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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:
supporting study
Study period:
1995-11-20 to 1995-12-22
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test is comparable to guideline study, although some experimental details are not reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
TIC at Day 0 was not reported
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): On 11/02/1996 sludge was sampled at an aerobic sewage treatment plant at Ossemeersen Gent. It had 4.45 g/l of total solids and 1.95 g/l of total ash after sieving over 200 um and a pH of 7.32.

- Pretreatment: Three litres of the sieved sludge was aerated for one week. Sludge settled and supernatant decanted. 800 ml of thickened sludge was diluted with 2.2 l freshly prepared mineral medium. 20ml of this anaerobically treated sludge was added to 5 l of freshly prepared mineral medium. The final total solids concentration was not determined. The DOC for the sludge at the start was 2.4 mg C/l while the DOC value of the mineral medium was 1.1 mg C/l.
Duration of test (contact time):
28 d
Initial conc.:
20 other: mgC/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: 20 ml aerobically treated sludge added to 5L mineral salts medium.

- Test temperature: 22 +/- 1 C

TEST SYSTEM
- Culturing apparatus: Penicillum flask, butyl rubber stoppers

- Test volume: 80 ml

- Head space: 40 ml

SAMPLING
- Sampling frequency: Day 7, 14, 21, 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
Reference substance:
aniline
Parameter:
% degradation (CO2 evolution)
Value:
92
Sampling time:
28 d
Results with reference substance:
The reference substance degraded by >60% after 14 days.

Table 1: Degradation kinetics

Type of suspension

Vessel no.

% degradation at sampling time (days)

7

14

21

28

Reference substance

39.04

74.86 

73.58

84.06

 

2

48.96

72.10

67.41

87.84

3

53.32

68.51

66.32

92.30

 

54.44

74.62

67.94

90.43

 

 

 

94.27

 

 

 

 

86.13

Mean 

48.94

72.52

66.82

89.17

 

 s

7.01

2.95

3.25

3.86

95% C.L.

11.15

4.69

5.17

4.05

Test sample

73.06

89.04 

78.65 

93.25

 

2

68.06

85.37

77.38 

92.81 

 

3

68.79 

88.86 

74.26 

92.52 

 

70.76

86.64 

76.04 

93.21 

 

 

 

 

89.04 

 

 

 

 

91.35 

Mean 

70.17

87.48 

76.58 

92.03 

 

 s

2.23 

1.78 

1.88 

1.62 

 

95% C.L.

3.55 

2.83 

2.99 

1.70 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
A GLP ready biodegradability study conducted according to the ISO ring test "CO2 headspace biodegradation test". The substance was found to be readily biodegradable meeting the ten day window.
The following validity criteria were fulfilled
(1) the reference substance degraded by >60% after 14 days and

(2) the total inorganic carbon (TIC) present in the blank controls at the end of the test was less than 15% of the organic carbon added initially as the test substance.

TIC at Day 0 was not reported.


Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Summary report only available. There is insufficient information reported to assess the validity of this test.
Qualifier:
according to guideline
Guideline:
other: RDA-Blok-Test equivalent to a two-phase closed bottle test
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: effluent of predominantly domestic sewage treatment plant
Duration of test (contact time):
30 d
Initial conc.:
50 mg/L
Based on:
COD
Value:
65 - 77
Sampling time:
30 d
Details on results:
15 days = 30-75%
30 days = 65-77%
Degradation data only reported for days 15 and 30.

The method used is suitable for poorly water-soluble compounds. No information is provided regarding the validity criteria.

This information is from a summary of the full report and reports test concentration as 100 mg COD/l in the test procedure and 50 mg COD/l in the results section.

Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: screening test, other
Remarks:
(BOD test)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: Assessed using methods based on OECD Guideline 301D (Closed Bottle Test) and Procedure C.4-E of the Annex to Directive 92/69/EEC
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Duration of test (contact time):
5 d
Parameter:
other: % of COD
Value:
62
Sampling time:
5 d
Details on results:
The mean BOD after 5 days was 1.50 gO2/g. The mean COD was 2.43 gO2/g. The BOD:COD ratio ranged from 49% to 74%.
The mean 5 day BOD of Kalcohl 0898 was 62% of its COD.
Parameter:
BOD5
Value:
1.5 g O2/g test mat.
Parameter:
COD
Value:
2.43 g O2/g test mat.
Parameter:
BOD5*100/COD
Value:
62 other: ranged from 49% to 74%
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Guideline study although some validation data not reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Initial conc.:
10 mg/L
Based on:
COD
Reference substance:
other: Sodium benzoate
Value:
59
Sampling time:
29 d
Details on results:
Kinetic of control substance:
3 days = 39%
8 days = 74%
15 days = 82%
29 days = 89%

The test substance degraded <60% over the test period and therefore cannot be considered readily biodegradable. However, significant degradation was observed therefore the substance is considered inherently biodegradable.

