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Environmental fate & pathways

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No purity; Limited information on test design
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
no
Remarks:
Non-GLP testing facility.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Station Epuration Gembloux 2nd Stage (28 Oct 2011)
- Concentration of sludge: > 10^6 CFU/mL
- Type and size of filter used: Glass fibre filter
Duration of test (contact time):
28 d
Initial conc.:
>= 10 - <= 20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Stock solution (prepared with High Quality Water = water containing less than 1 mg/L of DOC): Phospate buffer solutions pH 7.4, Magnesium sulphate solution 0.091 M, Calcium chloride solution 0.25 M, Ferric chloride solution 0.001 M
- Test temperature: 22 ± 2 °C
- Aeration of dilution water: Air free from carbon dioxide passes through the solutions using a flow of 50 to 100 mL/min.

TEST SYSTEM
- Culturing apparatus: 2 L brown glass flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: 2 L flasks were mounted with an aeration tube on a magnetic stirrer.
- Details of trap for CO2 and volatile organics if used: The released CO2 from biodegradation reacts with Ba(OH)2 and is trapped under Barium carbonate precipitate (in 250 mL flasks). Then the amount of CO2 is determined by back titration of the remaining Ba(OH)2 with HCl and phenolphthalein as indicator.

SAMPLING
- Sampling frequency: every two to three days for the first 10 days and every three to five days until the day 28
- Sampling method: At day 28, the pH of the solution in the container has to be measured, the 1 to 10 mL of concentrated HCl has to be added to the container in order to eject the CO2 from the test container. Last titration is carried out the day 29.
Reference substance:
other: Na Benzoate 20 mg C/L
Parameter:
% degradation (CO2 evolution)
Value:
94.6
Sampling time:
28 d
Details on results:
Within the 10-day time window the 60% level was passed. Therefore, the test substance is regarded as readily biodegradable.
Results with reference substance:
Degradation: 91.3% after 29 days, 60% pass level was reached.

Table 1: Degradation (%) of reference substance and test substance

Day

Reference 1

Reference 2

Reference Mean

Test 1

Test 2

Test mean

0

0.0

0.0

0.0

0.0

0.0

0.0

2

5.5

5.4

5.5

30.9

29.8

30.4

4

24.3

24.2

24.2

52.5

52.6

52.6

7

51.6

49.6

50.6

66.8

66.7

66.7

10

67.4

63.3

65.4

74.1

75.3

74.7

14

79.9

75.0

77.4

80.8

82.4

81.6

16

83.3

79.3

81.3

82.5

85.9

84.2

18

85.3

82.2

83.7

83.2

87.9

85.5

21

89.0

85.3

87.1

84.7

89.7

87.2

23

92.3

87.4

89.8

85.0

90.7

87.8

25

94.3

88.9

91.6

85.8

91.4

88.6

28

96.1

91.0

93.5

86.4

94.2

90.3

29

97.1

92.1

91.3

87.3

95.3

94.6

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions: Analytical purity of test substance is not stated. Temperature adjustment in the course of oxygen measurements.
Qualifier:
according to guideline
Guideline:
other: ISO/DIS method 10708: Water quality - Evaluation in an aqueous medium if the "ultimate" aerobic biodegradability of organic compounds - Method by determining the biochemical oxygen demand / two-phase closed bottle test)
Deviations:
yes
Remarks:
Temperature adjustment in the course of oxygen measurements
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Temperature adjustment in the course of oxygen measurements
GLP compliance:
no
Remarks:
But the study adhered GLP comparable quality conditions (DIN ISO 9001 certified laboratory)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sewage treatment plant Hochdahl predominantly municipal sewage serving for a population equivalent of approx. 27000.
- Preparation of inoculum for exposure: the mineral medium was inoculated with activated sludge and after addition of a magnetic stirrer stabilised under laboratory conditions for approx. one week. The bottles were aerated with compressed air until O2-saturation was reached and then spiked with a predetermined amount of test substance.
- Concentration of sludge: 2.5 - 3.5 mg/L test medium
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to OECD 301 guidelines prepared in deionised water
- Test temperature: 20 - 25 °C; before each oxygen measurement, the temperature was adjusted to 20 ± 0.5 °C half an hour before each measurement.


