Registration Dossier

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:
14 March 2017 - 13 April 2017
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 guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
according to guideline
other: ISO International Standard 9439 “Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test
Version / remarks:
according to guideline
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"
Version / remarks:
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Details on test material:
- Name of test material (as cited in study report): Tri butyl thiourea
- Appearance: Clear pale to medium brown liquid
- Storage conditions: At room temperature
Specific details on test material used for the study:
Since Tri butyl thiourea was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/L, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test item bottle A: 38.17 mg; test item bottle B: 38.03 mg and toxicity control bottle: 38.03 mg). To this end, 10 mL of Milli-RO water was added to each weighing bottle containing the test item. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test item and the test organisms.

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Storage conditions: Freshly obtained sludge was kept under continuous aeration until further treatment.
- Concentration of suspended solids: 4.4 g/L in the concentrated sludge.
- Preparation of inoculum for exposure: The sludge was allowed to settle (31 minutes) and the supernatant liquid was used as inoculum at the amount of 10 mL/L of mineral medium.
- Pretreatment: 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 overnight to purge the system of CO2.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
19 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: According to OECD 301B
- Test temperature: 22-24 °C
- pH: at start: 7.6, t=14d: 7.7, t=28d: 7.6
- pH adjusted: no
- Test media were excluded from light and the test vessels were brown coloured glass bottles.
- Test media were aerated and stirred continuously.

- Number of culture flasks/concentration: 2 for test suspension, 2 for inoculum blank, 1 for positive control, 1 for toxicity control.
- Method used to create aerobic conditions: Aeration with synthetic air (CO2 < 1 ppm).
- Details of trap for CO2 and volatile organics: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle. 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.

- Sampling frequency: Titrations were made on day 2, 5, 7, 9, 15, 19, 23, 27 and 29. Titrations for the positive and toxicity control were made over a period of at least 14 days.
- Sampling method: Titration of Ba(OH)2 in the gas scrubbing bottles.

- Inoculum blank: yes; contained only inoculum
- Toxicity control: contained test item, reference item, and inoculum
- Other: positive control; contained reference item and inoculum.
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
Mean of 2 test bottles
Details on results:
- Biodegradation: The relative biodegradation revealed 19% and 3% biodegradation of Tri butyl thiourea (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.
- Toxicity control: More than 25% biodegradation occurred within 14 days (35%, based on ThCO2). Therefore, the test item was assumed not to inhibit microbial activity.
- ThCO2: ThCO2 of Tri butyl thiourea was calculated to be 2.34 mg CO2/mg. The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

- Deviations from study plan:
1. There were no titrations between nominal day 9 and nominal day 15. This is not in line with the Study Plan, which states that there will be titrations at least every 5 days. Evaluation: Three CO2 traps are placed in line, in order to catch any breakthrough CO2. Furthermore, all validity criteria were met. Therefore, it can be concluded this deviation has had no impact on the validity or results of the test.
2. On nominal day 20 the gas bottles were empty, leading to a short breakdown in aeration. Evaluation: Such a short (<1 day) breakdown in aeration has no impact on the results. Furthermore, all validity criteria were met.
The study integrity was not adversely affected by the deviations.

BOD5 / COD results

Results with reference substance:
The positive control, sodium acetate, showed a normal biodegradation curve and reached 86% biodegradation after 14 days, which passes the validity criterium of at least 60% biodegradation within 14 days.

Any other information on results incl. tables

Acceptability of the test

1. The positive control item was biodegraded by at least 60% (86%) within 14 days.

2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 17%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (42.2 mg CO2 per 2 litres of medium, corresponding to 21.1 mg CO2/L).

4. The Inorganic Carbon content (IC) of the test item (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli-RO water (Millipore Corp., Bedford, Mass., USA, carbon levels < 500 ppb)), IC was less than 5% of TC (mainly coming from the test item, 12 mg TOC/L).

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Tri butyl thiourea was biodegraded significantly (19% and 3%) 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 Tri butyl thiourea was not readily biodegradable.