Kinetic of test substance (in %):
= 15 after 3 day(s)
= 43 after 8 day(s)
= 52 after 15 day(s)
= 59 after 29 day(s)

Cumulative CO2 production in the controls after 29 days (77.8 and 80.1 mgCO2) was within the acceptable range for this assay system (recommended maximum = 120 mgCO2 for a three litre  culture). The reference compound reached 

the pass level within 14 days and the parallel assays did not differ by more than 20%. No information is given on total 
inorganic carbon levels at the start of the test.

Mean cumulative CO2 production by the mixtures containing the test substance at 10 mgC/L was equivalent to 15% after
three days and 59% after 29 days.

Interpretation of results:
inherently biodegradable
Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Non-guideline study
Principles of method if other than guideline:
Method: other
GLP compliance:
not specified
Oxygen conditions:
anaerobic
Inoculum or test system:
other: digested sewage sludge diluted to 10%
Initial conc.:
50 mg/L
Based on:
DOC
Parameter:
% degradation (CH4 evolution)
Value:
75
Sampling time:
8 wk
Details on results:
Octanol was readily mineralized (> 75% of theoretical methane production).
Endpoint:
biodegradation in water: screening test, other
Remarks:
(BOD5 test)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Non-guideline study using acclimated inoculum. Whilst pre-acclimation would normally be considered a major methodological deficiency, in the case of the aliphatic alcohols which are naturally occurring in the environment and also produced by microorganisms, pre-acclimation of the culture is not considered to significantly affect the test outcome.
Principles of method if other than guideline:
The study used a BOD technique, with acclimated microbial culture capable of degrading alcohols and ketones as sole carbon and energy source.
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Mineral salts medium containing 250 ul/L of test chemical was inoculated with domestic sewage and incubated on a rotary shaker (120 rpm) at 21+/-3 deg C. These cultures were stored individually at 4 deg C in 0.2 M phosphate buffer (pH 6.8) containing 100 ul/L of the enrichment substrate.
Duration of test (contact time):
5 d
Details on study design:
Test chemical and 1ml of acclimated seed were added to 20 ml of dilution water in 300 ml BOD bottles. The bottles were then filled to capacity with dilution water, sealed, and incubated for 5d at 21C +/- 3 C. Initial concentrations of test chemical in the BOD bottles ranged from 0 to 3.2 mg/l 
and never exceeded the measured (or in some cases, estimated) water solubility of the chemical. BOD was determined by measurement of dissolved oxygen concentrations in the test vessels at the start and end of the test  period.
Parameter:
% degradation (O2 consumption)
Value:
54.1
Sampling time:
5 d
Remarks on result:
other: %ThOD
Parameter:
BOD5
Parameter:
COD
Parameter:
BOD5*100/COD

The primary purpose of this study was to determine a quantitative structure-biodegradability relationship for a series of alcohols.

Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The information reported is insufficient to assess the validity of this study.
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 835.3100 Aerobic Aquatic Biodegradation Test
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: no information on inoculum provided
Duration of test (contact time):
31 d
Initial conc.:
20 mg/L
Based on:
test mat.
Reference substance:
other: Sodium benzoate
Value:
60
Sampling time:
30 d
Details on results:
Kinetic of control substance:
4 days = 47.1%
10 days = 58.1%
17 days = 60.5%
24 days = 61.2%
31 days = 62.2%

The test substance attained <60% degradation during the test period.

Kinetic of test substance (in %):
= 39 after 4 day(s)
= 53 after 10 day(s)
= 57 after 17 day(s)
= 59 after 24 day(s)
= 60 after 31 day(s)

There is no information given on the validity criteria.

