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Biodegradation in water and sediment: simulation tests

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Endpoint:
biodegradation in water: sewage treatment simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-03-30 to 2005-06-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD 314B. Deviations, reliability, and validity evaluated against current OECD 314B (Oct. 3, 2008)
Deviations:
no
Principles of method if other than guideline:
Radiolabelled test material was dosed to freshly collected activated sludge in an open test system. Periodically subsamples were collected, lyophilised and extracted. The disappearance of parent and progression of metabolite formation and decay were monitored over time by thin layer chromatogrphy 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).
GLP compliance:
no
Remarks:
study conducted before 2008
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, industrial, non-adapted
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
Not applicable
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge mixed liquor was obtained from Fairfield Wastewater Treatment Plant (Fairfield, OH), which receives predominantly domestic wastewater.

- Storage length: None (radiolabeled test material was dosed to freshly collected activated sludge)

- Preparation of inoculum for exposure: Not reported

- Concentration of sludge: TSS of sludge at the time of collection is not provided in the report. However, the solids level of the mixed liquor suspended solids was adjusted to 2500 mg/L before use.

- Initial cell/biomass concentration: Not reported
Duration of test (contact time):
48 h
Initial conc.:
10 µg/L
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: Approx. 1 L

- Composition of medium: Each 2 L test flask contained: 1 L activated sludge; 0.05 µM of Tetradecanol.

- Test temperature: The test flasks were incubated at 20 ±2°C and gently mixed on shaker table.

- pH: 7

- Aeration of dilution water: No

- Suspended solids concentration: 2500 mg/L of TSS

- Continuous darkness: Not reported

TEST SYSTEM
- Culturing apparatus: 2 L flasks

- Number of culture flasks/concentration: One

- Method used to create aerobic conditions: Test was conducted in open test system.

- Measuring equipment: 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: Yes

SAMPLING
- Sampling frequency: At 0.02, 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5.5, 24, 48, and 144 h

- Sampling method: At each sampling, each sludge mixture was mixed thoroughly and sampled as follows:
a) For determination of mineralization: Triplicate 1 mL samples of the mixed sludge were removed and placed into 20 mL glass scintillation vials containing 1 mL of 0.5% HCI. acidified samples were incubated overnight.
b) For analysis of parent, metabolite and radioactivity associated with solids: 25 mL of sludge was collected from each treatment and transferred to a 35 mL screwtop centrifuge tube and immediately frozen in a dry ice acetone bath. The frozen samples were stored at -80°C until lyophilization on a Virtis bench-top model 3.3L freeze dryer.Analysis of sample is discussed in ‘Details on analytical method’ section.

- Sample storage before analysis: Not specified.

CONTROL AND BLANK SYSTEM
- Inoculum blank: No

- Abiotic sterile control: Yes (Sludge was autoclaved and amended with mercuric chloride (1g/L) to serve as an abiotic control)

- Toxicity control: No

STATISTICAL METHODS: For kinetic analysis, the data describing the disappearance of parent were fitted to various equations using Jandel TableCurve 2D software, Version 4.0. Based on statistical considerations and the visual quality of the fit, the two-compartment first order decay model was used to fit all data.
Reference substance:
not required
Test performance:
No data
Compartment:
other: sediment, material (mass) balance
% Recovery:
107.3
% Degr.:
76.7
Parameter:
CO2 evolution
Remarks:
(Mineralization)
Sampling time:
48 h
% Degr.:
21
Parameter:
other: % of radioactivity associated with solids
Sampling time:
48 h
% Degr.:
1.3
Parameter:
other: % of radioactivity as parent
Sampling time:
48 h
% Degr.:
8.3
Parameter:
other: % of radioactivity as metabolite
Sampling time:
48 h
Compartment:
other: Activated domestic sludge (non adapted)
DT50:
0.48 min
Type:
other: First order
Remarks on result:
other: Half-life of primary degradation
Other kinetic parameters:
first order rate constant
Transformation products:
yes
No.:
#1
No.:
#2
Details on transformation products:
- Formation and decline of each transformation product during test: Tetradecanoic acid reached a maximum level of 18.2% and polar metabolites reached their maximum level of 7.7% after 0.08 h. Concurrent with the loss of parent, there was the instantaneous appearance of fatty acids and polar metabolites, which peaked and subsequently declined.

- Pathways for transformation: Tetradecanol degradation involved two pathways:
oxidation of the alcohol to a fatty acid, which was beta oxidized to form carbon dioxide, and omega
oxidation of the methyl group to yield dioic acids, which undergo beta oxidation from either direction

- Other: The majority of the activity in abiotic control remained as parent, 8.6% of radioactivity was in the form of transformation products, which had chromatographic mobility similar to the two major metabolites observed in the biotic treatments.
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

TRANSFORMATION PRODUCTS: Please refer to the above section 'Details on transformation products'.

