Hydrocarbons, C18-C24, iso-alkanes <2% aromatics

Biodegradation in soil

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in soil: simulation testing

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

1. Hypothesis for the analogue approach:
The hypothesis for the analogue approach is that the test substance GTL Base Oil Distillates (C18-C50 branched, cyclic and linear hydrocarbons – Distillates / 848301-69-9 / 482-220-0) and GTL Base Oil 3 are produced in the same Fischer-Tropsch process as GTL Gasoil which is the starting material from which the registration substance is produced by fractional distillation. The source substance contains constituents of the target substance, Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics, although it covers a wider carbon number distribution. The substances therefore have qualitatively similar properties (RAAF Scenario 2 applies). See Endpoint Summary (CSR Section 5.6.3 for additional information).
2. Source and target chemical(s)
The source substance GTL Base Oil Distillates (Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear) is composed of linear, branched and cyclic hydrocarbons of chain length C18-C50
The source substance GTL Gasoil (C8-C26) (C8-C26 branched and linear hydrocarbons – Distillates) is the substance from which the registration substance is produced. GTL Gasoil is composed of linear, branched and cyclic hydrocarbons of chain length C8-C26.
The target substance, Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics, is composed of linear, branched and cyclic hydrocarbons of chain length C18-24.
3. Analogue approach justification
The constituents of the source and target substances are all hydrocarbons. Identical constituents have identical potential for biodegradation.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

DT50:

>= 11.4 - <= 22.4 d

Temp.:

>= 18 - <= 22 °C

Remarks on result:

other: aerobic soil, 1000 mg/kg

Remarks:

Test substance: Distillates (Fischer-Tropsch), C8-26, branched and linear (two studies)

DT50:

>= 35.6 - <= 73 d

Temp.:

>= 18 - <= 22 °C

Remarks on result:

other: aerobic soil, 1000 mg/kg

Remarks:

Test substance GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50–branched, cyclic and linear (two studies)

DT50:

ca. 35.8 d

Temp.:

>= 18 - <= 22 °C

Remarks on result:

other: aerobic soil, 1000 mg/kg

Remarks:

Test substance GTL Base oil 3 (one study)

Transformation products:

not measured

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 June 2011 and 14 October 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation

Qualifier:

according to guideline

Guideline:

other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.1 route of degradation, 7.1.1.2 rate of degradation, 7.1.1.2.1 laboratory studies - aerobic degradation

Deviations:

no

GLP compliance:

yes

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

aerobic

Soil classification:

other: Single representative field soil I (Site B2 Ingleby, Derbyshire; Loamy Sand) was used for the study.

Year:

2011

Details on soil characteristics:

Parameters
Site location: Ingleby (Site B2)
Batch: EF67

Soil characteristics:
pH: 5.48
Cation exchange capacity (meq/100g): 9.24
Organic carbon (% w/w): 1.4
Bulk Density (g/ml): 1.38
Soil type (according to USDA): Loamy Sand

Particle size analyses (% w/w) USDA :
2.00-0.050 mm (Sand) : 83
0.050-0.002 mm (Silt): 9
> 0.002 (Clay): 8

MWHC (g water/100 g soil) at pF 2.0: 11.9

Microbial Biomass (mg C/100 g):
Start of incubation: 13.48
End of incubation: 7.63

The soil was freshly sampled by Land Research Associates, Lockington, Derby, from permanent pasture land in Ingleby, Derbyshire, UK (SK 346269 (SK 34636 26943, 52°51' 19.8” N, 1° 29' 13.9” W) on 11 March 2011 and transported to Harlan Laboratories Ltd, Shardlow, UK shortly after sampling.

Soil Preparation
Prior to transportation to Harlan Laboratories Ltd, Shardlow, UK the soil had been sieved to 2mm by Land Research Associates, Lockington, Derby. The soil was stored at approximately 5 °C between sieving and delivery. Upon receipt, the soil was stored in a refrigerator until use and was watered if needed. The soil was characterized for particle size distribution*, moisture content at water holding capacity , pH*, bulk density*, organic carbon content*, cation exchange capacity* and microbial biomass. The soil parameters are shown in Table 1.