Interpretation of results:
inherently biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-03-17 to 2009-04-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
no
Remarks:
At the time of the study, this lab was in the process of attaining formal GLP status and did not hold certification. The work was conducted in accordance with GLP-principles (personal communication, 2010) and to high quality standards.
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): Fairfield Wastewater Treatment Plant, Fairfield, Ohio

- Preparation of media: The media was prepared one day prior to test initiation. The media consisted of the following reagents (1ml/l) in high quality deionised water: magnesium sulfate (2.25%), calcium chloride (2.75%), ferric chloride (0.025%) and phosphate buffer (10 ml/l). The phosphate buffer solution consisted of potassium dihydrogen phosphate (8.5 g/l), dipotassium hydrogen phosphate (21.75 g/l), disodium hydrogen phosphate dihydrate (33.4 g/l) and ammonium chloride (0.5 g/l).

- Preparation of inoculum for exposure: Activated sludge solids centrifuged for 20 minutes at 3000rpm and the supernatant decanted. Solids resuspended in media and homogenised in a blender for 1 minute. The solids were washed a second time as descripbed above and the TSS (total suspended solids) measured. Sufficient inoculum was added to the media to obtain a solids concentration of 15 mg/l. This mixture was adjusted to pH7 and aerated overnight with CO2-free air.

- Concentration of sludge: 15 mg solids/l.
Duration of test (contact time):
28 d
Initial conc.:
18.8 mg/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: test material, sludge inoculum and phosphate buffered media

- Test temperature: 22 C




TEST SYSTEM
- Culturing apparatus: 1 litre bottles

- Number of culture flasks/concentration: 18.8 mg/l. Two replicates.


SAMPLING
- Sampling frequency: 12h

- Sampling method: Conductivity probe immersed in 1% NaOH to measure production of CO2.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. Six replicates

- Reference substance: Sodium Benzoate. Three replicates
Parameter:
% degradation (CO2 evolution)
Value:
77.9
St. dev.:
3.4
Sampling time:
28 d
Details on results:
The final SOC levels ranged from 0.5 to 0.6 mg/l, and were <0.1 mg/L for reference substance.

Table 1: Degradation kinetics

Type of suspension

% degradation at sampling time (days)

0

1

2

3

6

8

10

14

16

20

22

24

27

28

 

 

 

 

 

 

 

 

 

 

Test sample (mean of 2 replicates)

0

0

7.50

20.76

50.59

65.51

71.75

77.65

79.87

78.35

78.37

76.47

77.86

77.93

 

 

 

 

 

 

 

 

 

 

 

Reference substance (mean of 3 replicates)

0

0

26.03

41.45

68.48

76.51

79.90

81.88

82.14

82.10

82.19

81.76

81.42

81.69

 

 

 

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
A reliable study conducted according to an appropriate test protocol determined the substance to be readily biodegradable.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1995-11-20 to 1995-12-22
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Test is comparable to guideline study, although some experimental details are not reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
yes
Remarks:
TIC at Day 0 was not reported
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): On 11/02/1996 sludge was sampled at an aerobic sewage treatment plant at Ossemeersen Gent. It had 4.45 g/l of total solids and 1.95 g/l of total ash after sieving over 200 um and a pH of 7.32.

- Pretreatment: Three litres of the sieved sludge was aerated for one week. Sludge settled and supernatant decanted. 800 ml of thickened sludge was diluted with 2.2 l freshly prepared mineral medium. 20ml of this anaerobically treated sludge was added to 5 l of freshly prepared mineral medium. The final total solids concentration was not determined. The DCO for the sludge at the start was 2.4 mg C/l while the DOC value of the mineral medium was 1.1 mg C/l.
Duration of test (contact time):
28 d
Initial conc.:
20 other: mgC/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: 20 ml aerobically treated sludge added to 5L mineral salts medium.

- Test temperature: 22 +/- 1 C

TEST SYSTEM
- Culturing apparatus: Penicillum flask, butyl rubber stoppers

- Test volume: 80 ml

- Head space: 40 ml

SAMPLING
- Sampling frequency: Day 7, 14, 21, 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
Reference substance:
aniline
Parameter:
% degradation (inorg. C analysis)
Value:
92
Sampling time:
28 d
Results with reference substance:
The reference substance degraded by >60% after 14 days.

Table 1: Degradation kinetics

Type of suspension

Vessel no.

