Octadecan-1-ol

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: sewage treatment simulation testing

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:

Good quality study but not conducted to GLP. Published paper based on a more complete unpublished study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 303 A (Simulation Test - Aerobic Sewage Treatment. A: Activated Sludge Units)

GLP compliance:

not specified

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Initial conc.:

4 mg/L

Based on:

test mat.

Details on study design:

Laboratory continuous activated sludge study.

Concentration: 4 mg/L of TS
Temperature: 20ºC
Hydraulic residence time (HRT) 6 h
Sludge retention time (SRT) 10 d

The feed to the sludge unit was of sterile synthetic sewage and AE concentrate and non-sterile tap water.

19 d acclimation was used, followed by 10 days of evaluation.

At the start the unit was seeded with sewage treatment plant (STP) activated sludge.

The unit was sampled several times per week, and the samples were analysed immediately.

Test performance:

Analytical recovery of the alcohols was high.

The results showed that the CAS unit was running in a similar way to a full scale STP.

% Degr.:

99.1

Parameter:

test mat. analysis

Sampling time:

30 d

Details on results:

Results are corrected for control values.

Alcohol Conc. in Conc. in % removal
effluent ng/L sludge µg/g
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

Total elimination of alcohols, correcting for control: 97.4% of input
Total alcohols in waste sludge solids 2.0% of input
Total alcohols in suspended solids 0% of input
Total alcohols dissolved in effluent 0.7% of input

This shows that most of that which does not degrade (itself a small amount) is in the solids.

Conclusions:

A very high degree of removal of C12-18 alcohols from a test substance constituting alcohols as part of a mixed alcohol ethoxylate test substance was demonstrated in a 30-day test using a continuous activated sludge simulation methodology. The findings are reliable as part of a weight of evidence.

Reference 2

Endpoint:

biodegradation in water: sediment simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-01-21 to 2010-04-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 314. Deviations, reliability, and validity evaluated against current OECD 314 (Oct. 3, 2008)

Deviations:

no

Principles of method if other than guideline:

1 ml samples of sediment with 100 µl of overlying water individually dosed with the test chemical and statically incubated in a sealed dessicator continuously purged with CO2 free air.

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.

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

natural sediment

Details on source and properties of sediment:

OHIO RIVER SEDIMENT
- Details on collection: The sediment was collected from the Ohio River near the Lawrenceburg Indiana area at approx. river mile 490 on Dec. 14, 2009. The sediment was collected from a boat using a corer in water depth of approximately 10 ft. Approx. the first 12 inches of sediment were used and the lower anaerobic fraction was discarded.

- Textural classification: The sediment consisted of 48% sand, 40% silt and 12% clay and was classified as Loam

- pH at time of collection: 7.2

- Organic content: 2.5%

- Total nitrogen: 0.1%

- Sediment samples sieved: Not reported

GREAT MIAMI RIVER (GMR) SEDIMENT
- Details on collection: The sediment was collected from the GMR bank at the confluence of the Fairfield Wastewater Treatment Plant effluent discharge point on Nov. 24, 2009. The sediment was collected using a dipper and sampled approximately to a depth of 10 inches.

- Storage conditions: The sediment filled a 2.5 gallon plastic bucket and was stored refrigerated prior to the test.

- Textural classification: The sediment consisted of 94% sand, 2% silt and 4% clay and was classified as Sand.

- pH at time of collection: 8.4

- Organic content : 0.4%

- Sediment samples sieved: Not reported

- Total nitrogen: 0.02%

The above collected sediment samples were characterized by the University of Wisconsin, Madison Soil & Plant Analysis Laboratory
Preparation of above collected sediments: During initial set-up, the collected sediment was spread in an even layer in a shallow pan and allowed to settle. The overlying water was removed, and small 1 mL core samples were taken using a 1 mL syringe barrel whose tapered end had been removed. These sediment cores were transferred to culture tubes.

Details on inoculum:

The "inoculum" was what was naturally present in the sediment samples collected from the rivers.

Duration of test (contact time):

60 d

Initial conc.:

336 other: µg/kg dry weight sediment (Ohio river sediment)

Based on:

act. ingr.

Initial conc.:

172 other: µg/kg dry weight sediment (Great Miami River sediment)

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

CO2 evolution

test mat. analysis

other: Biomass... (see attached file)

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: 1 mL
- Composition of medium: Each screw cap culture tubes contained:
1 mL sediment with 0.1 mL of overlying water
100 µL of the dosing solution
- Test temperature: Both the abiotic and biotic test systems were incubated at 22°C in a controlled temperature room.
- Aeration of dilution water: Yes, please see ‘Method used to create aerobic conditions’ below.

