Docosan-1-ol

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-01-23 to 2012-05-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: municipal wastewater treatment plant Breisgauer Bucht, Germany
- Laboratory culture: no
- Storage conditions: not mentioned
- Storage length: 1 day
- Preparation of inoculum for exposure: The activated sludge was washed twice by settling the sludge, decanting the supernatant and resuspending the sludge in tap water.
- Pretreatment: In the reactor vessels 6.9 mL activated sludge was filled up to 1500 mL with 1493.1 mL mineral medium corresponding to 30 mg/L dry solids, the system was sealed and aerated with CO2-free air at a rate of 50-100 ml/min overnight.
- Concentration of sludge: Dry solid of the activated sludge was determined as 6.53 g/L by weight measurements after 3 h drying at 110 °C (mean of triplicate measurements)
- Initial cell/biomass concentration: 30 mg dry solid/L

Duration of test (contact time):

29 d

Initial conc.:

20 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Composition of medium: according to the guideline
- Test temperature: 21 - 22 °C
- Suspended solids concentration: 30 mg/L
- Continuous darkness: no, diffuse light during test

TEST SYSTEM
- Culturing apparatus: Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septum were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by a magnetic stirrer with 2 cm stir bars.
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: aerated by passage of CO2-free air at a rate of 30 - 100 mL/min (1.6 - 5.5 bubbles/second)
- Measuring equipment: total carbon analyzer TOC-5000A, Shimadzu with an autosample ASI-5000 A using a non dispersive infrared (NDIR) detector
- Details of trap for CO2: The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH.


SAMPLING
- Sampling frequency: days 4, 7, 11, 14, 21, 28 and 28 after acidification and second absorber
- Sampling method: Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 vessels (without test substance with inoculum)
-Solvent control: The solvent control was prepared in the same way as the test vessels. 3.7 ml of trichloromethane were added into the control vessel. The vessel was slewed under the fume hood until the solvent had evaporated completely.
- Toxicity control: no

Reference substance:

benzoic acid, sodium salt

Preliminary study:

A first test was carried out without the addition of a solvent control. The mean degradation extent of the test item was 111.1% and it was assumed that that the solvent an influence on the CO2-evolution and on the degradation values of the test item. The test was therefore repeated with an additional solvent control.

Test performance:

The CO2-free air production system consisted of an air compressor, two 1000 mL gas wash bottles filled with dry soda lime, followed by one bottle filled with 0.1 M NaOH (sodium hydroxide) and one gas wash bottle filled with demineralised water. The CO2-free air was passed on to an air distributor with two input and 22 output channels and through PE-tubes. Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septums were used as reactors. The liquid volume was fixed as 1.500 mL each. Mixing was performed by a magnetic stirrer with 2 cm stir bars.
A stock solution of 10.1 g/l was prepared and 3.7 ml of the stock solution were added into the three test vessels (corresponding to a TOC concentration of 20 mg/l). From the reference compound 5.15 mL of a stock solution of 10 g/L were added to the reference vessels. 3.7 ml of the solvent was added into the solvent control vessel.
The activated sludge and the mineral medium were aerated with CO2-free air in separate containers overnight. On the next day, after complete evaporation of the solvent under the fume hood 6.9 ml activated sludge was filled up to 1500 ml with 1493.1 ml mineral medium and added to each vessel.
The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series, each filled with 200 mL 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 ml PE syringes. The amount of CO2 released from the reactors is calculated through IC-measurements in the CO2-absorber while considering the amount of CO2 removed for IC-measurement.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

87.5

Sampling time:

28 d

Remarks on result:

other: with consideration of the solvent control

Results with reference substance:

The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days.

Table 1. Ultimate biodegradation of docosan-1-ol (as % of ThCO₂) with consideration of the solvent control

Test vessel no.	Day:	0	4	7	11	14	24	28	29
10	Test	0	31.3	62.7	77.7	81.6	88.2	86.6	88.4
11	Test	0	22.5	54.9	73.6	80.8	86.2	84.8	88.7
12	Test	0	16.0	46.1	68.1	74.5	87.4	82.1	85.4
4	Reference	0	65.3	75.9	85.7	81.4	86.9	86.1	84.2
5	Reference	0	64.3	76.1	86.4	84.0	86.2	88.6	86.6
6	Reference	0	69.1	78.8	85.3	86.2	87.6	84.9	86.7

 

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The degradation of docosan-1-ol was 87.5 % after 28 days. The degradation rate reached more than 60% within the 10-day window and therefore the test substance is considered to be readily biodegradable.