% degradation at sampling time (days)

7

14

21

28

Reference substance

39.04

74.86 

73.58

84.06

 

2

48.96

72.10

67.41

87.84

3

53.32

68.51

66.32

92.30

 

54.44

74.62

67.94

90.43

 

 

 

94.27

 

 

 

 

86.13

Mean 

48.94

72.52

66.82

89.17

 

 s

7.01

2.95

3.25

3.86

95% C.L.

11.15

4.69

5.17

4.05

Test sample

73.06

89.04 

78.65 

93.25

 

2

68.06

85.37

77.38 

92.81 

 

3

68.79 

88.86 

74.26 

92.52 

 

70.76

86.64 

76.04 

93.21 

 

 

 

 

89.04 

 

 

 

 

91.35 

Mean 

70.17

87.48 

76.58 

92.03 

 

 s

2.23 

1.78 

1.88 

1.62 

 

95% C.L.

3.55 

2.83 

2.99 

1.70 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
A GLP ready biodegradability study conducted according to the ISO ring test "CO2 headspace biodegradation test". The substance was found to be readily biodegradable meeting the ten day window.
The following validity criteria were fulfilled
(1) the reference substance degraded by >60% after 14 days and

(2) the total inorganic carbon (TIC) present in the blank controls at the end of the test was less than 15% of the organic carbon added initially as the test substance.

TIC at Day 0 was not reported.


Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Non-guideline study.
Principles of method if other than guideline:
Method: other
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Initial conc.:
50 other: μl/l
Based on:
test mat.
Parameter followed for biodegradation estimation:
test mat. analysis
Parameter:
% degradation (test mat. analysis)
Value:
50
Sampling time:
1.9 h
Remarks on result:
other: The biodegradation rate constant for octan-1-ol was 0.36 h-1. This equates to a half-life of 1.9 hours.

Description of key information

Readily biodegradable: 92% in 28 d (ISO method); 77.9% (CO2) in 28 days (OECD 301B; not GLP) based on weight of evidence.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A reliable study (Federle, 2009), conducted according to an appropriate test protocol (OECD 301B), but not conducted according to GLP, determined the substance to be readily biodegradable (77.9% CO2 evolution in 28 days), meeting the ten day window. Trichloromethane was used as a solubilising agent in this study. The solvent was then evaporated under a gentle stream of N2 gas to deposit the test material as a film on the walls of the vessel.

This study (Federle, 2009), using a methodology with appropriate loading method for the low solubility of the substances, was carried out with a range of linear saturated alcohols from four carbon chain length (C4) to twenty-two carbon chain length (C22).

These results are significant and fit for purpose even though the study was not conducted to GLP. The study gave results of 76.1% (C4), 77.7% (C6), 77.9% (C8), 74.6% (C10), 69.0% (C12), 82.2% (C14), 82.4% (C16), 95.6% (C18), 88.4% (C20) and 87.9% (C22) in 28 days. All were readily biodegradable, meeting the ten-day window.

This evidence is presented as weight of evidence together with degradation of 92% in 28 days in a reliable, GLP-compliant test using ISO standard methodology similar to OECD 310 (P&G, 1996).

59% degradation was reported in a study using the modified Sturm test methodology (Huntingdon Life Sciences, 1996).

It is quite normal to observe some inter-laboratory variation in screening studies, particularly for substances where solubility limits may be a factor in degradation rates under the conditions of the testing. Due to the very diluted nature of the inoculum and its limited size, it may sometime happen that no degradation-competent microorganisms are present in a particular inoculum. This is evidenced by the variable mineralisation levels seen for standard reference substances under the conditions of OECD 301 (e.g. glucose, 55-90%; benzoates 61-95%) in studies collated by AISE/CESIO [AISE/CESIO company data, and the 'Study on the possible problems for the aquatic environment related to surfactants in detergents' (WRc Ref EC4294, May 1997)].

In the case where multiple reliable studies exist showing a range of extent of biodegradation in the course of standard tests, the normal approach is to base the interpretation on the higher degradation results, this is in line with ECHA guidance on information requirements and chemical safety assessment. An important piece of additional evidence to consider is the availability of ready biodegradation data from a series of tests conducted at the same laboratory at the same time, to examine degradability throughout the series of linear alcohols from C4-C22. Whilst at the time of the study by Federle (2009), the laboratory was not GLP-certified, the data are reliable and consistent throughout the homologous series. In this study (Federle, 2009) octan-1-ol (and all other chain lengths studied) was found to be readily biodegradable.