The soil moisture content was determined for triplicate sub-samples by oven drying and weighing. An adequate water content was obtained by adding purified water to reach final water contents of 6.8 g water per 100 g soil. This value corresponds to a pF value between pF2.0 and 2.5.
Several days before the start of the study, the soil was conditioned to room temperature.

Soil No.:

#1

Duration:

ca. 120 d

Soil No.:

#1

Initial conc.:

ca. 1 000 mg/kg soil d.w.

Based on:

test mat.

Details on experimental conditions:

Experimental Conditions
Biotic Test System
Samples of 100 g of soil, based on dry weight, were incubated under aerobic conditions in all-glass flasks in the dark. The flasks were fitted with a polyurethane bung which freely allowed air to pass into the systems. Each test system was uniquely identified.

Abiotic Test System
Samples of 100 g of soil, based on dry weight, were incubated in sterilized glass metabolism flasks under continuous ventilation with moistened air in the dark. Each test system was uniquely identified.

Temperatures
The soil samples were incubated in an air-conditioned room at a temperature of 18-22 °C, except during the periods: 27th to 28th June 2011, 6th to 7th July 2011 and 11th to 12th July 2011, the temperature exceeded the maximum of 22 °C and reached a maximum of 32 °C. The temperatures were continuously monitored. Although some of the temperatures were outside of the required range of 20 ± 2 deg C specified in the Study Plan, they were considered to have no adverse effect on the study due to the short time periods involved.

Moisture Content
Biotic systems
The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added where necessary.

Abiotic Systems
Abiotic sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilisation and/or bound residues.

The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of sterilization solution (an aqueous solution of cycloheximide and sodium azide) equal to that lost by evaporation was added where necessary.

Treatment and Sampling
Rationale for the Application Rate
The aim was to apply the test item, at two levels, to aliquots of fresh soil (100 g dry weight) at the target rates of 1000 mg/kg dry soil (100 mg/100 g dry soil) and 100 mg/kg dry soil (10 mg/100 g dry soil).

Preparation of the Application Solution
The test item was supplied as a liquid. Two stock solutions were prepared. Application solution 1 was prepared by diluting a nominal weight of test item (10 g) in 100 ml hexane. The nominal application solution concentration was 100000 mg/l. Application solution 2 was prepared by diluting a nominal weight of test item (1 g) in 100 ml hexane. The nominal application solution concentration was 10000 mg/l.
An aliquot (1 ml) of the application solutions was calculated to be applied to each sample to reach the target concentration (1000 and 100 mg/kg dry soil).

Treatment of the Test System
Aliquots were applied evenly onto the soil surface. The treated soil was then mixed thoroughly. The total amount of organic solvent added to the samples was 1.0% v/w.
Synthetic control samples and samples for the determination of the microbial biomass were treated with 1 ml of hexane and incubated under the same conditions as the treated samples.
After treatment, the biotic sample flasks were plugged with polyurethane bungs and were incubated in an air-conditioned room. The abiotic samples had microbial filter fitted to the inlet and outlet of each sample vessel and were connected to the air-flow system and incubated in an air-conditioned room.

Sampling

Soil Samples
Duplicate test samples of each test level biotic, abiotic and a single control sample were taken for extraction and analysis immediately after administration (Day 0), and after 7, 14, 28, 41, 56, 90, and 120 days.

Extraction of test item from Soil
Each sample was subjected to the following extraction procedure: Each vessel was emptied into a glass vessel and homogenised by placing on a flat bed shaker for 10 minutes at approximately 300 rpm.

The following weights were taken in to glass vessels:

Soil Method to follow below Weight of Soil aliquot to be taken for analysis (g)
Synthetic Control 1 10g
100 mg/Kg 1 10g
1000 mg/Kg 2 5g

100 mg/kg and Synthetic Control Soil Extraction (Method 1)
Tetrachloroethylene (15 ml) was then added to the soil and the samples were shaken for 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and passed though glass wool in to a measuring cylinder.

Tetrachloroethylene (15 ml) was then added to the soil and the samples and shaken manually to break up the soil cake. The samples were then shaken for a 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and fitltered though glass wool in to a measuring cylinder.
The samples were then placed under a stream of nitrogen and taken to a volume less than 10 ml. The final volume was adjusted to 10 ml with tetrachloroethylene and mixed thoroughly. The extracts were quantified by spectroscopic analysis.