TEST SYSTEM
- Culturing apparatus: 13 x 100 mm screw cap culture tubes
- Number of replicates: Three replicates per sampling intervals were prepared for biotic treatment and two replicates were prepared for abiotic treatment.
- Method used to create aerobic conditions: The dessicator was continuously purged with CO2 free air to maintain aerobic conditions. The abiotic samples were left open to the atmosphere.
- Measuring equipment: CO2 measuring apparatus is not reported in the study and mineralization to 14CO2 was determined indirectly by measuring the difference in total radioactivity between samples from the biotic and abiotic treatments.
- Test performed in open system: The abiotic samples were left open to the atmosphere while the biotic treatments were placed into a sealed dessicator.

SAMPLING
- Sampling frequency: Samples were collected immediately after dosing, as well as after 1, 2, 5, 9, 20, 30, 40, 50 and 60 d.
- Sampling method: At each sampling time, sample was removed from each tube and immediately flash frozen in a dry ice-acetone bath. These frozen samples were then lyophilized. Analysis of sample is discussed in ‘Details on analytical methods’ section.

- Sample storage before analysis: The samples for ‘chemical analysis’ were capped and stored at -80°C until analysis.
CONTROL AND BLANK SYSTEM
- Inoculum blank: No
- Abiotic sterile control: Yes (Sediment was heat sterilized for 1 h at 121°C and amended with 100 µL of 1% mercuric chloride to serve as an abiotic control)
- Toxicity control: No

STATISTICAL METHODS: For kinetic analysis the parent loss and mineralization data were fit to a variety of first order decay and production equations using nonlinear regression. For parent loss, the initial level of parent used in this analysis was the average percent of parent recovered from the abiotic treatments. For mineralization, the initial level of mineralization was assumed to be zero. Regression analysis was performed using Jandel Table Curve2D (version 4.01) software. The criteria used to judge the quality of the fit for the various equations were: 1) F-value 2) visual observation of the data fit on the graph 3) Examination of the error residuals for the regression model and 4) R2.
The best fit for parent loss and mineralization was 2 Compartment Decay Model and 2 Compartment Production Model respectively. The equations are shown below:
i) 2 Compartment Decay Model:
Y = (Ae(-k1t)) + (Be(-k2t))
where,
Y = % of initial 14C present as parent
t = Time
e = Base of the natural log
A = %Parent degraded at first order rate k1
B = %Parent degraded at first order rate k2
ii) 2 Compartment Mineralization Model:
Y = A(1-e(-k1t)) + B(1-e(-k2t))
where,
Y= %of the initial radioactivity recovered as 14CO2
A = %of the initial dose mineralized at first order rate k1
B = %of the initial dose mineralized at first order rate k2

Reference substance:

not required

Test performance:

No data

Compartment:

other: sediment, material (mass) balance

% Recovery:

85.6

St. dev.:

2.2

% Degr.:

61.1

Parameter:

CO2 evolution

Remarks:

(mineralization)

Sampling time:

60 d

Remarks on result:

other: Ohio River sediment (Normalized to 100% mass balance)

% Degr.:

11.6

Parameter:

other: % of radioactivity as parent

Sampling time:

60 d

Remarks on result:

other: Ohio River sediment (Normalized to 100% mass balance)

% Degr.:

5.5

Parameter:

other: % of radioactivity as metabolite

Sampling time:

60 d

Remarks on result:

other: Ohio River sediment (Normalized to 100% mass balance)

% Degr.:

21.8

Parameter:

other: % of radioactivity associated with solids

Sampling time:

60 d

Remarks on result:

other: Ohio River sediment (Normalized to 100% mass balance)

% Degr.:

71.6

St. dev.:

7.4

Parameter:

CO2 evolution

Remarks:

(mineralization)

Sampling time:

60 d

Remarks on result:

other: Great Miami River sediment

% Degr.:

10.4

St. dev.:

4.7

Parameter:

other: % of radioactivity as parent

Sampling time:

60 d

Remarks on result:

other: Great Miami River sediment

% Degr.:

5.2

Parameter:

other: % of radioactivity as metabolite

Sampling time:

60 d

Remarks on result:

other: Great Miami River sediment

% Degr.:

10

St. dev.:

1.5

Parameter:

other: % of radioactivity associated with solids

Sampling time:

60 d

Remarks on result:

other: Great Miami River sediment

Compartment:

sediment

DT50:

1.1 d

St. dev.:

0.1

Type:

other: Two Compartment First Order Model

Remarks on result:

other: Half life for primary degradation of readily bioavailable test material (sediment source: Ohio River) (Compartment 1)

Compartment:

sediment

DT50:

34.7 d

St. dev.:

5.2

Type:

other: Two Compartment First Order Model

Remarks on result:

other: Half life for primary degradation of less bioavailable test material (sediment source: Ohio River) (Compartment 2)

Compartment:

sediment

DT50:

1.7 d

St. dev.:

0.6

Type:

other: Two compartment first order model

Remarks on result:

other: Half life for mineralization of readily bioavailable test material (sediment source: Ohio River) (Compartment 1)

Compartment:

sediment

DT50:

13.9 d

St. dev.:

8.3

Type:

other: Two compartment first order model

Remarks on result:

other: Half life for mineralization of less bioavailable test material (sediment source: Ohio River) (Compartment 2)

Compartment:

sediment

DT50:

0.04 d

St. dev.:

0.01

Type:

other: Two compartment first order model

Remarks on result:

other: Half life for primary degradation of readily bioavailable test material (sediment source: Great Miami River) (Compartment 1)

Compartment:

sediment

DT50:

17.3 d

St. dev.:

3

Type:

other: Two compartment first order

Remarks on result:

other: Half life for primary degradation of less bioavailable test material (sediment source: Great Miami River) (Compartment 2)

Compartment:

sediment

DT50:

0.2 d

St. dev.:

0.09

Type:

other: Two compartment first order model

Remarks on result:

other: Half life for mineralization of readily bioavailable test material (sediment source: Great Miami River) (Compartment 1)

Compartment:

sediment

DT50:

13.9 d

St. dev.:

2.8

Type:

other: Two compartment first order model

Remarks on result:

other: Half life for mineralization of less bioavailable test material (sediment source: Great Miami River) (Compartment 2)

Other kinetic parameters:

other: Two compartment first order decay model (provided the best statistical fit): 0.6 ± 0.08 d-1 for primary degradation of readily bioavailable test mater... (see attached file)

Transformation products:

yes

No.:

#1

Details on transformation products:

Not reported

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS: Long chain fatty acids (see description of Extractable Residues below).

RESULTS OF EXPERIMENT 1 (sediment source: Ohio river)

EXTRACTABLE RESIDUES: All of the abiotic extracts contained a major peak (Rf 0.25-0.28) associated with parent and two relatively minor peaks that had Rf values of 0.02-0.04 and 0.70-0.75, the latter corresponding to long chain fatty acid. The average recovery of parent from the abiotic samples was 70.5%. The chromatograms from the biotic samples revealed the same three peaks as the abiotic samples aswell as two additional minor peaks at Rf 0.0 and 0.8.
In the biotic treatments, the level of parent decreased from approx.70% (time 0) to 27% (day 5) to < 10% (day 60). The Fatty acid metabolite reached a maximum level of 11% after Day 2 and decreased to < 5% after Day 40. The two polar intermediates were likely associated with the beta oxidation of the alkyl chain and decreased from a combined maximum level of 6% to 2 % by the end of the test. A second non polar metabolite observed at Rf 0.80-0.83 was transient and remained at <1% of the initial radioactivity after Day 30.
- % of applied amount at Day 0: 78% in active flask (parent and metabolite; sample time was 0 d)
- % of applied amount at end of study period: 14.4% in active flask (parent and metabolite; sample time was 60 d)

NON-EXTRACTABLE RESIDUES: The % radioactivity retained on solids increased over time and reached a maximum level of 19% after 40 d where it remained relatively constant for the remainder of the test. In the abiotic treatments, the percent of radioactivity retained on solids ranged from 5 to 11%.
- % of applied amount at Day 0: 10.1% in active flask (Solids; sample time was 0 d)
- % of applied amount at end of study period: 18.4% in active flask (Solids; sample time was 60 d)

MINERALISATION:
- % of applied radioactivity present as CO2 at end of study: The level of 14C-CO2 (mineralized test material) increased throughout the study, and on Day 60 accounted for 52% of the initial radioactivity dosed. For details, please refer to ‘Table 1’ in the ‘Any other information on results incl. tables’ section.

VOLATILIZATION: No volatilization of test material was observed as the TLC analysis of the abiotic control revealed that the parent test material remained intact.