The conclusion of ready biodegradability is consistent with evidence of rapid metabolism of long-chain fatty alcohols in fish, mammals and microorganisms(see IUCLID Sections 5.3.1, 7.1 and 6.1.4).

For these reasons, the lower degradation levels shown in the Vista, 1994 and Huntingdon Life Sciences, 1996a studies are not taken as Key.

In addition to the reliable standard protocol ready biodegradation studies discussed above, two 5-day biochemical oxygen demand study are available (Vaishnav 1987; Huntingdon Life Sciences, 1996b). REACH Guidance (Chapter R7b) states the following regarding this type of test:

"Information on the 5-day biochemical oxygen demand (BOD5) can be used for classification purposes only when no other measured degradability data are available. Thus, priority is given to data from ready biodegradability tests and from simulation studies regarding degradability in the aquatic environment. The BOD5 test is a traditional biodegradation test that is now replaced by the ready biodegradability tests. Therefore, this test should not be performed today for assessment of the ready biodegradability of substances. Older test data may, however, be used when no other degradability data are available".

This BOD5 studies are therefore selected as supporting studies. Both tests indicate readily biodegradable based on a BOD5/ThOD ratio of 0.62 and 0.54.

There are also two studies for which summary reports only are available. There is insufficient information reported to assess the validity of these tests. A summary report of a ready biodegradability study reports the substance to be readily biodegradable (65-77% in 30 days) (Henkel, 1999, RDA-Blok-test method). An inherent biodegradability test conducted according to US EPA OPPTS guideline found the substance to be inherently biodegradable based on 60% biodegradation (Vista, 1994).

A non-standard study published by Yonezawa and Urushigawa (1979) determined the reaction rate of octan-1-ol in the supernatant of a study culture. The half-life was found to be 1.9 hours.

>75% biodegradability under anaerobic conditions has also been demonstrated for octan-1 -ol (Shelton and Tiedje, 1984).

Discussion of trends in the Category of C6-24 linear and essentially-linear aliphatic alcohols:

Many biodegradation assays have been carried out on this family of alcohols. Studies generated on single carbon chain length alcohols for tests that conform most closely to ready test biodegradability methods (OECD 301 series) show that alcohols with chain lengths up to C22 are readily biodegradable. In all cases the inoculum was not acclimated. Older reliable data suggest that chain lengths above C18 are not readily biodegradable, however those studies used loading techniques which, while in general still reliable, did not make allowance for the reduced bioavailability caused by the low water solubility of these longest chain substances. Where the substances are introduced into the test vessels by coating onto the flask, very rapid biodegradation was confirmed at all chain lengths tested.

In the older supporting tests, alcohols with chain lengths up to C18 are readily biodegradable. At carbon chain lengths ≤ 14, most tests showed that pass levels for ready biodegradation were reached within the 10 day window. Chain lengths of C16-18 achieved ready test pass levels, but not within the 10 day window. The one test on a single carbon chain length greater than C18 (using docosanol) showed degradation of 37%.

Tests which allowed adaptation are considered to have significant methodological deficiencies in terms of REACH requirements for the present purpose, and are accordingly considered to be Klimisch reliability 3: Invalid. However these also consistently demonstrate extensive biodegradability. Aliphatic alcohols occur naturally in the environment and environmental organisms will be acclimated.

Reliable studies for decanol and tetradecanol that show low levels of degradation are considered to be unexplained outliers. It is usual in the interpretation of such data to take the highest levels of degradation as the key study.

Federle (2009) conducted ready biodegradation screening tests on even-numbered saturated single chain length alcohols (C6-C22) using an appropriate test method (OECD 301B). Although, the test was not conducted in compliance with GLP, the study was found to be consistent with other available data, reliable and acceptable for environmental assessment. All tests substances were found to behave in a similar way. The substances were found to be readily biodegradable meeting the ten day window after a brief lag period. A separate test using the same methodology has confirmed the ready biodegradability result, meeting the ten-day window, at the upper end of the carbon number range (docosan-1-ol) in a GLP-compliant study (Flach, 2012).