TOTAL UNIDENTIFIED RADIOACTIVITY (RANGE) OF APPLIED AMOUNT: Not reported

EXTRACTABLE RESIDUES
- % of applied amount at Day 0: 66.28% (parent and metabolite; sample time was 0.02 h)
- % of applied amount at end of study period: 9.63% (parent and metabolite; sample time was 48 h)

NON-EXTRACTABLE RESIDUES
- % of applied amount at Day 0: 17.84% in active flask (sample time was 0.02 h)
- % of applied amount at end of study period: 21% in active flask (sample time was 48h)

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: 76.7% in bioactive test flask (sample time was 48 h). CO2 evolution was not analyzed for abiotic flask as majority of the radioactivity in the abiotic control was recovered primarily as intact parent. For details, please refer to ‘table 1’ and '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 throughout the experiment.

Continuous activated sludge (CAS) study of Tetradecanol (study # 45535)

Table 1: Biotic flask

Sampling time (h)

Parent and less polar metabolite* (%)

Water extracted (polar metabolite)

Associated with solids (%)

CO2 (%)

Mass balance

0.02

60.76

5.52

17.84

30.18

114.3

0.08

36.48

7.65

19.88

NA

NA

0.25

34.04

7.57

19.64

40

101.25

0.5

27.76

6.24

19.98

38.92

92.89

1

24.76

4.43

22.21

44.68

96.08

1.5

25

4.64

20.62

48.62

98.88

2

NA

NA

NA

43.73

NA

3

14.88

9.6

19.88

52.92

97.27

4

14.28

7.65

23.7

53.3

98.94

5.5

11.56

7.36

23.08

53.37

95.37

24

9.4

4

21.57

58.95

93.92

48

7.6

2.03

21

76.67

107.29

144

6

1.6

12.9

77.67

98.17

* Extracted using methanol as solvent

 

Table 2: Abiotic flask

Sampling time (h)

Parent (%)

Metabolite (%)

Water extracted

Associated with solids (%)

CO2 (%)

Mass balance

48

88.6

8.3

0.3

2.1

NA

99.3

 

Table 3: Predicted removal of parent Tetradecanol from activated sludge as a function of the concentration of effluent solids

% Removed as a Function of Effluent Solids

Kd (L/kg)

0 mg/L

5 mg/L

20 mg/L

8486

99.81

99.80

99.77

Validity criteria fulfilled:
yes
Conclusions:
A reliable study conducted according to to generally accepted scientific principles determined the substance to degrade by 76.7% , 76.7% was mineralized, 21% was non-extractable (solids), 8.3% was metabolite, and 1.3% remained as parent after 48 hours. The rate constants for primary biodegradation and mineralisation in activated sludge were 86.5 and 3.4 h-1, respectively.
Executive summary:

A simulation of the biodegradation of Tetradecanol in activated sludge was conducted under aerobic conditions in accordance with the OECD 314B guideline. A solution of radiolabeled Tetradecanol (1-14C) was tested at 10 µg/L.  The inoculum was activated sludge obtained from Fairfield Wastewater Treatment Plant (Fairfield, OH), which receives predominantly domestic wastewater.

After 48 h, 76.7% was mineralized, 21% was non-extractable (solids), 8.3% was metabolite, and 1.3% remained as parent. The rate constants for biodegradation of Tetradecanol in activated sludge were:

Primary biodegradation: 86.5 h-1

Mineralization: 3.4 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.

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005-06-07 to 2005-12-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: other guideline: OECD 314D. Deviations, reliability, and validity evaluated against current OECD 314D (Oct. 3, 2008)
Deviations:
no
Principles of method if other than guideline:
One ml samples (biotic and abiotic) of sediment with 100µl of overlying water dosed with the radiolabelled test chemical and statically incubated in a sealed dessicator continuously purged with CO2 free air and connected to a gas trapping system to collect 14CO2.
GLP compliance:
no
Remarks:
study conducted before 2008
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
- Details on collection: Sediment was collected from the Ohio River near Cincinnati, Ohio area at approximately river mile 476-477 by using a
dredge and the top 2-3 cm layer of sediments was retained.

- Storage conditions: The sediment was placed in plastic jars and kept on ice during transport and then stored in a 4°C cold room prior to the test.

- Textural classification: The sediment consisted of 55% sand, 36% silt and 9% clay.

- pH at time of collection: 7.6

- Organic content: 2.4%

- Total nitrogen: 0.1%

- Percent dry weight: 63.1%

- Sediment samples sieved: Not reported; The sediment was 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 sample collected from the river.
Duration of test (contact time):
92 d
Initial conc.:
170 other: µg 14C-1- tetradecanol per kg dry weight 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: Approx. 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: Four replicate samples per sampling interval 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.

- Measuring equipment: To measure the 14CO2, internal base trap (50 mL beaker containing 20 mL of 1.5 N KOH) was used to trap any evolved
14CO2 in the headspace of the dessicator. The effluent gas was passed through a gas trapping system consisting of one empty trap followed by threebase traps containing 100 mL of 1.5 N KOH to recover any 14CO2 not collected by the internal trap.

