and inoculum (2 bottles), Inoculum blank: containing only inoculum (2 bottles),Positive control: containing reference item and inoculum (1 bottle), Toxicity control: containing test item, reference item and inoculum (1 bottle).
- Method used to create aerobic conditions: A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Measuring equipment: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany). Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days. Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator. On day 28, the pH of all test suspensions was measured and 1 mL of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
Volume of test vessels: 2000 mL
Volume test solution: 2000 mL
Test duration: 28 days (last CO2-measurement on day 29)

- Inoculum blank: 2 bottles containing only inoculum
- Toxicity control: 1 bottle containing test item, reference item and inoculum
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Preliminary study:
No information about a preliminary study are available.
Test performance:
No unusual observations during test period observed.
% Degradationopen allclose all
Key result
% degradation (CO2 evolution)
Bottle A (based on ThCO2)
Sampling time:
28 d
Remarks on result:
other: The criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
Key result
% degradation (CO2 evolution)
Bottle B (based on ThCO2)
Sampling time:
28 d
Remarks on result:
other: The criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.
Details on results:
Theoretical CO2 Production:
The ThCO2 of (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was calculated to be 1.56 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

In the toxicity control, more than 25% biodegradation occurred within 14 days (48%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference item sodium acetate, which showed a normal biodegradation curve.

BOD5 / COD results

Results with reference substance:
The positive control item was biodegraded by at least 60% (72%) within 14 days (validity criterion fulfilled).

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
(S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was biodegraded significantly (35% and 30%) during the test period. However, since at least 60% biodegradation was not reached within 10 days immediately following the attainment of 10% biodegradation (10-day window), the criterion for ready biodegradability was not met. Thus, under the conditions of this test (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)benzenecarbothioate was not readily biodegradable.
Executive summary:

The objective of the study was to evaluate the non-volatile test item (S)-(4,5-dihydro-5 -thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge; Carbon dioxide (CO2) evolution test (modified Sturm test). The study procedures described in this report were in compliance with the OECD guideline No. 301 B, 1992, under GLP. In addition, the procedures were designed to meet the test methods of the ISO standard 10634, 1995.

The relative biodegradation values calculated from the measurements performed during the test period revealed 35% and 30% biodegradation of (S)-(4,5-dihydro-5-thioxo-1,3,4 -thiadiazol-2-yl) benzenecarbothioate (based on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

In the toxicity control, (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

In conclusion, (S)-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl) benzenecarbothioate was designated as not readily biodegradable.