Some variability is seen in the ultimate percentage degradation over the course of the study (see Table below). It is quite normal to observe some inter-laboratory variation in screening studies, particularly for substances where solubility limits may be a factor in degradation rates under the conditions of the testing. As discussed above, due to the very diluted nature of the inoculum and its limited size, it may sometime happen that no degradation-competent microorganisms are present in a particular inoculum. This is evidenced by the variable mineralisation levels seen for standard reference substances under the conditions of OECD 301. In the case where multiple reliable studies exist showing a range of extent of biodegradation in the course of standard tests, the normal approach is to base the interpretation on the higher degradation results, this is in line with ECHA guidance on information requirements and chemical safety assessment, and consistent with the availability of ready biodegradation data from a series of tests conducted at the same laboratory at the same time, to examine degradability throughout the series of linear alcohols from C6-C22. Whilst at the time of the study (P&G, 2009), the laboratory was not GLP-certified, the data are reliable and consistent throughout the homologous series. In this study (Federle, 2009) and all other chain lengths studied were found to be readily biodegradable.

Biodegradation under anaerobic conditions

The anaerobic biodegradability of a range of chain lengths within the category has been investigated (C8, C16 alcohols (two studies), and C16-18 and C18 unsaturated alcohols). All test substances were anaerobically degradable. Results from available studies are presented in the table below.

Biodegradation by algae

Rapid degradation in water is indicated by the difficulties encountered in aquatic toxicity tests (chronic Daphnia reproduction) for long chain aliphatic alcohols (Section 6.1.4). Alcohols in the range C10-C15 were found to be rapidly removed from the test medium. This was attributed to metabolism by algae present as a food source in tests, and in later stages of the 21-day tests to bacterial degradation by microbes adsorbed onto the carapace of the test daphnids, despite daily cleaning of the animals.

Natural occurrence

It is important for context to note the findings from studies in the EU and US which consistently show that anthropogenic alcohols in the environment are minimal compared to the level of natural occurrence. Using stable isotope signatures of fatty alcohols in a wide variety of household products and in environmental matrices sampled from river catchments in the United States and United Kingdom, Mudge et al.(2012) estimated that 1% or less of fatty alcohols in rivers are from waste water treatment plant (WWTP) effluents, 15% is fromin situproduction (by algae and bacteria), and 84% is of terrestrial origin. Further, the fatty alcohols discharged from the WWTP are not the original fatty alcohols found in the influent. While the compounds might have the same chain lengths, they have different stable isotopic signatures (Mudgeet al., 2012).

In conclusion, the environmental impact of these studies is that it has confirmed that the fatty alcohols entering a sewage treatment plant (as influent) are partly derived from detergents, but these are not the same alcohols as those in the effluent which arise fromin-situbacterial synthesis. In turn, the fatty alcohols found in the sediments near the outfall of the WWTP are derived from natural synthesis and are not the same alcohols as those in the effluent.

Ready biodegradation data on single constituent alcohols

CAS

Chemical Name

Comment

Method

Result

% degradation

Result

10 day window

Reliability

Reference

111-27-3

1-Hexanol

 

301B

77.7% in 28 days at 17 mg/L

69.8%

2

Federle 2009

111-27-3

1-Hexanol

 

OECD 301-D

77% in 30 days at 2 mg/L

61% in 30 days at 5 mg/L

>60% in 14 days 

2

Richterich, 2002a

111-27-3

1-Hexanol

 

Non-standard

- half life of 8.7 hours

-

2

Yonezawa and Urushigawa 1979

111-87-5

1-Octanol

 

301B

77.9% in 28 days at 18.8 mg/L

79.2%

2

Federle 2009

111-87-5

1-Octanol

 

ISO ring test (CO2 headspace biodegr. test)

92% in 28 days at 20 mg/L

>60%

2

Procter & Gamble, 1996

111-87-5

1-Octanol

 

OECD 301-B

59 % in 29 days at 10 mgC/L

-

2

Huntingdon Life Sciences Ltd. 1996a

111-87-5

1-Octanol

 

Non-standard

- half life of 1.9 hours

-

2

Yonezawa and Urushigawa 1979

112-30-1

1-Decanol

 

 

74.6% in 28 days at 15.1 mg/L

68.6%

2

Federle 2009

112-30-1

1-Decanol

 