- Test performed in closed vessels: Yes, the biotic treatments were placed in a sealed dessicator.

- Details of trap for CO2 if used: Following two traps were used: Internal trap: One 50 mL glass beaker (placed in sealed in dessicator) containing 20 mL 1.5 N KOH was used to trap evolved 14CO2 in headspace of the dessicator; External trap: Three glass bottles containing 100 mL of 1.5 N KOH to recover any 14CO2 not collected by the internal trap

SAMPLING
- Sampling frequency: Biotic treatment was sampled at 0.01, 0.25, 0.50, 1, 2, 3, 7, 10, 15, 36, 92 and 188 d and abiotic treatment was sampled less
frequently at 0.01, 1, 2, 3, 7, 10, 15 and 188 d.

- Sampling method: At each sampling time, each sediment treatment was sampled as follows: 1) For analysis of trapped 14CO2 in headspace: Triplicate 1 mL subsamples are removed from the beaker containing KOH in dessicator and analyzed. KOH solution was replaced with fresh solution after each sampling. 2) For analysis of evolved 14CO2 in effluent gas: Triplicate 1 mL sample was removed at each sampling time from first base trap, placed in separate vials and analyzed. The two remaining traps were moved one slot closer to the flask and a new trap was added to the third slot. 3) For analysis of dissolved 14CO2 in the sediment samples: Dissolved 14CO2 was measured by placing replicate sediment samples inside a 25 mmx150 mm culture tube with a septum containing screw cap and a 2-inch piece of 10 mm thick glass rod with 5 mL of 1.5N KOH in the bottom. After sealing the tube, 1 mL of 50% HCI was injected using a syringe directly into the sediment tube through the septum to volatilize any dissolved 14CO2. The acidified samples were incubated for several days and subsequently, a 1 mL sample of the 1.5 N KOH was removed from the bottom of the culture
tube for analysis 4) For chemical analysis: Sample was removed from each tube and immediately flash frozen in a dry ice-acetone bath. These frozen samples were then lyophilized, extracted in methanol, vortexed and centrifuged until the supernatant was clear.
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 (The samples were autoclaved for 90 min and amended with 50 µL of a solution containing 2% mercuric chloride to serve as abiotic control)

- Toxicity control: No

STATISTICAL METHODS: For kinetic analysis the parent loss and mineralization data were both fit to a 2 compartment first order decay and production equations using nonlinear regression. Regression analysis was performed using Jandel Table Curve 2D (version 4.01) software. The criteria used to judge the quality of the fit for the observed data are: 1) F-value, 2) visual observation of the data fit on the graph, 3) Examination of the error residualsfor the regression model and 4) R2. 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:
80.4
St. dev.:
13.5
% Degr.:
76.5
Parameter:
CO2 evolution
Sampling time:
92 d
Remarks on result:
other: Normalized to 100% mass balance
% Degr.:
3.7
Parameter:
other: other: % remaining as parent
Sampling time:
92 d
Remarks on result:
other: Normalized to 100% mass balance
% Degr.:
17
Parameter:
other: % of radioactivity associated with solids
Sampling time:
92 d
Remarks on result:
other: Normalized to 100% mass balance
% Degr.:
2.9
Parameter:
other: % as metabolite
Sampling time:
92 d
Remarks on result:
other: Normalized to 100% mass balance
% Degr.:
96.4
Parameter:
other: Total removal (mineralized + metabolite + incorporated into solids)
Sampling time:
92 d
Remarks on result:
other: Normalized to 100% mass balance
Compartment:
sediment
DT50:
0.04 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for primary degradation of readily bioavailable test material (compartment 1)
Compartment:
sediment
DT50:
11.4 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for primary degradation of less bioavailable test material (compartment 2)
Compartment:
sediment
DT50:
0.15 d
Type:
other: Two Compartment First Order Mode
Remarks on result:
other: Half life for mineralization of readily bioavailable test material (compartment 1)
Compartment:
sediment
DT50:
34.9 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for mineralization of less bioavailable test material (compartment 2)
Other kinetic parameters:
other: Two compartment first order decay model (provided the best statistical fit) 8.9 ± 1.8 d-1 for primary degradation of readily bioavailable test materia... (see attached file)
Transformation products:
yes
No.:
#1
Details on transformation products:
Please refer to 'Transformation products' under 'Details on results' section.
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

TRANSFORMATION PRODUCTS:
Fatty acid represented approximately 6% of the radioactivity during the first 2 d and then decreased in abundance to <2% after Day 92. A total of 3
metabolites were identified by TLC:
Rf (TLC) Substance
<0.00 Polar metabolite
0.05 Polar metabolite
0.23 Parent
0.66 Long-chain fatty acid


EXTRACTABLE RESIDUES:
All of the abiotic extracts contained a major peak (Rf 0.23) associated with parent and two minor polar peaks that had Rf values of <0.0 and 0.05 that remained relatively constant with time. This finding indicated that the Tetradecanol degraded slightly before the test initiation but had remained biologically inactive through the experiment. The average recovery of parent from the abiotic samples was 58.1%. Biotic samples revealed the presence of same three peaks as observed in abiotic treatment, as well as a peak corresponding to long chain fatty acid. However, these polar metabolites in the biotic treatment decreased with time. In the biotic treatments, primary degradation of the parent started immediately with metabolites accounting for 18.7% of the radioactivity in the 15 min sample (normalized to 100% mass balance). After 1 d, only 24.7% of the dosed radioactivity was recovered as parent (normalized to 100% mass balance).