RESULTS OF EXPERIMENT 2 (sediment source: Great Miami River)

EXTRACTABLE RESIDUES: The Rad-TLC analysis of the abiotic extracts reveled almost identical results to those of the Ohio River sediments, a consistently high level of parent and the same two minor metabolites. The average recovery of parent from the abiotic samples was 88%. The initial level of parent in the biotic treatment was approx. 65%, decreasing to <25% after Day 9 and <11% after Day 60. The subsequent metabolic pattern was very similar to that observed in the Ohio River samples.
- % of applied amount at Day 0: 90.1% in active flask (parent and metabolite; sample time was 0 d)
- % of applied amount at end of study period: 15.6% in active flask (parent and metabolite; sample time was 60 d)

NON-EXTRACTABLE RESIDUES: The fraction of radioactivity associated with the extracted biotic solids increased over time reaching a maximum level of 16% after 40 d and decreased to 10% by Day 60 indicating that 14C biomass was being converted to 14CO2. In the abiotic treatments, the percent of radioactivity associated with the extracted solids ranged from 3 to 5%.
- % of applied amount at Day 0: 8.1% in active flask (Solids; sample time was 0 d)
- % of applied amount at end of study period: 10% in active flask (Solids; sample time was 0 d)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: The calculated % mineralized increased throughout the study and accounted for 72% of the initial radioactivity dosed on Day 60.
For details, please refer to ‘Table 2’ in the ‘Any other information on results incl. tables’ section.

VOLATILIZATION: No volatilization of test material was observed as the TLC analysis of the abiotic control revealed that the parent test material remained intact.

Results with reference substance:

Not applicable

Biodegradation of [1 -14C]-Octadecanol in River Sediment (Study # 66844)

Table 1: Percent of dosed radioactivity recovered as parent, metabolites, associated with extracted solids and mineralised to 14CO2 in dosed Ohio River sediments

Time (Day)	Percent of dosed radioactivity recovered as:						Mass balance
	Parent (Rf 0.25-0.28)	Polar Metabolite (Rf <0.00)	Polar Metabolite (Rf 0.04)	Long Chain Fatty Acid (Rf 0.70-75)	Solids	CO2
0	69.4 ± 2.1	0.2 ± 0.2	5.5 ± 1.0	2.8 ± 1.2	10.1 ± 1.1	NC	88
1	49.5 ± 1.9	0.4 ± 0.6	5.1 ± 0.9	7.5 ± 0.6	14.7 ± 1.4	7.2 ± 1.3	84.4
2	35.5 ± 1.8	1.0 ± 0.4	5.9 ± 1.8	11.1 ± 0.3	15.3 ± 2.3	15.5 ± 2.0	84.3
5	27.1 ± 3.5	0.3 ± 0.4	2.8 ± 1.3	9.6 ± 0.8	14.4 ± 3.4	35.4± 12.2	89.6
9	22.5 ± 1.6	0.5 ± 0.7	3.5 ± 1.4	8.5 ± 1.2	13.6 ± 5.4	34.0 ± 8.8	82.6
20	14.5 ± 1.4	0.6 ± 0.3	2.7 ± 1.7	7.3 ± 0.9	17.2 ± 2.7	40.4 ± 2.7	82.7
30	13.0 ± 1.0	0.5 ± 0.1	2.4 ± 0.6	6.0 ± 1.4	16.4 ± 5.1	47.2 ± 3.3	85.5
40	13.7 ± 1.4	0.5 ± 0.3	2.0 ± 0.8	4.2 ± 0.5	19.2 ± 0.3	50.8 ± 10.2	90.4
50	10.2 ± 0.1	0.3 ± 0.0	2.2 ± 0.2	4.5 ± 0.5	18.6 ± 0.4	48.2 ± 1.2	84
60	9.8 ± 2.1	0.0 ± 0.0	1.4 ± 0.0	3.2 ± 0.3	18.4 ± 1.6	51.5 ± 3.3	84.3
Mean							85.58±2.2
Abiotic avg (n = 8)	70.5±1.8	ND	4.9±0.9	4.1±1.2	7.1±1.9	NC	85.9±3.5

Table 2: percent of dosed radioactivity recovered as parent, metabolites, associated with extracted solids and mineralised to 14CO2 in dosed Great Miami River sediments