1000 mg/kg Soil Extraction (Method 2)
Tetrachloroethylene (10 ml) was then added to the soil and the samples were shaken for a 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and passed though glass wool in to volumetric.

Tetrachloroethylene (10 ml) was then added to the soil and the samples and shaken manually to break up the soil cake. The samples were then shaken for 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and filtered though glass wool in to a volumetric.

The final volume was adjusted to 20 ml with tetrachloroethylene. The extracts were quantified by spectroscopic analysis.

Soil No.:

#1

% Degr.:

ca. 64

Parameter:

test mat. analysis

Remarks:

Biotic (1000 mg/Kg)

Sampling time:

120 d

Soil No.:

#1

% Degr.:

ca. 71

Parameter:

test mat. analysis

Remarks:

Abiotic (1000 mg/Kg)

Sampling time:

120 d

Soil No.:

#1

% Degr.:

ca. 65

Parameter:

test mat. analysis

Remarks:

Biotic (100 mg/Kg)

Sampling time:

120 d

Soil No.:

#1

% Degr.:

ca. 78

Parameter:

test mat. analysis

Remarks:

Abiotic (100 mg/Kg)

Sampling time:

120 d

Soil No.:

#1

DT50:

ca. 73 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Biotic (1000 mg/Kg)

Soil No.:

#1

DT50:

ca. 62.9 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Abiotic (1000 mg/Kg)

Soil No.:

#1

DT50:

ca. 26 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Biotic (100 mg/Kg)

Soil No.:

#1

DT50:

ca. 30.2 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Abiotic (100 mg/Kg)

Transformation products:

not measured

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

RESULTS
Rate of Test Item Degradation
The results are summarised in Tables 2 – 5 in terms of percentage of fortification and in Appendix 2 - 5 in mg equivalents per kg dry soil. The rate of degradation is shown graphically in Figure 1 – 4.

The percentage fortification was established for each sampling interval. On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as mean values.

The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) and day 120 is shown in the table below:

Recovery at Day 0 (% Fortification) Recovery at Day 120 (% Fortification)
Sample 100 mg/kg 1000 mg/kg 100 mg/kg 1000 mg/kg
Biotic 100 92 35 36
Abiotic 134 98 22 29

The measured amounts of the test item in Table 2 - 5 were subjected to first-order reaction kinetics. The following DT50, DT75 and DT90 values were calculated for the test item, based on first-order kinetics:

100 mg/kg 1000 mg/kg
Disappearance
Rate (days) Biotic Abiotic Biotic Abiotic
DT50 26.0 30.2 73.0 62.9
DT75 57.5 75.5 150.0 130.2
DT90 99.1 139.5 254.3 221.8
r2 0.8708 0.8064 0.9382 0.882

Microbial Biomass
The microbial biomass of the biotic soil was determined to be 13.48 mg C/ 100 g dry soil prior to the start of incubation. At the end of the incubation period, the microbial biomass was determined to be 7.63 mg C/ 100 g dry soil (Table 1). These results demonstrate that the biotic soil remained viable during the study.

The results for the abiotic soil at the beginning and end of the study showed no microbial activity.

Table1              Soil Characteristics

 

Parameters	Soil
Site location:	Ingleby (Site B2)
Batch:	EF67
Soil characteristics:
pH	5.48[1]
Cation exchange capacity (meq/100g)	9.24^†
Organic carbon (% w/w)	1.4^†
Bulk Density (g/ml)	1.38*
Soil type (according to USDA)	Loamy Sand
^[2]Particle size analyses (% w/w)USDA^[3]:
2.00-0.050 mm (Sand)	83
0.050-0.002 mm (Silt)	9
> 0.002 (Clay)	8
MWHC^[4](g water/100 g soil) at pF 2.0	11.9*
Microbial Biomass (mg C/100 g):
Start of incubation	13.48
End of incubation	7.63

Table2              Pattern ofDegradation of the Test Item(100 mg/kg Biotic)

 

100 mg/kg Biotic	Replicate	Incubation Time (Days)
		0	7	14	28	41	56	90	120
Amount of Test Item                 (% Fortification)	A	113	109	71	32	32	22	19	[1]
	B	86	94	68	35	17	24	12	35
	Mean	100	102	70	34	25	23	16	35