301-D

88% in 30 days at 2 mg/L

>60%

2

Richterich, 2002c

112-30-1

1-Decanol

 

301-B

29 % after 29 day(s) at 10 mg/L COD

-

2

Huntingdon Life Sciences Ltd. 1996b

112-53-8

1-Dodecanol

 

301B

69% in 28 days at 15.4 mg/L

63%

2

Federle 2009

112-53-8

1-Dodecanol

 Supporting

301-D

79% in 28 days at 2 mg/L

>60% in 14 days

1

Werner, 1993

112-72-1

1-Tetradecanol

 

301B

82.2% in 28 days at 15.9 mg/L

77.2%

2

Federle 2009

112-72-1

1-Tetradecanol

 

BODIS ~ISO 10708

92% in 28 days at 100 mg/L COD

>60%

1

Henkel, 1992d

112-72-1

1-Tetradecanol

 

301-B

28 % after 28 day(s) at 25.4 mg/L

-

1

Mead 1997b

36653-82-4

1-Hexadecanol

 

301B

82.4% in 28 days at 15.3 mg/L

75.2%

2

Federle 2009

36653-82-4

1-Hexadecanol

 

301B

62% after 28 days at 17.1 mg/L

<60%

1

Mead, 1997c

36653-82-4

1-Hexadecanol

 

BODIS

76 % after 28 day(s) at 100 mg/L COD

<60% after 14 d

2

Henkel KGaA 1992a

112-92-5

1-Octadecanol

 

301B

95.6% in 28 days at 14.5 mg/L

90.2%

2

Federle 2009

112-92-5

1-Octadecanol

 Supporting

301D

38% in 29 days at 5 mg/L

69% in 29 days at 2 mg/L

<60%

1

Henkel, 1992f

629-96-9

1-Eicosanol

 

301B

88.4% in 28 days at 15.6 mg/L

83.4%

2

Federle 2009

661-19-8

1-Docosanol

 

301B

87.5% in 28 days at 20 mg/L

75.6%

1

Flach, 2012

661-19-8

1-Docosanol

 

301B

87.9% in 28 days at 15.3 mg/L

83%

2

Federle 2009

661-19-8

1-Docosanol

 

301B

37% after 28 days at 12.4 mg/L

<60%

1

Mead, 2000

 

Anaerobic degradation of alcohols

CAS

Chemical name

Comment

Method

Source of sludge

Concentration of test substance

Duration

% degradation at end of test

Reliability

Reference

111-87-5

1-Octanol

 

Serum bottle, gas production + GC analysis

1oor 2odigesters

50µg/ml

8 weeks

>75%

2

Sheltonand Tiedje, 1984

36653-82-4

1-Hexadecanol

 

Batch test using14C labelled test material

Municipal digester sludge fortified with activated sludge

1 mg/L

28 days

90%

2

Nuck and Federle, 1996

36653-82-4

1-Hexadecanol

 

Batch test using14C labelled test material

Municipal sewage digester

10 mg/L

28 days

97%

2

Steber and Wierich, 1987

68002-94-8

Alcohols, C16-18 and C18 unsaturated

Supporting

ECETOC screening test

Municipal sewage digester

50 mg/L

8 weeks

89%

2

Steberet al. 1995



A study by Rorije et al. (1998) on structural requirements for anaerobic biodegradation of organic chemicals is relevant. The study used a computer-automated structure evaluation program (MCASE) to analyse rates of aquatic anaerobic biodegradation of a set of diverse organic compounds, and developed a predictive model. Primary alcohols were one of the most important fragments linked to biodegradability (biophore). The authors discuss how the presence of a biophore indicates a possible site of attack for microbes to follow a metabolic pathway for anaerobic biodegradation.

Biodegradation in STP-simulation tests

Other recent data on ethoxylated alcohols also suggest that the rate of degradation could be higher than usually assigned to readily-biodegradable substances. In an OECD 303A study of the fate of alcohol ethoxylate homologues in a laboratory continuous activated sludge unit (Wind,et al., 2006) useful data about the properties and environmental exposures of alcohols are presented, although the paper describes mainly the properties of alcohol ethoxylates (AE). The waste water organisms were exposed principally to ethoxylates, but the alcohols would be generated by the degradation of the ethoxylates. The test substance comprised a 2:1 mixture of two commercial alcohol ethoxylate surfactants with chain lengths of C12-C15 (odd and even numbered) and C16-C18 (even numbered), respectively. The test substance was dosed at a concentration of 4 mg/L in the influent.

