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Biodegradation in water: screening tests

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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:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Temperature ranged from 17.7-24.2 °C. The pH of the sodium acetate control was not measured after 14 d. The amount of K2HPO4 in the nutrient solution was 21.7 g. None of these deviations are thought to have influenced the results.
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Oxidation ditch in Hazerswoude, the Netherlands
- Storage conditions: Aerated until use.
- Storage length: 5 days
- Concentration of sludge: 5 mL/L
Duration of test (contact time):
28 d
Initial conc.:
1.08 mg/L
Based on:
test mat.
Initial conc.:
2.16 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: potassium dihydrogen phosphate 8.5 mg/L, dipotassium hydrogen phosphate 21.7 mg/L, disodium monohydrogen phosphate heptahydrate 33.4 mg/L, ammonium chloride 0.5 mg/L, magnesium sulphate heptahydrate 22.5 mg/L, calcium chloride dihydrate 36.4 mg/L, iron (III) chloride hexahydrate 0.25 mg/L
- Test temperature: 17.7 - 24.2 °C
- pH: 6.2-7.3
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 293 mL BOD bottles
- Number of culture flasks/concentration: 16
- Method used to create aerobic conditions: aeration of mineral medium
- Measuring equipment: oxygen electrode
- Test performed in closed vessels due to significant volatility of test substance: yes

SAMPLING
- Sampling frequency: Days 0, 7, 14, 21, and 28
- Sampling method: oxygen electrode

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Toxicity control: yes

STATISTICAL METHODS: Average oxygen consumption and standard deviations were calculated.
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
28
Sampling time:
28 d
Remarks on result:
other:
Remarks:
1.08 mg/L test substance
Key result
Parameter:
% degradation (O2 consumption)
Value:
19
Sampling time:
28 d
Remarks on result:
other:
Remarks:
2.16 mg/L test substance
Details on results:
Inoculum blanks showed no more than 1.50 mg O2/L degradation. The inoculum activity control showed complete biodegradation. The toxicity control showed 38-70% degradation, and thus was valid.
Key result
Parameter:
BOD5
Value:
0.47 g O2/g test mat.
Remarks on result:
other:
Remarks:
1.08 mg/L test concentration
Key result
Parameter:
BOD5
Value:
0.4 g O2/g test mat.
Remarks on result:
other:
Remarks:
2.16 mg/L test concentration
Results with reference substance:
The reference substance results were valid with over 60% degradation in 10 days.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance was only 19-28% biodegraded in 28 days, and thus is not readily biodegradable.
Executive summary:

The test substance was tested for biodegradability in a 28-day closed bottle test using concentrations of 1.08 and 2.16 mg/L. The results of the toxicity controls, reference substance, inoculum controls, and inoculum activity controls were valid. The test substance was only 19-28% degraded in 28 days. The test substance is therefore not readily biodegradable.

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:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to
Guideline:
other: ISO Standard 9439: Water Quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - carbon dioxide evolution test
Version / remarks:
1999
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Freshly obtained from the municipal sewage treatment plant "Waterschaf de Maaskant", 's-Hertogenbosch, The Netherlands
- Method of cultivation: The sludge was kept under continuous aeration.
- Pretreatment: Before use, the sludge was allowed to settle (60 min) and the liquid was decanted for use as inoculum at the amount of 10 mL/L of mineral medium.
- Concentration of sludge: 3.8 g suspended solids/L
Duration of test (contact time):
29 d
Initial conc.:
19 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline (prepared in tap water treated by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges)
- Test temperature: 21.7 - 22.2 °C
- pH: 7.6 - 7.9
- pH adjusted: no
- Aeration of dilution water: During the test, test media were aerated continuously.
- Other: 0.0125 M Ba(OH)2 was stored in a sealed vessel to prevent absorption of CO2 from the air.

TEST SYSTEM
- Culturing apparatus: 2 L all-glass brown colored bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Mineral medium was aerated during exposure (mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle containing 0.5-1 L 0.0125 M Ba(OH)s solution to trap CO2; The synthetic air was sparged through the scrubbing solutions at a rate of approx. 1-2 bubbles per second (30-100 mL/min).
- Measuring equipment: Produced CO2 was mesured by titration of the remaining Ba(OH)2 wiith 0.05 M HCl.
- Details of trap for CO2 and volatile organics if used: CO2 was bsorbed using barium hydroxide.

SAMPLING
- Sampling frequency: Titrations were made every second or third day during the first 10 d and thereafter at least evey fifth day. In the positive and toxicity control titrations were made at least every 14 d.
- Sampling method: Each time a CO2-absorber bottle was removed for titration. The removed bottle was replaced with a new CO2-absorber at the far end of the series of absorber bottles. On day 28 the pH was measured in each test suspension and 1 mL of concentrated HCl was added to the bottles of the inoculum blank and test suspension. Bottles were aerated overnight to drive off CO2 present in the test suspension. Final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, two bottles
- Abiotic sterile control: no
- Toxicity control: yes, one bottle
- Other: reference substance, one bottle
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
26
Sampling time:
29 d
Remarks on result:
other: Mean of two replicates
Results with reference substance:
The reference substance was degraded to 72% after 14 days confirming the suitability of the inoculum.

The toxicity control degraded to 19% after 14 d indicating slight inhibiting effects of the test item to the inoculum.

Table 1: Degradation of the test item, toxicity control and reference substance

Day

Biodegradation [%]

Bottle A

Bottle B

Mean

Toxicity control

Reference substance

2

0

1

0

2

13

5

0

5

3

9

44

7

0

7

3

14

57

9

1

9

5

16

64

14

6

13

10

19

72

19

16

18

17

-

-

23

20

21

20

-

-

27

23

22

22

-

-

29

27

25

26

-

-

29

27

25

26

-

-

29

27

25

26

-

-

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable

Description of key information

Not readily biodegradable: 19 - 28% after 28 d (OECD 301D); 26% after 28 d /OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Two experimental studies are available investigating the biodegradation of fatty acids C16-18 (even numbered), mono, di and triesters with sucrose. Both studies are considered as key studies since they are of equal quality and reliability.
The first study (Mitsubishi, 2003) was performed according to OECD 301D (GLP). Activated sludge from a sewage treatment plant was used and two concentrations were tested (1.08 and 2.16 mg/L). Since the test item was only degraded to 19-28% after 28 d based on O2 consumption it is not considered to be readily biodegradable according to the OECD criteria.
This result is confirmed by the second experimental study which was performed according to OECD 301B (Sisterna, 2009). The test item was exposed to domestic activated sludge at a concentration of 19 mg/L (corresponding to 12 mg/L TOC) and the CO2 evolution was measured for 28 d. On day 28, 1 mL of concentrated hydrochloric acid was added to the bottles of the inoculum blank and test suspension. Bottles were aerated overnight to drive off CO2 present in the test suspension. Final titration was made on day 29. The substance was degraded to 26% at the end of the test. Thus, the substance is not considered to be readily biodegradable according to the OECD criteria as well.