- % of applied amount at Day 0: 81.2%% in active flask (parent or metabolite; sample time was 0.01 d; normalized to 100% mass balance).

- % of applied amount at end of study period: 6.6% in active flask (parent or metabolite; sample time was 92 d; normalized to 100% mass balance).

NON-EXTRACTABLE RESIDUES:
The fraction associated with the extracted solids initially increased over time reaching a maximum level of 25.9% at day 2 (32.9%, normalized for 100% mass balance), and decreased to 14.2% by Day 92 (17.0%, normalized for 100% mass balance) in biotic treatment. In the abiotic treatments, the
percent of radioactivity associated with the extracted solids ranged from 2.4 to 7.2%.

- % of applied amount at Day 0: 4.0% in active flask (Solids; sample time was 0.01 d; normalized to 100% mass balance).

- % of applied amount at end of study period: 17.0% in active flask (Solids; sample time was 92 d; normalized to 100% mass balance).

MINERALISATION:
Production of 14CO2 increased throughout the study and accounted for 76.5% of the initial radioactivity dosed on Day 92 (normalized for 100% mass balance). 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.

NOTE: the test was conducted for 188 d, and the results at 188 d are presented in the Tables below. However, the results reported in this robust summary are at 92 d, because the % of radioactivity as parent, as metabolites, and as long chain fatty acid was quantified at Day 92. At Day 188, these endpoints were not analyzed because of the low level of radioactivity in the extract.
Results with reference substance:
Not applicable

Table 1: Percent of Dosed Radioactivity recovered as Parent, Metabolites, associated with Extracted Solids and mineralised to CO2 in the Biotic samples.

Time (days)

Parent (Rf 0.23)

Polar metabolite (Rf <0.00)

Polar metabolite (Rf 0.05)

Long Chain Fatty Acid (Rf 0.66)

Solids

CO2

Total Recovery/Mass balance

0.01

75.0 ± 5.1

4.0 ± 0.2

10.5 ± 2.0

7.9 ± 0.2

4.8 ± 0.1

17.8 ± 2.4

120.1 ± 1.4

0.25

21.8 ± 1.3

2.2 ± 0.7

3.3 ± 0.9

5.1 ± 0.9

22.7 ± 0.8

25.6 ± 2.2

80.8 ± 1.4

0.5

18.9 ± 5.0

1.3 ± 0.2

1.8 ± 0.1

5.9 ± 1.2

25.2 ± 1.5

24.0 ± 1.5

77.0 ± 6.1

1

20.2 ± 0.9

1.0 ± 0.2

2.2 ± 0.3

6.8 ± 0.9

25.0 ± 0.2

26.6 ± 0.4

81.7 ± 1.7

2

15.7 ± 0.3

1.3 ± 0.5

1.9 ± 0.2

6.4 ± 0.3

25.9 ± 1.4

27.5 ± 0.1

78.8 ± 2.2

3

14.9 ± 1.8

1.0 ± 0.1

2.1 ± 0.0

4.8 ± 0.8

25.6 ± 1.4

28.7 ± 1.0

77.2 ± 2.1

7

9.2 ± 0.7

0.8 ± 0.0

1.9 ± 0.5

3.8 ± 1.0

22.2 ± 0.7

33.7 ± 0.2

71.7 ± 0.8

10

9.5 ± 0.8

1.0 ± 0.3

1.8 ± 0.1

3.1 ± 0.1

20.2 ± 0.5

35.5 ± 0.9

71.2 ± 2.6

15

8.7 ± 2.8

1.3 ± 0.8

1.6 ± 0.6

2.8 ± 0.2

18.7 ± 1.0

36.7 ± 0.4

69.8 ± 3.7

36

8.1 ± 2.0

1.0 ± 0.3

1.5 ± 1.0

2.4 ± 0.1

17.3 ± 0.3

39.6 ± 0.2

69.9 ± 0.4

92

3.1 ± 0.0

0.4 ± 0.4

0.4 ± 0.5

1.6 ± 0.5

14.2 ± 0.4

63.9 ± 1.7

83.5 ± 1.8

188

NA

NA

NA

NA

13.3 ± 0.6

67.3 ± 0.4

83.2 ± 1.9

NA = Not Analysed due to low level of radioactivity in extract

Mean   = 80.4 ± 13.5

Sediment collected from Ohio River (test conducted under aerobic conditions).