Executive summary:

Docosan-1-ol was tested for ready biodegradation according to OECD 301B. The degradation of the test item was 87.5 % within 28 days (with reference to the solvent control). The biodegradation of the test item reached the criterion for ready biodegradation.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting 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

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.:

15.3 mg/L

Based on:

test mat.

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: 15.4 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

Reference substance:

benzoic acid, sodium salt

Parameter:

% degradation (CO2 evolution)

Value:

87.9

St. dev.:

6.4

Sampling time:

28 d

Results with reference substance:

A ready biodegradation rate of 83% was obtained for the 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	12.54	58.02	72.02	77.49	84.39	85.92	88.42	87.30	87.51	88.23	87.87
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 ready biodegradation value of 87.9% was obtained for the test substance using an appropriate test procedure. The result was considered reliable.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to an appropriate OECD test guideline and EU test method and national standard method, and in compliance with GLP. Not key study: Other studies (same reliability score) but with higher degradation rate are available.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Duration of test (contact time):

28 d

Initial conc.:

12.4 mg/L

Based on:

test mat.

Reference substance:

other: Sodium benzoate

Value:

37

Sampling time:

28 d

Details on results:

The test material degraded <60% over the test period therefore it cannot be considered readily biodegradable.

Kinetic of test substance (in %):
= 16 after 8 day(s)
= 23 after 10 day(s)
= 29 after 14 day(s)
= 33 after 22 day(s)
= 37 after 28 day(s)

Results with reference substance:

Kinetic of control substance:
8 days = 59%
10 days = 63%
14 days = 64%
22 days = 64%
28 days = 74%

The following validity criteria were met: 
(1) The IC/TC ratio of the test material suspension in the mineral   medium at the start of the test was below 5%, 
(2) the total CO2 evolution in the control vessels on day 28 was 37.85  mg/l (= 113.55 mg/3 l), 
(3) degradation of reference substance reached pass level within 14 days, 
(4) toxicity control (KALCOL 220-80 and sodium benzoate) degraded by 42%  after 14 days, and 
(5) results of parallel assay did not differ from each other by more than  20%.

Interpretation of results:

not readily biodegradable

Description of key information

Readily biodegradable: 87.5% (CO2) in 28 days (OECD 301B; GLP), supported by consistent findings for analogous alcohols of comparable chain lengths.

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 (87.9% CO₂evolution 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 N₂gas 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.

The result of this study is supported by reliable read-across ready biodegradation data for analogous alcohols of shorter (C18 and C20) chain lengths. C18 alcohol (octadecan-1-ol, CAS 112 -92 -5) in the same study gave results of 95.6% biodegradation in 28 days (Federle, 2009).

A study using the same methodology but with GLP gave results of 87.5% biodegradation in 28 days for docosan-1 -ol (Flach, 2012). As a GLP-compliant test this value is used as Key for this substance.

A third reliable study with docosan-1-ol (Mead, 2000) conducted to appropriate test protocols (OECD 301B; EU method C.4; EPA OPPTS 835.3110) found the substance to be not readily biodegradable (37% in 28 days CO2 evolution). The result from this study is considered as an unexplained outlier.

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. Furthermore, the very low limit of solubility of the registration substance is an important consideration. The studies in which lower levels of degradation were achieved did not use adapted methodology to avoid overloading the test system. 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) docosan-1-ol (and all other chain lengths studied) was found to be readily biodegradable.

For these reasons, the Mead 2000 study is not selected as key study. In the same way, the lower degradation levels shown in the Richterich, 1992, Henkel, 1992, Mead, 1997 and Vista, 1994 studies of the C18 analogous alcohol (octadecan-1-ol, CAS 112-92-5) are not taken as Key.

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).

A study of biodegradability of a multiconstituent saturated/unsaturated alcohol (Alcohols, C16 -18 and C18-unsaturated, CAS 143-28-2) in an anaerobic test system showed 88.6% biodegradation over a period of 84 days. This provides useful further supporting information for the rapid and complete degradation of the longer chain alcohol substances, such as C22, under a range of conditions.

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. 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) 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.

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 from in-situ bacterial 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.

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 Table 3 below:

Table 3. 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

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.

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.