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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:

Test material

Constituent 1
Reference substance name:
Reaction product aqueous Phosphoric Acid, 1-Methoxypropan-2-ol and 4,4'-Isopropylidenediphenol-Epichlorohydrin
EC Number:
Molecular formula:
Not possible to assign for a complex UVCB substance
Reaction product aqueous Phosphoric Acid, 1-Methoxypropan-2-ol and 4,4'-Isopropylidenediphenol-Epichlorohydrin
Test material form:

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium. The concentration of suspended solids (SS) was determined to be 3.0 g/L in the concentrated sludge as used for the test. The magnetically stirred sludge was used as inoculum at the amount of 5 mL/L of mineral medium, leading to a SS concentration of 15 mg/L.

Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
18 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: Standard as per OECD guideline
- Test temperature: 22 - 23°C
- pH: 7.6
- pH adjusted: No
- Aeration of dilution water: yes
- Continuous darkness: yes

- Number of culture flasks/concentration: 2 bottles (2 Litre coloured glass bottles)
- 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 passed through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 mL/min).
- Test performed in open system: No

Preparation: At the start of the test (day 0), test and reference item were added to the bottles containing the microbial organisms and mineral components.The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before. 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.

Experimental CO2 production: 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)).

Measurements: 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 item. 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 were 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) was used as pH-indicator.

On the penultimate day, the pH of respective test suspensions was measured and 1 mL of concentrated HCl (37%) 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 15 (positive and toxicity control) and on day 29 (remaining vessels).

- Inoculum blank: yes, 2 bottles
- Abiotic sterile control: no
- Toxicity control: yes, 1 bottle
- Positive control: yes , 1 bottle

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
Acceptability of the Test
1. The positive control item was biodegraded by at least 60% (90%) within 14 days.
2. The difference of duplicate values for %-degradation of the test item was always less than 20 (≤ 10%).
3. The total CO2 release in the blank at the end of the test did not exceed 40 mg/L (61.5 mg CO2 per 2 litres of medium, corresponding to 30.8 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, 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.
% Degradation
% degradation (CO2 evolution)
>= 15 - <= 16
Sampling time:
28 d
Remarks on result:
other: Sampling time actually Day 29, but microbial activity ended on Day 28 by addition of HCl
Details on results:
The relative biodegradation values calculated from the measurements performed during the test period revealed 15% and 16% biodegradation of the test item (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, the test item was found not to inhibit microbial activity. Since all criteria for acceptability of the test were met, this study was considered to be valid.

BOD5 / COD results

Results with reference substance:
90% degradation after 14 days

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The test item was not readily biodegradable under the conditions of the modified Sturm test presently performed.

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