Table3         Pattern ofDegradation of the Test Item (100 mg/kg Abiotic)

100 mg/kg Biotic	Replicate	Incubation Time (Days)
		0	7	14	28	41	56	90	120
Amount of Test Item                 (% Fortification)	A	135	88	113	56	17	28	43	20
	B	132	119	102	44	*	23	25	23
	Mean	134	104	108	50	17	26	34	22

 

Table4         Pattern ofDegradation of the Test Item (1000 mg/kg Biotic)

100 mg/kg Biotic	Replicate	Incubation Time (Days)
		0	7	14	28	41	56	90	120
Amount of Test Item                 (% Fortification)	A	91	80	91	64	59	66	33	41
	B	93	84	84	72	52	45	39	30
	Mean	92	82	88	68	56	56	36	36

 

Table5         Pattern ofDegradation of the Test Item (1000 mg/kg Abiotic)

100 mg/kg Biotic	Replicate	Incubation Time (Days)
		0	7	14	28	41	56	90	120
Amount of Test Item                 (% Fortification)	A	96	85	93	43	50	16	71	23
	B	99	87	96	90	40	23	16	34
	Mean	98	86	95	67	45	20	44	29

 

[1]Data shared with Harlan Laboratories Ltd project number 41005208

^[2]Parameters as determined by CEM Analytical Services, Glendale Park, Fernbank Road, North Ascot,  Berkshire, UK (Study Number: CEMS-4995)

^[3]According to USDA soil texture classification system

^[4]Moisture content at water holding capacity

[5]<LOQ result considered anomalous therefore not included in mean or disappearance rate calculations

 

Conclusions:

The rate of degradation of the test item at in a single soil type incubated in the dark under aerobic conditions at 18 ± 32°C was investigated. The test item has a DT50 degradation rate ranging from 26.0-30.2 days at 100 mg/kg and 62.9-73.0 days at 1000 mg/kg at
18 ± 32°C.

As the microbial activity confirmed that sterility was maintained, the losses from the sterile system were therefore thought to be due to abiotic factors.
After reviewing the system design the losses were considered to be due to inconsistent apparatus used to incubate the bioitc and abiotic test systems. It is considered from the results of a subsequent study performed (Harlan Laboraotries Ltd., project number: 41200180), which utilized the same incubation apparatus for the abiotic and abiotic test systems, that the loss of the test item seen in this study in the abiotic vessels was due to the air flow being pulled over the soil, therefore causing losses possibly due to volatilization.

Executive summary:

Introduction.The purpose of the study was to determine the rate of degradation of the test item in one soil incubated in the dark under aerobic conditions at 18 ± 32°C.

The work was designed to be compatible withOECD Guideline 307 for testing chemicals: Aerobic - Anaerobic Transformation in Soil (April 2002), SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation and Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.1 route of degradation, 7.1.1.2 rate of degradation, 7.1.1.2.1 laboratory studies - aerobic degradation.

It should be noted that this test was designed to evaluate the toxicity of single substances of low volatility and not complex substances such as the test item (GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-C50 –branched, cyclic and linear) which is considered a UVCB substance.[1]  This means that greater care is required in the interpretation of the data and the study design was altered by the inclusion of abiotic (sterile) controls as described below. In effect for the types of substances evaluated in this study the term degradation has been used for the aerobic systems although it should be noted that this is a combination of biological degradation and losses due to physical (abiotic) factors. The sterile controls were used to ascertain a disappearance rate to account for abiotic loss processes such as losses due to volatilisation or due to significant binding to soil (non extractable residues).

Method.The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand.

The freshly collected soil was supplied sieved to 2 mm. The test item was then applied to soil samples (100 g dry weight) at a concentration of approximately 100 mg/kg and

1000 mg/kg dry soil.The soil moisture, of biotic samples, content was adjusted with purified water. Abiotic sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilisation and/or bound residues. The treated soil samples were incubated at 18 to 32ºC in the dark. Prior to treatment and at the end of the incubation period, the microbial biomass was determined for each soil. The results show that the biotic soil remained viable during the study.

