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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
Study period:
2009-03-17 to 2009-04-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to an appropriate OECD test guideline. It was not compliant with GLP.
Qualifier:
according to
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.:
15.9 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: 15.9 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:
82.2
St. dev.:
1.7
Sampling time:
28 d
Details on results:
The final SOC levels ranged from 0.3 to 0.4 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

0

9.07

48.86

63.11

70.05

77.82

80.12

81.93

81.94

82.12

82.03

82.15

 

 

 

 

 

 

 

 

 

 

 

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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Not key study: Other studies (same reliability score) but with higher degradation rate are available.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Initial conc.:
25.4 mg/L
Based on:
test mat.
Reference substance:
other: Sodium benzoate
Value:
28
Sampling time:
28 d
Details on results:
Kinetic of control substance:
1 days = 20%
10 days = 66%
20 days = 91%
28 days = 105%

The test substance attained <60% degradation over the test period, therefore it cannot be considered readily biodegradable.

Kinetic of test substance (in %):
= 2 after 1 day(s)
= 10 after 10 day(s)
= 23 after 20 day(s)
= 28 after 28 day(s)

The following validity criteria were met 
(1) Parallel assays did not differ by more than 20%, 
(2) the reference substance degraded by >60% during the 14 day window, 
(3) CO2 evolution in the inoculum blank did not exceed 40 mg/l at the end of the test, 
(4) IC content of the test substance suspension in mineral medium at the start of the test was less than 5% of the total carbon.

Interpretation of results:
other: not readily biodegradable
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.
Principles of method if other than guideline:
Method: other: US EPA OPPTS 835.3100 Aerobic Aquatic Biodegradation Test
GLP compliance:
not specified
Oxygen conditions:
aerobic
Inoculum or test system:
other: no information provided on inoculum
Duration of test (contact time):
31 d
Initial conc.:
20 mg/L
Based on:
test mat.
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium benzoate
Value:
57
Sampling time:
31 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 over the test period, therefore it cannot be considered readily biodegradable.

Kinetic of test substance (in %):
= 28 after 4 day(s)
= 47 after 10 day(s)
= 54 after 17 day(s)
= 56 after 24 day(s)
= 57 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:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
he study was conducted according to an appropriate international test guideline and in compliance with GLP.
Qualifier:
according to
Guideline:
other: BOD-test for Insoluble Substances (BODIS) ISO 10708
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge, predominantly domestic
Details on inoculum:
- Concentration of sludge: 30mg dry matter/l
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: Mineral medium with activated sludge. Stabilized for one week at 20 - 25 C. Aerated until 02 saturation reached.

- Test volume: 200ml

- Test temperature: 20 - 25 C


TEST SYSTEM
- Culturing apparatus: 300 ml bottles

- Number of culture flasks/concentration: 12 flasks. 100 mg/l.


SAMPLING
- Sampling frequency: 7, 14, 21 and 28 d

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
Reference substance:
other: Sodium acetate
Test performance:
The validity criteria were fulfilled: (1) degradation rate of reference has reached level of 60% within 14 days; (2) Parallel assays did not differ by more than 20%; (3) total oxygen consumption in blanks after first week was lower than 3 mg O2 and lower than 1 mg O2 in the following weeks; and (4) residual concentration of O2 in the test bottles did not fall below 0.5 mg/l.
Key result
Parameter:
% degradation (O2 consumption)
Value:
92
Sampling time:
28 d
Results with reference substance:
Degradation rate of reference reached level of 60% within 14 days

Table 1: Degradation kinetics

Type of suspension

Vessel no.

% degradation at sampling time (days)

7

14

21

28

Blank (inoculated test medium)

 Mean (n=3)

8.62

9.27

9.29

9.27 

 

 

  

 

 

 

Reference substance 100 mg/l

  73

 83

 83 

 83 

 

  76

 87

 87

 87 

             c

 75

 85

 87

 87

mean 

 75

 85 

 86 

 86

 

 

 

 

 

Test sample 100 mg/l

a

  70

  87

 89

 93

b

  63

  80

 92

 86

c

  69

 86

 94

 96

mean

   67

 84

 88

 92

 

 The test substance attained >60% degradation within the 10-day window, therefore it can be considered readily biodegradable.

Interpretation of results:
readily biodegradable
Conclusions:
The substance was determined to be readily biodegradable, meeting the ten day window, in a reliable study conducted according to an appropriate test protocol, and in compliance with GLP.

Description of key information

Readily biodegradable: 92% (COD) in 28 d (BOD test for insoluble substance: ISO 10708); 82.2% (CO2) in 28 days (OECD 301B; not GLP)

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 (82.2% 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 (P&G, 2009).

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 (Richterich, 1992).

Two further studies using standard methodology demonstrated a lower level of degradation over 28 days: 28% degradation (Mead, 1997, using OECD 301B methodology) and 57% in 31 days (Vista, 1994, using a US EPA standard method) were observed in these studies.

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) tetradecanol (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 Mead, 1997 and Vista, 1994 studies are not taken as Key.

There is also a study for which only a summary report is available. There is insufficient information reported to assess the validity of this test. An inherent biodegradability test conducted according to US EPA OPPTS guideline found the substance to be inherently biodegradable based on 57% biodegradation (Vista, 1994).

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 the 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 (Federle, 2009), the laboratory was not GLP-certified, the data are reliable and consistent throughout the homologous series. In this study (Federle, 2009), all 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 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 from in situ production (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 (Mudge et 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 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.

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.

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

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

Yonezawa, Y. and Urushigawa, Y. 1979. Chemico-biological interactions in biological purification systems. V. Relation between biodegradation rate constants of aliphatic alcohols by activated sludge and their partition coefficients in a 1-octanol-water system. Chemosphere 3:139-142.

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.

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