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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): EMCA
- Physical state: Colourless liquid
- Analytical purity: 99.3 %
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: The source of the test organisms was activated sludge freshly obtained from a municipal sewage treatment plant "Waterschap de Maaskant', 's-Hertogenbosch, the Netherlands, receiving predominantly domestic sewage.
- Laboratory culture: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 4.2 mg/L in the concentrated sludge (information obtained from municipal sewage treatment plant).
- Method of cultivation: Before use sludge was allowed to settle (65 minutes) and the liquid was decanted for use as inoculum at the amount of 10mL/L mineral medium.
- Preparation of inoculum for exposure: Mineral components, Milli-RO water (Ca 80% total volume) and inoculum (1% final volume) were added to each bottle. The mixture was aerated with synthetic air to purge the system of CO2.
- Water: Tap water purified by reverse osmosis (Milli-Ro) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q) were used.
Duration of test (contact time):
28 d
Initial conc.:
30.5 mg/L
Based on:
other: Corresponding to 12mg TOC/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium contained 10 mL of solution a), 1 mL of solutions b) to D and Milli-RO water.
Stock solution of mineral components:
a)8.50 g KH2PO4
21.75 g K2HPO4
67.20 g Na2HPO4*12H2O
0.50 g NH4Cl
dissolved in Milli-Q water and made up to 1 L, pH 7.4 ± 2
b) 22.50 g MgSO4*7H2O dissolved in Milli Q water and made up to 1 L.
c) 36.40 g CaCl2*H2O dissolved in Milli Q water and made up to 1L.
d) 0.25 FeCl3*6H2O dissolved in MilliQ water and made up to 1 L.

- Additional substrate: Barium hydroxide 0.0125M stored in sealed vessels to prevent absorption of CO2 from the air.
- pH: 7.5-7.8 in all vessels including blanks, positive controls and toxicity control.
- pH adjustment: no.
- Temperature: 21.0-22.1 ºC.
- Aeration of dilution water: During the test period the test media was continuously aerated and stirred.
- Suspended solids concentration: 4.2 g/L in the concentrated slude, before use sludge was allowed to seetle for 65 minutes and the liquid was decanted for use as inoculum at the amont of 10mL/L of mineral medium.


TEST SYSTEM
- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: During the test period the test media was continuously aerated and stirred.
- Measuring equipment: Titration.
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: The subtance and positive controls were added to the bottles containing the microbial organisms and mineral components (Ca 80% total volume). The volumes of suspensions were made up to 2L with Milli-RO water. Three CO2 absorbers (bottles filled with 100mL 0.0125 M Ba(OH)2 were connected in series to the exit air line of each test bottle.
- Other: The CO2 produced in each bottle reacted with 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.


SAMPLING
- Sampling method: Titrations on day 2, 5, 7, 9, 14, 19, 23, 27, 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 bottles
- Toxicity control: 1 bottle (containing test substance, reference substance and inoculum)
- Positive control (reference substance): 1 bottle

Reference substance:
acetic acid, sodium salt
Remarks:
40 mg/L (12mg TOC/L).
Test performance:
The relative degradation values calculated from the measurements performed during test period, revealed 14 and 34 % degradation of EMCA for the duplicate bottles tested. The 10-day window (at leasty 60% biodegradation) was not met, therefore the subtance can not be considered ready biodegradable.
In the toxicity bottle more than 25 % degradation occured within 14 days (44 % based on ThCO2), therefore the test subtance was not assumed to inhibtio microbial activity.
Parameter:
% degradation (CO2 evolution)
Remarks:
replicate #1
Value:
14
Sampling time:
29 d
Remarks on result:
other: The % degradation was calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test subtance (88.5 mg/CO2/2L).
Parameter:
% degradation (CO2 evolution)
Remarks:
replicate #2
Value:
34
Sampling time:
29 d
Remarks on result:
other: The % degradation was calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test subtance (88.5 mg/CO2/2L).
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
(average)
Value:
24
Sampling time:
29 d
Details on results:
The test subtance was not ready biodegradable based on the determination of produced CO2 by titration of barium hydroxide with standardized HCl0.05 M solution.
Results with reference substance:
The positive control substance was degraded by 70 % within 14 days.

It was noted that for a very short period zero air was used (synthetic air including CO2). It was noted that the contents of a Ba(OH)2 bottle preceding the test series was renewed.

On days 27 and 29 the difference of duplicate values for % degradation of EMCA was on the borderline of the criterion (20%). This borderline was considered to have no effect on the outcome of the study.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test revealed 14 and 34 % degradation for each of the duplicatle bottless after 29 days. The test item is not readily biodegradable.
Executive summary:

A screening test for biodegradation in water was conducted in accordance to OECD 301 B CO2 evolution (Modified Sturm test) under GLP conditions with the test substance Carbamic chloride, N-ethyl-N-methyl-. The test included 2 replicate of blank controls, 2 replicates of the test suspension, a positive control (sodium acetate) and a toxicity control. Three CO2 absorbers (bottles filled with 0.0125 BA(OH)2 were connected in series to the exit air line of each test bottle. The CO2 evolution was followed by titrating the remaining BA(OH)2 with 0.05 M standardized HCl. Titrations were made every 3 days until day 10 and then at least every fifth day. The last titration was performed on day 29. The relative degradation calculated from cumulative measurements performed until day 29 of the test revealed 14 and 34 % degradation of the test substance in the replicates. The substance is not ready biodegradable. However, the average of degradation (24 %) could be regarded as evidence of inherent primary biodegradability.

Description of key information

Key study. Test method according to OECD 301 B (Modified Sturm test). GLP study. The test item is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria

Additional information

A screening test for biodegradation in water was conducted in accordance to OECD 301 B - CO2 evolution (Modified Sturm test) under GLP conditions with the test item. The test included 2 replicate of blank controls, 2 replicates of the test suspension, a positive control (sodium acetate) and a toxicity control. The relative degradation calculated from cumulative measurements performed until day 29 of the test revealed 14 and 34 % degradation of the test substance in the replicates. The substance cannot be regarded as ready biodegradable. However, the average of degradation (24 %) could be regarded as evidence of inherent primary biodegradability.