Samples were taken from each soil immediately after treatment with test item (Day 0)and after 7, 14, 28, 41, 56, 90 and120 days. Each sample was exhaustively extracted bysolvent extraction at room temperature using tetrachloroethylene. The extracts were then subjected to spectrographic analysis.

Results.The percentage fortification recovered was established for each sampling interval.

The total mean recovery in terms of percentage fortification immediately after treatment   (Day 0) is shown in the table below:

	Recovery at Day 0 (% Fortification)		Recovery at Day 120 (% Fortification)
Sample	100 mg/kg	1000 mg/kg	100 mg/kg	1000 mg/kg
Biotic	100	92	35	36
Abiotic	134	98	22	29

The calculated DT₅₀, DT₇₅and DT₉₀values for the test item based on first-order kinetics are presented below:

	100 mg/kg		1000 mg/kg
Disappearance Rate (days)	Biotic	Abiotic	Biotic	Abiotic
DT50	26.0	30.2	73.0	62.9
DT75	57.5	75.5	150.0	130.2
DT90	99.1	139.5	254.3	221.8
r^2[2]	0.8708	0.8064	0.9382	0.882

 

Conclusion.The rate of degradation of the test item at in a single soil type incubated in the dark under aerobic conditions at 18 to 32°C was investigated. The test item has a DT₅₀degradation rate ranging from 26.0-30.2 days at 100 mg/kg and 62.9-73.0 days at
1000 mg/kg at 18 to 32°C.

As the microbial activity confirmed that sterility was maintained, the losses from the sterile system were therefore thought to be due to abiotic factors.

After reviewing the system design the losses were considered to be due to inconsistent apparatus used to incubate the bioitc and abiotic test systems. It is considered from the results of a subsequent study performed (Harlan Laboraotries Ltd., project number: 41200180), which utilized the same incubation apparatus for the abiotic and abiotic test systems, that the loss of the test item seen in this study in the abiotic vessels was due to the air flow being pulled over the soil, therefore causing losses possibly due to volatilization.

[1]UVCB substances: Substances of Unknown or Variable composition, Complex reaction products or Biological materials. (REACH Technical Guidance for identification and naming of substances).

 

[2]coefficient of determination

Reference 3

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-02-14 to 2012-08-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Read-across is considered to be reliability 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation

Qualifier:

according to guideline

Guideline:

other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.1 route of degradation, 7.1.1.2 rate of degradation, 7.1.1.2.1 laboratory studies - aerobic degradation

Deviations:

no

GLP compliance:

yes

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

aerobic

Soil classification:

other: Single representative field soil I (Site B2 Ingleby, Derbyshire; Loamy Sand) was used for the study.

Year:

2011

Details on soil characteristics:

Parameters
Site location: Ingleby (Site B2)
Batch: EF67

Soil characteristics:
pH: 5.48
Cation exchange capacity (meq/100g): 9.24
Organic carbon (% w/w): 1.4
Bulk Density (g/ml): 1.38
Soil type (according to USDA): Loamy Sand

Particle size analyses (% w/w) USDA :
2.00-0.050 mm (Sand) : 83
0.050-0.002 mm (Silt): 9
> 0.002 (Clay): 8

MWHC (g water/100 g soil) at pF 2.0: 11.9

Microbial Biomass (mg C/100 g):
Start of incubation: 13.48
End of incubation: 7.63

The soil was freshly sampled by Land Research Associates, Lockington, Derby, from permanent pasture land in Ingleby, Derbyshire, UK (SK 346269 (SK 34636 26943, 52°51' 19.8” N, 1° 29' 13.9” W) on 11 March 2011 and transported to Harlan Laboratories Ltd, Shardlow, UK shortly after sampling.

Soil Preparation
Prior to transportation to Harlan Laboratories Ltd, Shardlow, UK the soil had been sieved to 2mm by Land Research Associates, Lockington, Derby. The soil was stored at approximately 5 °C between sieving and delivery. Upon receipt, the soil was stored in a refrigerator until use and was watered if needed. The soil was characterized for particle size distribution*, moisture content at water holding capacity , pH*, bulk density*, organic carbon content*, cation exchange capacity* and microbial biomass. The soil parameters are shown in Table 1.