TEST SYSTEM
- Culturing apparatus: glass test bottles with a total volume of approx. 300 mL. The test vessels have a known volume of aqueous test mixture (2/3) and air (1/3).
- Number of culture flasks/concentration: 3
- Measuring equipment: O2-electrode (WTW; FRG; microprocessor oximeter OXI 2000 with electrode model TriOxmatic EO 200)
- Test performed in closed vessels due to significant volatility of test substance: performed in closed bottles
- Other: The bottles were aerated with compressed air by means of a sintered glass tube until O2 saturation was reached and then spiked with the test material. The bottles were stoppered and incubated on a shaker. The chemical oxygen demand (COD) was determined in a variant of the ISO method 6060 (closed system with a pressure equaliser / Kelkenberg method, Z. f. Wasser und Abwasserforschung 8, 146 (1975)).


SAMPLING
- Sampling frequency: sampled on day 7, 14, 21 and 28


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 3 replicates
- Abiotic sterile control: no
- Toxicity control: no
Reference substance:
acetic acid, sodium salt
Remarks:
concentration: 100 mg ThOD/L
Parameter:
% degradation (O2 consumption)
Value:
90
Sampling time:
28 d
Remarks on result:
other: Mean from all test bottles
Details on results:
Within the 14-day time window the 60% level was passed. Therefore, the test substance is judged as readily biodegradable.
Results with reference substance:
92% of the reference substance was degraded after 7 days, 101% after 14 and 21 days and 102% after 28 days. The results with the reference substance show that the study is valid according to the ISO Guideline 10708.

Table 1: Oxidation of the test substance

sample

% oxidation

 

day 7

day 14

day 21

day 28

a)

83

91

95

96

b)

71

80

86

86

c)

69

82

87

88

mean

74

84

89

90

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Parameter:
% degradation (O2 consumption)
Value:
90
Sampling time:
28 d
Remarks on result:
other: Mean from all test bottles
Details on results:
Within the 14-day time window the 60% level was passed. Therefore, the test substance is judged as readily biodegradable.
Executive summary:

Within the 14-day time window the 60% level was passed. Therefore, the test substance is judged as readily biodegradable (90% within 28 d, OECD 301 D).

Description of key information

Readily biodegradable (94.6% within 28 d, OECD 301 B, CO2 evolution)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Two studies are available, in which the ready biodegradability of the target substance Fatty acids, C18-unsaturated, 2-hexyldecyl ester was investigated according to internationally accepted OECD testing guidelines. The key study was conducted with the target substance and the supporting study was conducted with the structurally related source substance fatty acids, C16-18, 2-hexyldecyl esters (CAS 101227-09-2). The source substance is characterized by similar fatty acid and alcohol components and is therefore considered a suitable representative for the assessment of the ready biodegradability of the target substance. Read-across was conducted in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5 and a detailed justification of the analogue approach is provided in IUCLID section 13.

In the key study, the target substance (10 – 20 mg/L DOC) was inoculated with activated sludge (> 10E+06 CFU/mL) for 28 d according to OECD guideline 301 B. The amount of CO2 produced by the degradation of the substance was trapped with Ba(OH)2 and precipitated as BaCO3 and quantified by back titration of the remaining Ba(OH)2 in the absorber flasks using HCl and phenolphthalein as indicator. Biodegradability was expressed in percent CO2 of theoretical CO2 (ThCO2).

The 60% level was reached within the 10-d window. Biodegradation reached a mean of 93.5% by Day 28 and 94.6% by Day 29. Therefore, the test substance is regarded as readily biodegradable according to the OECD criteria.

In the supporting study, 100 mg/L test item (based on ThOD/L) was inoculated with 2.5 – 3.5 mg/L activated sludge for 28 d in a closed bottle test according to OECD guideline 301 D. Biodegradation was quantified and expressed in terms of O2 consumption.

The 60% pass level was attained within the 14-d window and a mean degradation of 90% was recorded after 28 d. Therefore, the substance is readily biodegradable according to the OECD criteria.

Based on the structural similarities between the target and source substance and the reliable results on the target substance, it can be concluded that Fatty acids, C18-unsaturated, 2-hexyldecyl ester is readily biodegradable according to OECD criteria.