Time (Days)	% of Dosed Radioactivity Recovered as:							Mass balance
	Parent (Rf 0.25 -0.28)	Polar Metabolite (Rf <0.00)	Polar Metabolite (Rf <0.04)	Polar Metabolite1 (Rf 0.20)	Long Chain Fatty Acid (Rf 0.70-75)	Solids	CO2
0.02	64.8 ± 10.4	2.1± 3.0	5.4 ± 1.0	12.2	5.6 ± 0.4	8.1 ± 0.4	6.2 ± 0.6	104.4
1	41.7 ± 9.9	0.9 ± 0.8	4.8 ± 1.3	6.2	12.1 ± 0.6	13.4 ± 2.6	20.7 ± 7.5	99.8
2	43.2 ± 4.8	0.6 ± 0.4	5.0 ± 0.6	0	12.6 ± 0.3	12.4 ± 1.1	25.1 ± 2.4	98.9
5	38.7 ± 1.9	0.6 ± 0.5	3.6 ± 0.2	0	11.4 ± 2.8	14.9 ± 2.1	24.6 ± 6.6	93.8
9	23.5 ± 4.4	1.6 ± 1.1	3.0 ± 0.8	7.2	10.1 ± 0.6	14.6 ± 1.4	38.7 ± 2.3	98.7
20	18.3 ± 9.7	0.3 ± 0.0	2.2 ± 0.9	1.3	7.7 ± 3.3	14.7 ± 4.7	55.6 ± 6.0	100.1
30	9.2 ± 2.5	0.5 ± 0.2	1.6 ± 0.6	2.3	4.1± 0.1	15.2 ± 0.3	64.4 ± 3.2	97.3
40	13.0 ± 2.4	0.6 ± 0.1	1.3 ± 0.4	1.9	5.1 ± 0.8	15.9 ± 5.1	62.2 ± 3.4	100
50	12.1 ± 2.3	0.0 ± 0.0	1.1 ± 0.7	0	3.5 ± 1.2	11.8 ± 1.3	70.0 ± 3.7	98.5
60	10.4 ± 4.7	0.0 ± 0.0	1.0 ± 0.5	1	3.2 ± 1.1	10.0 ± 1.5	71.6 ± 7.4	97.2
Mean								98.87 ±1.7
Abiotic avg (n=8)	88.0 ± 4.5	ND	5.2 ± 0.8	ND	4.5 ± 1.5	3.9 ± 0.4	NC	100.9±6.5

NC = Not calculated

ND = Not detected

Table 3: Kinetic parameters describing the primary degradation and mineralisation of the test substance in biotic sediments using a two compartment first order model:

Sample	Pool A (%)	K­1 ­(hrs­^-1)	Half-life Pool A (days)	Pool B(%)	K­2 ­(hrs­^-1)	Half-life Pool B (days)
OHIO RIVER SEDIMENTS
Primary degradation	44.6 ± 2.4	0.6 ± 0.08	1.1 ± 0.1	25.2 ± 2.1	0.02 ± 0.003	34.7 ± 5.2
Mineralisation	23.8 ± 8.9	0.4 ± 0.13	1.7 ± 0.6	28.8 ± 7.0	0.05 ± 0.03	13.9 ± 8.3
GREAT MIAMI RIVER SEDIMENTS
Primary degradation	42.9 ± 3.7	16.5 ± 6.0	0.04 ± 0.01	42.7 ± 3.3	0.04 ± 0.007	17.3 ± 3.0
Mineralisation	19.4 ± 1.5	2.8 ± 1.2	0.2 ± 0.09	53.2 ± 2.7	0.05 ± 0.01	13.9 ± 2.8

Primary degradation of parent in both sediments was well described by the two compartment model (r² > 0.99). This process was biphasic with two pools of material exhibiting different degradation rates. These pools may represent readily bioavailable and less

bioavailable (e.g. sorbed) parent molecule. Octadecanol was easily recovered from the sandy GMR sediments (100% recovered in abiotic samples) but was more tightly bound to the loamy OR sediments and the reason that heat was added to the extraction process (86% recovered from abiotic treatment).

Validity criteria fulfilled:

yes

Conclusions:

The removal of Octadecanol was 86.8-88.4% (normalised) in two sediment die-away tests (60 d). The Octadecanol was mineralized (61.1-71.6%), incorporated into solids (21.8-10%), transformed into a metabolite (5.2-5.5%), or was still parent (10.4-11.6%), after 60 d. (See Executive Summary for rate constants.)

Executive summary:

The biodegradation of Octadecanol was evaluated in two sediment die-away studies conducted under aerobic conditions in accordance with the OECD 314 guideline. Radiolabeled Octadecanol [1-14C] was tested at 336 and 172 µg/kg dry wt in exp. 1 and 2 respectively. The sediment die-away tests either used sediment collected from the Ohio River (exp. 1) or the Great Miami River (exp. 2). Each biotic test treatment was measured in triplicate per sampling interval, while two replicates per sampling interval were used for abiotic treatments. The tests were continued for 60 days.

Experiment 1 (Ohio River sediment): 88.4% of Octadecanol was removed (60 d). After 60 d, 61.1% was mineralized, 21.8% was non-extractable (solids), 5.5% was metabolite, and 11.6% remained as parent. The rate constants for biodegradation of Octadecanol in Ohio River sediment were:

Primary biodegradation: 0.6 and 0.02 d-1 for readily bioavailable and less bioavailable test material respectively (2 compartment model).

Mineralization: 0.4 and 0.05 d-1 for readily bioavailable and less bioavailable test material respectively (2 compartment model).