Table 2: Percent of Dosed Radioactivity recovered as Parent, Metabolites, and associated with Extracted Solids in the Abiotic samples.

Time (days)

Parent (Rf 0.23)

Polar metabolite (Rf <0.03)

Polar metabolite (Rf 0.03)

Long Chain Fatty Acid (Rf 0.66)

Solids

Total Recovery/Mass balance

0.01

72.6 ± 8.1

3.3 ± 1.2

6.9 ± 1.1

ND

2.4 ± 0.1

85.2 ± 10.5

1

76.8 ± NC

2.7 ± NC

7.0 ± NC

ND

2.4 ± 0.1

86.2 ± 3.5

2

67.3 ± 5.2

2.9 ± 0.3

7.9 ± 0.6

ND

3.5 ± 0.3

81.5 ± 4.7

3

72.5 ± 1.8

2.6 ± 0.4

6.8 ± 0.5

ND

3.7 ± 0.1

85.6 ± 2.6

7

73.2 ± 6.7

1.8 ± 0.5

4.2 ± 0.7

ND

3.9 ± 0.3

83.0 ± 6.1

10

79.2 ± 1.2

1.4 ± 0.0

4.6 ± 0.5

ND

3.9 ± 0.3

83.0 6± 6.1

15

77.7 ± 0.4

0.9 ± 0.1

2.6 ± 0.6

ND

4.0 ± 0.1

85.3 ± 0.3

188

58.1 ± 13.0

3.1 ± 0.9

6.8 ± 6.0

ND

7.2 ± 0.4

78.3 ± 2.7

ND = Not Detected            NC = Not Calculated

Mean = 84.3 ± 3.3

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

Sample

Pool A (%)

1 ­(hrs­-1)

Half-life Pool A (days)

Pool B(%)

2 ­(hrs­-1)

Half-life Pool B (days)

Primary degradation

68.4

8.9

0.04

18.7

0.03

11.4

Mineralisation

26.0

4.7

0.15

42.2

0.02

34.9

Validity criteria fulfilled:
yes
Conclusions:
A reliable study conducted according to to generally accepted scientific principles determined the substance to degrade or mineralised by 76.5% (normalised result) over 92 d, 17.0% was incorporated into solids, 2.9% was metabolite, and 3.7% remained as parent. (See Executive Summary for rate constants.)
Executive summary:

The biodegradation of Tetradecanol was evaluated in an aerobic sediment die-away study in accordance with the OECD 314 guideline. Radiolabeled Tetradecanol [1-14C] was tested at 170 µg/kg dry wt. The sediment was collected from the Ohio River near Cincinnati, Ohio. Four replicates samples per sampling interval were used for biotic test treatments, while two replicate samples per sampling interval were used for abiotic test treatments.

After 92 d, 76.5% was mineralized, 17.0% was non-extractable (solids), 2.9% was metabolite, and 3.7% remained as parent.

The rate constants for biodegradation of Tetradecanol in sediment (two compartment model) were:

Primary biodegradation: 8.9 d-1 for readily bioavailable tetradecanol (compartment 1), and 0.03 d-1 for less bioavailable tetradecanol (compartment 2).

Mineralization: 4.7 d-1 for readily bioavailable tetradecanol (compartment 1), and 0.02 d-1 for less bioavailable tetradecanol (compartment 2).

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

Endpoint:
biodegradation in water: sediment simulation testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004-09-07 to 2005-06-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: other guideline: OECD 314. Deviations, reliability, and validity evaluated against current OECD 314 (Oct. 3, 2008)
Deviations:
no
Principles of method if other than guideline:
One ml samples (biotic and abiotic) of sediment with 100µl of overlying water dosed with the radiolabelled test chemical and statically incubated in a sealed dessicator continuously purged with CO2 free air and connected to a gas trapping system to collect 14CO2.
GLP compliance:
no
Radiolabelling:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
natural sediment
Details on source and properties of surface water:
Not applicable
Details on source and properties of sediment:
- Details on collection: Sediment was collected from Lytle Creek located in Wilmington, Ohio. Surficial (top 2-3 cm) sediment and overlying water was collected using a plastic scoop at a site immediately below the Wilmington Wastewater Treatment Plant outfall. This STP receives predominantly domestic waste.

- Storage conditions: The sediment was placed in plastic jars and kept on ice during transport and then stored in a 4°C cold room prior to the test.

- Textural classification: The sediment type was sandy comprised of 95% sand, 2% silt and 3% clay.

- pH at time of collection: 7.9

- Organic content: 2.1%

- Sediment samples sieved: Not reported

- Total nitrogen: 0.02%

- Percent dry weight: 62.5%
The collected sediment was 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 sample collected from the river.
Duration of test (contact time):
149 d
Initial conc.:
0.08 other: mg 14C-1- tetradecanol per kg dry weight 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: Approx. 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: Four replicates samples per sampling interval were prepared for biotic treatment and two replicates samples were prepared for abiotic treatment

- Method used to create aerobic conditions: The dessicator was continuously purged with CO2 free air to maintain aerobic conditions.