The soil moisture content was determined for triplicate sub-samples by oven drying and weighing. An adequate water content was obtained by adding purified water to reach final water contents of 6.8 g water per 100 g soil. This value corresponds to a pF value between pF2.0 and 2.5.
Several days before the start of the study, the soil was conditioned to room temperature.

Soil No.:

#1

Duration:

ca. 120 d

Soil No.:

#1

Initial conc.:

ca. 1 000 mg/kg soil d.w.

Based on:

test mat.

Details on experimental conditions:

Experimental Conditions
Biotic Test System
Samples of 100 g of soil, based on dry weight, were incubated under aerobic conditions in all-glass flasks in the dark. The flasks were fitted with a polyurethane bung which freely allowed air to pass into the systems. Each test system was uniquely identified.

Abiotic Test System
Samples of 100 g of soil, based on dry weight, were incubated in sterilized glass metabolism flasks under continuous ventilation with moistened air in the dark. Each test system was uniquely identified.

Temperatures
The soil samples were incubated in an air-conditioned room at a temperature of 18-22 °C, except during the periods: 27th to 28th June 2011, 6th to 7th July 2011 and 11th to 12th July 2011, the temperature exceeded the maximum of 22 °C and reached a maximum of 32 °C. The temperatures were continuously monitored. Although some of the temperatures were outside of the required range of 20 ± 2 deg C specified in the Study Plan, they were considered to have no adverse effect on the study due to the short time periods involved.

Moisture Content
Biotic systems
The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added where necessary.

Abiotic Systems
Abiotic sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilisation and/or bound residues.

The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of sterilization solution (an aqueous solution of cycloheximide and sodium azide) equal to that lost by evaporation was added where necessary.

Treatment and Sampling
Rationale for the Application Rate
The aim was to apply the test item, at two levels, to aliquots of fresh soil (100 g dry weight) at the target rates of 1000 mg/kg dry soil (100 mg/100 g dry soil) and 100 mg/kg dry soil (10 mg/100 g dry soil).

Preparation of the Application Solution
The test item was supplied as a liquid. Two stock solutions were prepared. Application solution 1 was prepared by diluting a nominal weight of test item (10 g) in 100 ml hexane. The nominal application solution concentration was 100000 mg/l. Application solution 2 was prepared by diluting a nominal weight of test item (1 g) in 100 ml hexane. The nominal application solution concentration was 10000 mg/l.
An aliquot (1 ml) of the application solutions was calculated to be applied to each sample to reach the target concentration (1000 and 100 mg/kg dry soil).

Treatment of the Test System
Aliquots were applied evenly onto the soil surface. The treated soil was then mixed thoroughly. The total amount of organic solvent added to the samples was 1.0% v/w.
Synthetic control samples and samples for the determination of the microbial biomass were treated with 1 ml of hexane and incubated under the same conditions as the treated samples.
After treatment, the biotic sample flasks were plugged with polyurethane bungs and were incubated in an air-conditioned room. The abiotic samples had microbial filter fitted to the inlet and outlet of each sample vessel and were connected to the air-flow system and incubated in an air-conditioned room.

Sampling

Soil Samples
Duplicate test samples of each test level biotic, abiotic and a single control sample were taken for extraction and analysis immediately after administration (Day 0), and after 7, 14, 28, 41, 56, 90, and 120 days.

Extraction of test item from Soil
Each sample was subjected to the following extraction procedure: Each vessel was emptied into a glass vessel and homogenised by placing on a flat bed shaker for 10 minutes at approximately 300 rpm.

The following weights were taken in to glass vessels:

Soil Method to follow below Weight of Soil aliquot to be taken for analysis (g)
Synthetic Control 1 10g
100 mg/Kg 1 10g
1000 mg/Kg 2 5g

100 mg/kg and Synthetic Control Soil Extraction (Method 1)
Tetrachloroethylene (15 ml) was then added to the soil and the samples were shaken for 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and passed though glass wool in to a measuring cylinder.

Tetrachloroethylene (15 ml) was then added to the soil and the samples and shaken manually to break up the soil cake. The samples were then shaken for a 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and fitltered though glass wool in to a measuring cylinder.
The samples were then placed under a stream of nitrogen and taken to a volume less than 10 ml. The final volume was adjusted to 10 ml with tetrachloroethylene and mixed thoroughly. The extracts were quantified by spectroscopic analysis.