Experiment 2 (Great Miami River sediment): 86.8% of Octadecanol was removed (60 d). After 60 d, 71.6% was mineralized, 10% was non-extractable (solids), 5.2% was metabolite, and 10.4% remained as parent. The rate constants for biodegradation of Octadecanol in Great Miami River sediment were:

Primary biodegradation: 16.5 and 0.04 d-1 for readily bioavailable and less bioavailable test material respectively (2 compartment model).

Mineralization: 2.8 and 0.05 d-1 for readily bioavailable and less bioavailable test material respectively (2 compartment model).

This biodegradation simulation test satisfied the guideline requirements for the OECD 314 simulation test guideline.

Reference 3

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH
The hypothesis is that the category members have similar structures and properties (very rapid biodegradability), which are consistent across the category (Scenario 6 in the RAAF). The consistency of this property across the category is discussed in the endpoint summary.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to the test material identity information within each endpoint study record and in the endpoint summary. The source chemicals and the target chemical are linear aliphatic alcohols which are members of the long chain linear aliphatic alcohol Category.

The long chain linear aliphatic alcohol Category has at its centre an homologous series of increasing carbon chain length alcohols. The category members are structurally very similar. They are all primary aliphatic alcohols with no other functional groups. The category members are linear or contain a single short-chain side-branch at the 2-position in the alkyl chain, which does not significantly affect the properties (‘essentially linear’). The category members have saturated alkyl chains or contain a small proportion of naturally-occurring unsaturation(s) which does not significantly affect the properties. The branched and unsaturated structures are considered to have such similar properties that their inclusion in the category is well justified.
Impurities: Linear and/or ‘essentially linear’ long chain aliphatic alcohols of other chain lengths may be present. These are not expected to contribute significantly to the properties in respect of this endpoint due to consistent properties (see point 3).
There are no impurities present at or above 1% which are not category members or which would affect the properties of the substance.

3. CATEGORY JUSTIFICATION
The category members are structurally very similar (see point 2) and are biochemically very similar. The metabolic synthesis and degradation pathways are well established. This Category is associated with a consistency and predictability in the physicochemical, environmental, and toxicological property data across its members.

The consistency of observations in this property across the range of chain lengths covered by this Category is described in the Endpoint Summary and in the Category Report attached in Section 13.

In this registration, the information requirement is based on read-across from hexadecan-1-ol, a member of the category with similar chain length and physicochemical properties, for which the comparability is strongest (in view of the similar physico-chemical properties of the source and target substances).

4. DATA MATRIX
A data matrix for the C6-24 alcohols Category is attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 314D. Deviations, reliability, and validity evaluated against current OECD 314D (Oct. 3, 2008)

Deviations:

no

Principles of method if other than guideline:

Radiolabelled test material was dosed to freshly collected river water and inoculum. Samples collected periodically were assayed for 14C activity by Liquid Scintillation Counting (LSC).

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: River water supplemented with domestic activated sludge

Details on inoculum:

- Source of inoculum/activated sludge: Activated sludge collected from the Downingtown Regional Water Pollution Control Center, Downingtown, Pennsylvania

- Storage length and conditions: The sludge was held overnight with aeration

- Storage length: Overnight

- Preparation of inoculum for exposure: The sludge was screened to remove large clumps and a TSS level was determined. Based on this reading the sludge was added to two semi-continuous activated sludge units (SCAS) at a target solids level of 2,500 mg/L. The sludge was diluted to this concentration with tap water. Approx. 300 mL of mixed liquor was collected from each of the duplicate SCAS units, composited and homogenized at medium speed in a blender for 2 min. The homogenized sample was poured into a beaker and allowed to settle for 30 min. The supernatant was decanted and added to the flasks at a concentration of 0.1% v/v.

- Concentration of sludge: The sludge was adjusted to a target total suspended solids level of 2500 mg/L

Duration of test (contact time):

31 d

Initial conc.:

100 µg/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

TEST CONDITIONS
- Volume of test solution/treatment: Approx. 1L

- Composition of medium: Each 2 L test flask contained:1 L river water; 1 mL activated sludge; 241 µL dosing solution; - Test temperature: 20.6 - 22.5 °C

- pH: Not reported

- Aeration of dilution water: The flasks were placed on a rotary platform shaker and aerated continuously with a CO2 free air source.

- Suspended solids concentration: 2500 mg/L

- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L Erlenmeyer glass flasks

- Number of culture flasks/concentration: Two per concentration

- Method used to create aerobic conditions: The flasks were placed on a rotary platform shaker (100 to 150 rpm) and aerated continuously with a CO2 free air source. Air was purged through the scrubbing train at a constant rate which is adequate to provide 1-2 bubbles/second in the alkali traps. The CO2 scrubbing apparatus consisted of: (a) One empty 1 L plastic bottle, to prevent backflow; (b) Five 1 L plastic bottles containing 700 mL 10 N NaOH and (c) One empty 1 L plastic bottle to prevent overflow of alkali into the test containers connected in series with Tygon tubing to a pressurized air source (approx. 10-15 psi).