- Measuring equipment: To measure the 14CO2, internal base trap (50 mL beaker containing 20 mL of 1.5 N KOH) were used to trap any evolved 14CO2 in the headspace of the dessicator. The effluent gas was passed through a gas trapping system consisting of one empty trap followed by three base traps containing 100 mL of 1.5 N KOH to recover any 14CO2 not collected by the internal trap.

- Test performed in closed vessels: Yes, the biotic treatment was placed in a sealed dessicator.

- Details of trap for CO2 if used: Following two traps were used:
Internal trap: One 50 mL glass beaker sealed dessicator) containing 20 mL 1.5 N KOH was used to trap evolved 14CO2 in headspace of the dessicator. External trap: Three glass bottles containing 100 mL of 1.5 N KOH to recover any 14CO2 not collected by the internal trap

SAMPLING
- Sampling frequency: Biotic treatment was sampled at 0.02, 1, 2, 3, 6, 9, 14, 34, 62 and 149 d and abiotic treatment was sampled less frequently at 2, 6, 9, 14 and 149 d.

- Sampling method: At each sampling time, each sediment treatment was sampled as follows:
1) For analysis of trapped 14CO2 in headspace: Triplicate 1 mL subsamples are removed from the beaker containing KOH in dessicator and analyzed. KOH solution was replaced with fresh solution after each sampling.

2) For analysis of evolved 14CO2 in effluent gas: Triplicate 1 mL sample was removed at each sampling time from first base trap, placed in separate vials and analyzed. The two remaining traps were moved one slot closer to the flask and a new trap was added to the third slot.

3) For analysis of dissolved 14CO2 in the sediment samples: Dissolved 14CO2 was measured by placing replicate sediment samples inside a 25 mmx150 mm culture tube with a septum containing screw cap and a 2-inch piece of 10 mm thick glass rod with 5 mL of 1.5N KOH in the bottom. After sealing the tube, 1 mL of 50% HCI was injected using a syringe directly into the sediment tube through the septum to volatilize any dissolved 14CO2. The acidified samples were incubated for several days and subsequently, a 1 mL sample of the 1.5 N KOH was removed from the bottom of the culture tube for analysis.

4) For chemical analysis: Sample was removed from each tube and immediately flash frozen in a dry ice-acetone bath. These frozen samples were then lyophilized, extracted in methanol, vortexed and centrifuged until the supernatant was clear.
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 (The samples were autoclaved for 90 min and amended with 50 µL of a solution containing 2% mercuric chloride to serve as abiotic control)
- Toxicity control: No

STATISTICAL METHODS: For kinetic analysis the parent loss and mineralization data were both fit to a 2 compartment first order decay and production equations using nonlinear regression. Regression analysis was performed using Jandel Table Curve 2D (version 4.01) software. The criteria used to judge the quality of the fit for the observed data are: 1) F-value, 2) visual observation of the data fit on the graph, 3) Examination of the error residualsfor the regression model and 4) R2. 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:
83.7
St. dev.:
7.6
% Degr.:
83.6
St. dev.:
0.6
Parameter:
CO2 evolution
Remarks:
(mineralization)
Sampling time:
149 d
Remarks on result:
other: Normalized to 100% mass balance
% Degr.:
7.1
St. dev.:
1.4
Parameter:
other: % of radioactivity as metabolite
Sampling time:
149 d
Remarks on result:
other: Normalized to 100% mass balance
% Degr.:
9.4
St. dev.:
0.7
Parameter:
other: % of radioactivity associated with solids
Sampling time:
149 d
Remarks on result:
other: Normalized to 100% mass balance
Parameter:
other: % of radioactivity remaining as parent
Sampling time:
149 d
Remarks on result:
other: Below detection
% Degr.:
ca. 100
Parameter:
other: Total removal (mineralized + metabolite + incorporated into solids)
Sampling time:
149 d
Remarks on result:
other: Normalized to 100% mass balance
Compartment:
sediment
DT50:
0.04 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for primary degradation of readily bioavailable test material (compartment 1)
Compartment:
sediment
DT50:
11.4 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for primary degradation of less bioavailable test material (compartment 2)
Compartment:
sediment
DT50:
0.4 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for mineralization of readily bioavailable test material (compartment 1)
Compartment:
sediment
DT50:
23.3 d
Type:
other: Two Compartment First Order Model
Remarks on result:
other: Half life for mineralization of less bioavailable test material (compartment 2)
Other kinetic parameters:
other: Two compartment first order decay model (provided the best statistical fit) 17.1 ± 1.4 d-1 for primary degradation of readily bioavailable test materi... (see attached file)
Transformation products:
yes
No.:
#1
Details on transformation products:
Please refer to ‘Transformation products’ under ‘Details on results’ section.
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, appearance of fatty acids at the origin corresponded with the initial disappearance of parent Tetradecanol. Fatty acid represented approximately 10% of the radioactivity during the first 3 d and they decreased in abundance until it was not detected on Day 149.