 

Results are shown in the table below:

Removal of alcohols during an activated sludge test on alcohol ethoxylates

Alcohol

Conc in effluent ng/L

Conc in sludge µg/g

%removal

C12

18

0.6

98.6

C13

21

0.7

99.5

C14

5.5

0

99.6

C15

2.9

1.1

99.8

C16

1.6

0.01

99.5

C18

58

0.7

99.1

Total

130

2

99.4

 

This shows that most of the alcohol which does not degrade (itself a small amount) was found in the solids in recovery at the end of the study.This study is important in that it indicates that the extent of removal of alcohols is high, from an exposure route that can realistically be anticipated based on the known life cycle.

References:

EU Commission, DGIII, Study on the possible problems for the aquatic environment related to surfactants in detergents, WRc, EC 4294, February, 1997

Flach, F., 2012. Biodegradability in the CO2-evolution test according to OECD 301b (July 1992). Hydrotox laboratory, report number 737, company study number 8571, Sasol, 2 May 2012.

Federle (2009). Ready Biodegradability Test, The Procter and Gamble Co., Study number 65522, 27thApril 2009

Mudge, S.M, Deleo, P.C., Dyer, S.D. (2012). Quantifying the anthropogenic fraction of fatty alcohols in a terrestrial environment. Environmental Toxicology and Chemistry, Vol. 31, No. 6, pp. 1209–1222.

Nuck, B.A. and Federle, T.W. 1996. Batch test for assessing the mineralization of 14C-radiolabeled compounds under realistic anaerobic conditions. Environ. Sci.. 30:12, 3597-3603.

Rorije E, Peunenburg WJGM, Klopman G (1998) Structural requirements for anaerobic biodegradation of organic chemicals: A fragment model analysis. Environmental Toxicology and Chemistry, Vol. 17, No. 10, pp. 1943 -1950.

Shelton, D.R. and Tiedje, J.M. 1984. General method for determining anaerobic biodegradation potential. Applied and Environmental Microbiology 850-857.

Steber, J., Herold, C.P. and limia, J.M. 1995. Comparative evaluation of anaerobic biodegradability of hydrocarbons and fatty derivatives currently used as drilling fluids. Chemosphere 31:4, 3105-3118.

Steber, J. and Wierich, P. 1987. The anaerobic degradation of detergent range fatty alcohol ethoxylates. Studies with 14C-labelled model surfactants. Water Research. 21:6, 661-667.

Wind, T., R.J. Stephenson, C.V. Eadsforth, A. Sherren, R. Toy. (2006) Determination of the fate of alcohol ethoxylate homologues in a laboratory continuous activated sludge unit. Ecotox and Environ Safety, 64: 42-60.

Richterich, K. 2002a. 1-Hexanol: Ultimate biodegradability in the closed bottle test.Final report R 0200259.

Procter & Gamble. 1996. Final report: ISO ring test CO2 headspace biodegradation test. Study ECM ETS 554/02.

Huntingdon Life Sciences Ltd.(HLS).1996a. Report No. 96/KAS217/0325.

Richterich, K. 2002c.Final report R 0200257.

Huntingdon Life Sciences Ltd.(HLS).1996b. Report No. 96/KAS223/0327.

Richterich. 1993. 1-Dodecanol: Aerobic biodegradation: Closed bottle test. Biological Research and Product Safety/Ecology: Unpublished results; test substance registration no. SAT 910724, Henkel KGaA; Report No. RE 920247 (With English summary report no. R9901416)

Henkel KGaA.1992d.  Report No. 920026 (test substance registration no. SAT 910723, test run no. 118).5 Marz 1992.

Mead, C. 1997b. Safepharm Laboratories, SPL Project Number 140/598.

Mead, C. 1997c. Safepharm Laboratories, SPL Project Number 140/543.

Henkel KGaA. 1992a.Biological Research and Product Safety/Ecology: Report No. RE 920102; test substance registration No. SAT 910721, test run No. 120.26 Juni 1992.

Henkel KGaA.1992f. Report No.RE920246, 18 December 1992.

Mead, C. 2000. Safepharm Laboratories, SPL Project Number 140/1002.