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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:
18 April 2016 - 19 May 2016
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 9439: Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous med ium - carbon dioxide evolution test
Version / remarks:
according to guideline
other: ISO 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:
yes (incl. QA statement)
certificate date: 3 Nov 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
The substance is a UVCB, of which a major component can be represented by the following: [C20H33O2]2Zn
Test material form:
Details on test material:
- Appearance: Brown coloured, brittle solid
- Storage condition of test material: At room temperature
- Chemical name: Zinc modified rosinate, hydrogenated zinc rosinate
- CAS No.: 68425-02-5
Specific details on test material used for the study:
Stability at higher temperatures: yes, maximum temperature: 150 °C
Solubilityin water: not yet measured
Stability in water: not indicated
Volatile: not indicated

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):
The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment
plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic s
- Storage conditions: The freshly obtained sludge was kept under continuous aeration until further
- Storage length: not indicated
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (34 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.
- Concentration of sludge: 4.3 g/Lin the concentrated sludge
- Water used: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
12 other: mg TOC/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: mineral medium according to OECD 301
- Test temperature: 21.9 - 22.5 °C
- pH: start: 7.6; end: 7.8 - 8.2
- pH adjusted: no
- Aeration of dilution water: continuously, except for a temporary breakdown on nominal test days 10 and
11 due to empty synthetic air gas cylinders. However, a relative short breakdown of the aeration (≤ 1 d
ay) was considered to have no effect on the outcome of the study.
- Suspended solids concentration: 10 mL supernatant liquid/L (for more details see section 'Details on i
- Continuous darkness: yes

- Culturing apparatus: 2 litre glass brown coloured bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration with synthetic air
- Measuring equipment: The amount of CO2 produced was determined by titrating Ba(OH)2 with 0.05 M
standardized HCl (1:20 dilution from 1 M HCl). Phenolphthalein (1% solution in ethanol, Merck) was used
as pH-indicator.
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: 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 mo
ved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the

- Sampling frequency: titrations were made every second or third day during the first 10 days, and thereaf
ter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the po
sitive and toxicity control were made over a period of at least 14 days
- Sampling method: titration
- Sample storage before analysis: not applicable

- Inoculum blank: yes, 2 replicates
- Abiotic sterile control: yes, 1 replicate
- Toxicity control: yes, 1 replicate

Reference substance
Reference substance:
acetic acid, sodium salt
Purity: 99.1%; 12 mg TOC/L

Results and discussion

% Degradation
Key result
% degradation (CO2 evolution)
Sampling time:
28 d
Remarks on result:
other: Result based on mean value of duplicate test bottles (17% and 34%)
Result based on mean value of duplicate test bottles (17% and 34%)
Details on results:
- The ThCO2 of the test item was calculated to be 2.62 mg CO2/mg, the ThCO2 of sodium acetate was
calculated to be 1.07 mg CO2/mg.
- The relative biodegradation values calculated from the measurements performed during the test period
revealed 17% and 34% biodegradation of Resin acids and Rosin acids, hydrogenated, zinc salts (based
on ThCO2), for the duplicate bottles tested. Thus, the criterion for ready biodegradability (at least 60% b
iodegradation within a 10-day window) was not met.
- In the toxicity control, more than 25% biodegradation occurred within 14 days (32%, 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:
62% biodegradation within 14 days.

Any other information on results incl. tables

Validity criteria:

1. The positive control item was biodegraded by at least 60% (62%) within 14 days.

2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤19%).

3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (52.8 mg CO2 per 2

litres of medium, corresponding to 26.4 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 tapwater

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)

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

Applicant's summary and conclusion

Validity criteria fulfilled:
See: ''Any other information on results'' section
Interpretation of results:
under test conditions no biodegradation observed
Under the conditions of the modified Sturm test, according to OECD 301B, the test item was found to
biodegrade for 17% and 34% (for the duplicate bottles tested) after 28 days of incubation. Therefore, it
cannot be termed as readily biodegradable.
Executive summary:

In a test performed according to OECD 301B (modified Sturm test) and GLP, the test item did not reach the

pass level of 60% for ready biodegradability, neither within the 10-d window nor after 28 days of incubation.

Therefore, it is designated as not readily biodegradable. All the criteria for acceptability of the test were met,

so the study is considered valid without constrictions.