EXTRACTABLE RESIDUES: All chromatograms of the abiotic treatments exhibited a single peak associated with parent. The average recovery of parent from the abiotic samples was 59.7% on Day 149. The chromatograms from the biotic samples revealed three peaks corresponding to the presence of parent (Rf 0.23), long chain fatty acids (Rf 0.66), and a peak at the origin representing very polar metabolites (Rf 0.03). Primary degradation of the parent started immediately with metabolites accounting for 23% of the radioactivity in the 15 min sample. After 1 d, only 15% of the dosed radioactivity was recovered as parent Tetradecanol. The polar materials at the origin only represented 1 to 3% of the total radioactivity except on Day 149, when they represented 6%.
- % of applied amount at Day 0: 99% in active flask (parent and metabolite; sample time was 0.02 d)
- % of applied amount at end of study period: 6.0% in active flask (parent and metabolite; sample time was 149 d)

NON-EXTRACTABLE RESIDUES: The fraction associated with the extracted solids initially increased over time reaching a maximum level of 25% and then declined to 8% by Day 149 in biotic treatments. In the abiotic treatments, the percent of radioactivity associated with the extracted solids ranged from 1 to 6%.
- % of applied amount at Day 0: 2.7% in active flask (Solids; sample time was 0.02 d)
- % of applied amount at end of study period: 8.0% in active flask (Solids; sample time was 149 d)

MINERALISATION:
- % of applied radioactivity present as CO2 at end of study: Production of 14CO2 increased throughout the study and accounted for 71% of the initial radioactivity dosed on Day 149. 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 with reference substance:
Not applicable

Biodegradation of Tetradecanol in River Sediment (Study # 67826)

Table 1: Percent of Dosed Radioactivity recovered as Parent, Metabolites, associated with Extracted Solids and mineralised to CO2 in the Biotic samples.

Time (days)

Parent (Rf 0.23)

Polar metabolite (Rf <0.03)

Long Chain Fatty Acid (Rf 0.66)

Solids

CO2

Total Recovery

0.02

77.4 ± 1.9

11.5 ± 0.63

10.2 ± 3.8

2.7 ± 1.0

NA

101 ± 0.0

1

14.9 ± 6.2

1.6 ± 0.2

9.6 ± 1.1

24.7 ± 1.6

30.3 ± 0.4

80.8 ± 0.4

2

12.5 ±0.4

1.6 ± 0.1

10.0 ± 0.3

25.3 ± 1.0

33.2 ± 0.5

82.7 ± 0.5

3

16.3 ± 1.3

1.9 ± 0.1

9.9 ± 1.1

25.2 ± 0.8

35.7 ± 1.7

89.0 ± 1.7

6

12.3 ± 4.6

1.7 ± 0.1

7.6 ± 0.4

23.4 ± 1.0

40.3 ± 1.1

85.2 ± 1.1

9

4.8 ± 1.1

3.3 ± 3.0

3.8 ± 1.0

20.8 ± 0.6

43.6 ± 0.1

76.4 ± 0.1

14

7.8 ± 0.9

1.4 ± 0.1

5.7 ± 0.8

18.5 ± 0.8

49.2 ± 0.4

82.6 ± 0.4

34

3.2 ± 0.4

0.8 ± 0.3

3.1 ± 0.3

14.5 ± 1.4

56.2 ± 0.1

77.8 ± 0.1

62

1.9 ± 0.4

0.7 ± 0.1

1.4 ± 0.1

10.2 ± 1.1

62.0 ± 0.2

76.2 ± 0.2

149

ND

6.0 ± 1.2

ND

8.0 ± 0.6

70.9 ± 0.5

84.8 ±0.5

ND = Not Detected            NA = Not Analysed

Mean = 83.7 ± 7.6  Biodegradation of test material in river sediment was conducted under aerobic conditions. 

Table 2: Percent of Dosed Radioactivity recovered as Parent, Metabolites, and associated with Extracted Solids in the Abiotic samples.

 Time (Day)

Parent

(Rf 0.23)

Metabolite (Rf 0.03)

Fatty Acid (Rf 0.66)

Solids

Mass balance

2

51.8 ± 9.6

ND

ND

2.9 ± 0.7

54.7 ± 10.3

6

74.1 ± 0.7

ND

ND

2.4 ± 0.2

76.5 ± 0.5

9

69.7 ± 7.4

ND

ND

1.4 ± 0.5

71.1 ± 7.9

14

71.3 ± 2.4

ND

ND

3.2 ± 0.1

74.5 ± 2.4

149

59.7 ± 4.9

ND

ND

6.0 ± 0.3

65.8 ± 5.2

Mean

68.5 ± 8.7

Sediment collected from Lytle Creek, Wilmington, Ohio.

