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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-08-23 to 2003-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
no
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge: Aeration tank of a municipal biological waste water treatment plant, non-adapted, not pre-conditioned.
- Preparation of inoculum for exposure: Washed twice with tap water.
- Concentration of sludge: 0.2 g/L dry matter in the final mixture

Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Aerobic mineral salts medium prepared with double destilled water (conductivity: < 1.5 µS/cm; DOC: < 0.3 mg/L)
- Additional substrate: No, test substance or procedure control as sole organic carbon sources
- pH adjustment: Yes, periodically adjusted to pH 6.5-8.0 with H2SO4, if necessary.
- Test temperature: 22 +/- 0.5 °C
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Temperature -controlled dark room
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Aeration with CO2-free air; test vessels were stirred (100 rpm) and aerated with sythetic CO2-free air for a maximum test period of 28 days.
- Measuring equipment: TOC analyzer
- Test performed in closed vessels: Yes
- Details of trap for CO2: Gas-absorption bottles filled with NaOH

SAMPLING
- Sampling frequency: At regular intervals: DOC determinations daily during the first phase of the test, than at least once a week and on the 27th and 28 th day; actually: at the beginning of the test, after 3 hours (0.125 days) and on days 1, 4 , 7, 11, 14, 19, 21, 25, 27, 28
- Sampling method: Not specified
- Sample storage before analysis: No, samples were analysed directly after sampling


CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Procedure control: Containing diethylene glycol as reference substance
- Toxicity control: Not performed
Reference substance:
diethylene glycol
Preliminary study:
None
Key result
Parameter:
% degradation (DOC removal)
Value:
85
Sampling time:
28 d
Details on results:
More than 80% of the initial concentration of the test item (based on DOC) was biodegraded between day 0 and 7 (without significant lag phase). The highest biodegradation occured after 27 days. In addition to DOC-determination, biodegradation of the test item was followed by CO2 evolution and calculated as % ThCO2. In result, biodegradation determined by CO2 evolution reached 62% after 28 days, which shows that the test item was predominantly transformed into CO2. In conclusion, as more than 70% biodegradation was observed after 28 days, the test item is regarded to be ultimately biodegradable.
Results with reference substance:
99% biodegradation after 14 days of incubation was observed, confirming the suitability of the test system.

Table 1: DOC concentrations of the test suspension, inoculum blank and procedure control and calculation of

degradation data

Inoculum blank (B) Procedure control with diethylene glycol ® Test suspension with test material (T)*
Time (days) DOC (mg/L) DOC (mg/L) DOC net. (mg/L) Degradation (%) DOC (mg/L)  DOC net. (mg/L) Degradation (%)
0 0.1 49.8 49.7 - 49 48.9 -
0.125 1.2 50.3 49.2 2 54.9 53.7 0
1 0.7 49 48.3 4 24.2 23.5 56
4 1.3 27.7 26.4 48 12.3 11 80
7 1.9 2.5 0.6 99 11.7 9.8 82
11 1.9 2.6 0.7 99 11.4 9.4 82
14 1.8 2.4 0.6 99 10.7 8.9 83
19 1.8 2.2 0.4 99 10.9 9.1 83
21 2 2.5 0.5 99 10.7 8.7 84
25 2.6 2.7 0.1 100 11.2 8.6 84
27 2.5 2.6 0 100 10.4 7.9 85
28 2.4 2.5 0.1 100 10.4 8 85

* Mean of two replicates

Table 2: IC concentrations, calculated from the concentrations in the gas absorption bottles, of the test suspension, inoculum blank and procedure control and

corresponding degradation data

Time (days) Test suspension IC (mg/L) Procedure control IC (mg/L) Inoculum blank IC (mg/L) Biodegradation Test suspension (% ThCO2) Biodegradation Procedure control (% ThCO2)
0 nd nd nd - -
7 22.0 50.0 11.7 20 77
14 36.8 62.1 21.5 30 82
21 54.6 70.2 29.3 50 83
28 71.0 80.5 39.4 62 83
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable, fulfilling specific criteria
Conclusions:
In an inherent biodegradability test according to OECD Guideline 302B, 85% of the initial test item concentration was biodegraded after a contact time of 28 days. Thus, the test item is regarded to be ultimately biodegradable.
Executive summary:

To assess the inherent biodegradability of the test item, a Zahn-Wellens Test / EMPA Test according to OECD Guideline 302 B was conducted under aerobis static conditions and in compliance with ISO/IEC 17025. Non-adapted and not pre-conditioned activated sludge of a municipal biological waste water treatment plant, non-adapted (final concentration 0.2 g/L test solution) was esposed to an initial test item concentration of 50 mg/L (2 replicates), along with a blank control (2 replicates) and a procedure control with the reference substance diethylene glycol (1 replicate) for an contact time of 28 days. Biodegradation was followed by DOC depletion (analysed with a TOC analyser) and, additionally, by CO2 evolution (CO2 trap: gas-absorption bottles filled with NaOH). The DOC content was determined at the beginning of the test, after 3 hours (0.125 days) and on days 1, 4 , 7, 11, 14, 19, 21, 25, 27, 28. In result, more than 80% of the initial concentration of the test item (based on DOC) was biodegraded between day 0 and 7 (without significant lag phase). The highest biodegradation occured after 27 days. Based on CO2 evolution the biodegradation reached 62% after 28 days, which shows that the test item was predominantly transformed into CO2. In the procedure control, 99% biodegradation after 14 days of incubation was observed, confirming the suitability of the test system. In conclusion, as more than 70% biodegradation of the test item was observed after 28 days, the test item is regarded to be ultimately biodegradable. All validity criteria of the test guidelein were fulfilled.

Description of key information

Biodegradation in water: screening tests

In a Zahn-Wellens Test / EMPA Test according to OECD Guideline 302 B, the test item was determined to be ultimately biodegradable (85% biodegradation after 28 days).

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria
Type of water:
freshwater

Additional information

Key study

To assess the inherent biodegradability of the test item, a Zahn-Wellens Test / EMPA Test according to OECD Guideline 302 B was conducted under aerobis static conditions and in compliance with ISO/IEC 17025. Non-adapted and not pre-conditioned activated sludge of a municipal biological waste water treatment plant, non-adapted (final concentration 0.2 g/L test solution) was exposed to an initial test item concentration of 50 mg/L (2 replicates), along with a blank control (2 replicates) and a procedure control with the reference substance diethylene glycol (1 replicate) for an contact time of 28 days. Biodegradation was followed by DOC depletion (analysed with a TOC analyser) and, additionally, by CO2 evolution (CO2 trap: gas-absorption bottles filled with NaOH). The DOC content was determined at the beginning of the test, after 3 hours (0.125 days) and on days 1, 4 , 7, 11, 14, 19, 21, 25, 27, 28. In result, more than 80% of the initial concentration of the test item (based on DOC) was biodegraded between day 0 and 7 (without significant lag phase). The highest biodegradation occured after 27 days. Based on CO2 evolution the biodegradation reached 62% after 28 days, which shows that the test item was predominantly transformed into CO2. In the procedure control, 99% biodegradation after 14 days of incubation was observed, confirming the suitability of the test system. In conclusion, as more than 70% biodegradation of the test item was observed after 28 days, the test item is regarded to be ultimately biodegradable. All validity criteria of the test guideline were fulfilled.