- Test performed in closed vessels: Yes

- Details of trap for CO2 and volatile organics if used: Glass bottles approx. 4 oz size containing 100 mL of 1.5 N KOH.

- Other: The study was terminated on Day 31 by adding 1 mL concentrated HCl to the flasks. Following an incubation of 3 d, duplicate 10 mL water samples were withdrawn from each flask and counted in Triton-X cocktail. Duplicate 1 mL samples from all three alkali traps were collected and counted in 20 mL Cab-O-Sil cocktail.

SAMPLING
- Sampling frequency: On Days 1, 3, 5, 7, 10, 14, 21, 28 and 31

- Sampling method: The first 14CO2 alkali trap in the individual trains was removed and a 1mL aliquot was counted in 20 mL of Cab-O-Sil. The remaining traps were moved one slot closer to the test flask and a new trap was added to the third slot. At the same time 10 mL water samples are withdrawn by syringe from each test flask and filtered through 0.2 µm filters. The filters were washed with 5 mL IPA/water, air dried, and counted in 20 mL of 3A cocktail to quantitate radioactivity in the biomass. The filtrate was treated as described in "Details on analytical methods" above.

- Sample storage before analysis: Not specified

CONTROL AND BLANK SYSTEM
- Inoculum blank: No

- Abiotic sterile control: No

- Toxicity control: No

STATISTICAL METHODS: Percent 14CO2 production vs time was analysed by the following empirical model: 14CO2 = a (1 - e(-k(t-c)))where,a = Extent of 14CO2 production (%)k = First order rate constant (day-1)t = Time of incubation (days)c = Lag period, if any (days). The constants a and k along with 95 % confidence intervals were generated for each treatment

Compartment:

other: water, material (mass) balance

% Recovery:

100

Remarks on result:

other: reported as 105% in source.

% Degr.:

ca. 92.4 - ca. 97.5

Parameter:

CO2 evolution

Sampling time:

31 d

Remarks on result:

other: based on range of results in two replicates

Other kinetic parameters:

first order rate constant

Transformation products:

not measured

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

yes

Details on results:

TEST CONDITIONS
- Aerobicity , moisture, temperature and other experimental conditions maintained throughout the study: Yes

MAJOR TRANSFORMATION PRODUCTS: Not determined in the study.

MINOR TRANSFORMATION PRODUCTS: Not determined in the study.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 97.5 and 92.4% for Flask 1 and 2 respectively. For details see ‘Table1’ and 'Table 2' below in the ‘Any other information on results incl. tables’ section.

Results with reference substance:

Not applicable

14 CO2 production of Cetyl Alcohol in river water (Study # 35425)

Day	Indirect % T 14CO2	Direct % T 14CO2	Biomass % 14C	Solution % 14C	Mass balance
1	66	47	23.9	10.1	81
3	68.7	56.4	20.2	11.1	87.7
5	68	65.3	22.7	9.3	97.3
7	73.9	73.8	19.1	7	99.9
10	74.7	80.4	19	6.3	105.7
14	80.5	80.9	13.3	6.2	100.4
21	85.8	93	9.8	4.5	107.3
28	89.9	96	7	3.1	106.1
31	91.7	97.5	6	2.2	105.7

 

 

Table 2: Flask 2

Day	Indirect % T 14CO2	Direct % T 14CO2	Biomass % 14C	Solution % 14C	Mass balance
1	60.2	40.8	28.9	10.9	80.6
3	69.6	53.9	19.9	10.5	84.3
5	66.2	63.4	23.4	10.4	97.2
7	71.8	69.9	18.5	9.7	98.1
10	73.1	78.8	19	7.8	105.6
14	81.8	81.2	11	7.2	99.4
21	87.9	86.1	7.3	4.8	98.2
28	91.6	91.5	5.4	3	99.9
31	93.6	92.4	4.3	2.1	98.8

Validity criteria fulfilled:

yes

Conclusions:

A reliable study conducted according to generally accepted scientific principles determined the substance to achieved a percentage degradation of 95.0% (CO2 produced) over 31 days, and rate constant for mineralization in surface water was 0.34 day-1. Results are the average of 2 test flasks. A similar result would be expected for the Target alcohol substance.

Key value for chemical safety assessment

Half-life in freshwater:

2.1 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

6 d

at the temperature of:

12 °C

Additional information

In accordance with Column 2 of REACH Annex IX, the simulation test on ultimate degradation in surface water and the sediment simulation test (required in Sections 9.2.1.2 and 9.2.1.4 respectively of the REACH regulation) do not need to be conducted as the substance is readily biodegradable. Identification of degradation products (required in Section 9.2.3 of the REACH regulation) is also not necessary because extensive mineralisation has been demonstrated.