Table 3: Kinetic Parameters describing the Primary Degradation and Mineralisation of the test substance in Biotic Sediment using a Two Compartment First Order Model:

Sample

Pool A (%)

1 ­(hrs­-1)

Half-life Pool A (days)

Pool B(%)

2 ­(hrs­-1)

Half-life Pool B (days)

Primary degradation

61.4

17.1

0.04

16.0

0.06

11.4

Mineralisation

34.2

1.74

0.40

35.8

0.03

23.3

Validity criteria fulfilled:
yes
Conclusions:
A reliable study conducted according to to generally accepted scientific principles determined the substance to degrade or mineralized by 83.6% , 9.4% was incorporated into solids, and 7.1% was transformed into a metabolite after 149 days. The amount remaining as parent was below detection. (See Executive Summary for rate constants.)
Executive summary:

The biodegradation of Tetradecanol was evaluated in an aerobic sediment die-away study in accordance with the OECD 314 guideline. Radiolabeled Tetradecanol [1-14C] was tested at 80 µg/kg dry wt. The sediment was collected from Lytle Creek located in Wilmington, Ohio. Four replicates per sampling interval were used for biotic test treatments, while two replicates per sampling interval were used for abiotic treatments.

After 149 d, 83.6% was mineralized, 9.4% was non-extractable (solids), 7.1% was metabolite, and no parent was detected. The rate constants for biodegradation of Tetradecanol in sediment were:

Primary biodegradation: 17.1 and 0.06 d-1 for readily bioavailable and less bioavailable Tetradecanol respectively (2 compartment model).

Mineralization: 1.74 and 0.03 d-1 for readily bioavailable and less bioavailable Tetradecanol respectively (2 compartment model).

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

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Non-GLP studies conducted to a high standard.
Principles of method if other than guideline:
Effluent monitoring of waste water treatment plants receiving predominantly municipal effluent. Concentration of alcohols and alcohol ethoxylates were measured.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Twenty-four hour composite samples of influent and effluent were collected from each of the locations from three days. They were preserved with formalin at the time of collection. These were composited in proportion to flow.
% Degr.:
>= 98.4 - <= 100
Parameter:
other: BOD removal
Remarks on result:
other: removal within WWTPs (residence time not stated)
Details on results:
Influent (In), effluent (Eff) values in ug/l, and % removal of alcohols are indicated in the table below, with alcohol data considered in two groups. The State in which the WWTP is found is indicated by the usual 2-letter abbreviation.

C12-15 OH C16-18 OH
WWTP type In eff % in eff %
TX Lagoon 297 2 99.3 92.7 2.4 97.4

NJ Oxidation 249 0.7 99.7 181 0.8 99.6
Ditch

OH Rotating 157 0.1 100 77 0.07 99.9
biological
contactor

IA Trickling 499 2.0 99.6 354 2.3 99.4
filter

MO Trickling 532 4.9 99.1 315 9 97.3
filter

KS Lagoon 67.5 1.1 98.4 35.4 2.2 93.8

CA Activated 20.05 0.2 99.9 169 0.4 99.8
sludge

OR Activated 92.9 0.2 99.8 133 0.6 99.5
sludge

AZ Oxidation 702 0.3 100 394 0.5 99.9
ditch

Results for the carbon number groups are considered alongside each other to enable the context of every data point to be seen. In the overall interpretation of the data, the results have been used with those from other studies to determine the contribution of measured alcohol concentrations from various sources.
Validity criteria fulfilled:
not applicable
Conclusions:
A very high level of biodegradation and removal was demonstrated in a study of alcohol concentrations in influent and effluent of several wastewater treatment plants in a reliable study.
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 with acceptable restrictions
Remarks:
Good quality study but not conducted to GLP. Published paper based on a more complete unpublished study report.
Qualifier:
according to
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.:
99.6
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.
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 interpolated based on read-across from members of the category with shorter and longer chain length, providing evidence of consistency in behaviour irrespective of variation in physico-chemical properties of specific category member substances.

4. DATA MATRIX
A data matrix for the C6-24 alcohols Category is attached in Section 13.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
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:
105
% 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.

Description of key information

Primary degradation is rapid with evidence of rates in the range 0.7 - 17 d- ¹ in the dissolved phase.

Key value for chemical safety assessment

Half-life in freshwater:
2.1 d
at the temperature of:
12 °C
Half-life in freshwater sediment:
0.6 d
at the temperature of:
12 °C

Additional information

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). A similar study is also available for an analogous saturated alcohol (described below).

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).

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 tetradecanol.

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

Sediment simulation testing

The degradation of C18 linear alcohol in sediments was determined in a study similar to that described above for tetradecan-1-ol, in accordance with OECD 314 and using radiolabelled 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 a study using activated sludge and radiolabelled (14C) test substance, 66.3% mineralisation of C16 to CO2 after 48h was determined in accordance with OECD 314B (Federle, 2005). 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 CO2was 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).