Measured degradation in sediment data are available for octadecan-1-ol and also for an analogous saturated alcohol (described below).

The degradation of C18 linear alcohol in sediments was determined in a study, conducted in accordance with OECD 314 test method and using radiolabelled (¹⁴C) test substance (Itrich, 2010). After 60 days, 61.1% mineralisation to CO2 was obtained using sediment from Ohio River, and 71.6% mineralisation to CO2 after 60 days was obtained using Great Miami River sediments. The radiochemical analytical results for sediment-associated and aqueous alcohols in this sediment degradation study indicated that there are two pools of substance, understood to represent the strength of adsorption of the alcohol to sediment particles, which degrade at different rates.

It is notable that significant technical difficulties were encountered during method development for a recent study of adsorption/desorption (OECD 106, Wildlife, 2015), with the structurally analogous substance decan-1-ol (CAS 112-30-1), using natural standard soils, in that it was not possible to detect sufficient substance and establish equilibrium in non-sterilised soil samples. Refer to the Discussion under Section 5.2.3 for a full description of the relevant findings. Half-lives in non-sterilised test soils were in the range approximately 15 minutes to 2 hours. The polar degradation product is most likely the corresponding carboxylic acid, though it was not definitively identified. The chromatograms show that decan-1-ol was effectively fully removed in all four soil types by the 24 h time point (in the case of 2 of the soil types, within 2 hours). Though sediments were not studied in this test, similar instability is to be expected and similar findings would be anticipated for octadecan-1-ol.

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

Sediment simulation testing

The degradation of C14 linear alcohol in sediments was determined in two studies conducted in accordance with OECD 314 test method and using radiolabelled (¹⁴C) test substance. After 92 days, 76.5% mineralisation to CO₂ was obtained using sediment from Ohio River near Cincinnati, Ohio area (Federle T W and Itrich N R, 2010a). After 149 days, 83.6% mineralisation to CO₂ was obtained using Lytle Creek sediments from Wilmington, Ohio (Federle T W and Itrich N R, 2010b).

The degradation of C18 linear alcohol in sediments was determined in a similar study, conducted in accordance with OECD 314 test method and using radiolabelled (¹⁴C) test substance (Itrich, 2010). After 60 days, 61.1% mineralisation to CO₂ was obtained using sediment from Ohio River, and 71.6% mineralisation to CO₂ after 60 days was obtained using Great Miami River sediments.

The radiochemical analytical results for sediment-associated and aqueous alcohols in these three sediment degradation studies indicated that there are two pools of substance, understood to represent the strength of adsorption of the alcohol to sediment particles, which degrade at different rates.

In another study, using activated sludge and radiolabelled (¹⁴C) test substance, 76.7% mineralisation of C14 to CO₂ after 48 h was determined in accordance with OECD 314B (Federle, 2005). In the same study, 66.3% mineralisation of C16 to CO₂ after 48h was determined. Another OECD 314 test using activated sludge and using radiolabelled (¹⁴C) test substance, indicates 95% mineralisation of C16 to CO₂ in 31 days (Federle, 1993).

Activated sludge simulation testing

In another study, an activated sludge simulation test using similar methods to the sediment studies described above, was conducted with radiolabelled C12 alcohol.

 

A simulation of the biodegradation of Dodecanol in activated sludge was conducted under aerobic conditions in accordance with the OECD 314B guideline (Federle, 2005). A solution of radiolabeled Dodecanol (1-¹⁴C) was tested at 9.8 µg/L.  The inoculum was activated sludge obtained from Fairfield Wastewater Treatment Plant (Fairfield, OH), which receives predominantly domestic wastewater. The disappearance of parent and progression of metabolite formation and decay were monitored over time by thin layer chromatography with radioactivity detection. Production of CO₂ was determined by comparing total radioactivity in a bioactive treatment compared to that in an abiotic control using liquid scintillation counting (LSC).

 

After 48 h, 74% was mineralized, 20.7% was non-extractable (solids), 9.4% was metabolite, and 0.8% remained as parent. The rate constants for biodegradation of Dodecanol in activated sludge were:

Primary biodegradation: 113 h^-1

Mineralization: 11 h^-1

 

This biodegradation simulation test satisfied the guideline requirements for the OECD 314 B simulation tests to assess the biodegradability of chemicals discharged in wastewater.

Another reliable study conducted according to ISO 11733:1995 and comparable to OECD 303 determined 99.5% DOC removal in 30 days for pentadecanol (Battersby N J, Sherren A J, Bumpus R N, 1999).