1000 mg/kg Soil Extraction (Method 2)
Tetrachloroethylene (10 ml) was then added to the soil and the samples were shaken for a 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and passed though glass wool in to volumetric.

Tetrachloroethylene (10 ml) was then added to the soil and the samples and shaken manually to break up the soil cake. The samples were then shaken for 30 minutes at approximately 200rpm. The soil was allowed to settle and the supernatant was removed and filtered though glass wool in to a volumetric.

The final volume was adjusted to 20 ml with tetrachloroethylene. The extracts were quantified by spectroscopic analysis.

Soil No.:

#1

DT50:

35.6 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: aerobic soil (1000 mg/Kg)

Soil No.:

#1

DT50:

101.9 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: sterile soil (1000 mg/Kg)

Transformation products:

not measured

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

RESULTS
Rate of Test Item Degradation
The results are summarised in Tables 2 – 5 in terms of percentage of fortification and in Appendix 2 - 5 in mg equivalents per kg dry soil. The rate of degradation is shown graphically in Figure 1 – 4.

The percentage fortification was established for each sampling interval. On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as mean values.

The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) and day 120 is shown in the table below:

Recovery (% Fortification)
Day 0 Day 120
Sample 1000 mg/kg 1000 mg/kg
Biotic 98 15
Abiotic 105 54

The measured amounts of the test item in Table 2 -3 were subjected to first-order reaction kinetics. The following DT50, DT75 and DT90 values were calculated for the test item, based on first-order kinetics:

1000 mg/kg
Disappearance
Rate (days) Biotic Abiotic
DT50 35.6 101.9
DT75 72.1 217.4
DT90 120.3 >1 year
r2 0.893 0.889

Microbial Biomass
The microbial biomass of the biotic soil was determined to be 13.48 mg C/ 100 g dry soil prior to the start of incubation. At the end of the incubation period, the microbial biomass was determined to be 7.63 mg C/ 100 g dry soil (Table 1). These results demonstrate that the biotic soil remained viable during the study.

The results for the abiotic soil at the beginning and end of the study showed no microbial activity.

Table 1: Soil Characteristics

 

Parameters	Soil
Site location:	Ingleby (Site B2)
Batch:	EF67
Soil characteristics:
pH	5.48[1]
Cation exchange capacity (meq/100g)	9.24^†
Organic carbon (% w/w)	1.4^†
Bulk Density (g/ml)	1.38*
Soil type (according to USDA)	Loamy Sand
^[2]Particle size analyses (% w/w)USDA^[3]:
2.00-0.050 mm (Sand)	83
0.050-0.002 mm (Silt)	9
> 0.002 (Clay)	8
MWHC^[4](g water/100 g soil) at pF 2.0	11.9*
Microbial Biomass (mg C/100 g):
Start of incubation	13.48
End of incubation	7.63

Table 2: Pattern ofDegradation of the Test Item (1000 mg/kg Biotic)

1000 mg/kg Biotic	Replicate	Incubation Time (Days)
		0	7	14	27	59	90	120
Amount of Test Item (% Fortification)	A	104*	79	79	64	44	9	17
	B	91*	70	94	59	26	11	12
	Mean	98	75	87	62	35	10	15

 

Table 3: Pattern ofDegradation of the Test Item (1000 mg/kg Abiotic)

1000 mg/kg sterile	Replicate	Incubation Time (Days)
		0	7	14	27	59	90	120
Amount of Test Item (% Fortification)	A	115^+*	90	92	82	78	47	48
	B	105*	85	91	75	69	52	60
	Mean	105	88	92	79	74	50	54

 +result not included in mean calculation

*Day 0 samples re-analysed as original results deemed anomalous

 

Conclusions:

The rate of degradation of the test item at in a single soil type incubated in the dark under aerobic conditions at 20 ± 2°C was investigated. The test item has a DT50 degradation rate of 35.6 days at 1000 mg/kg was obtained.

As the microbial activity confirmed that sterility was maintained, the losses from the sterile system were therefore thought to be due to abiotic factors.
After reviewing the system design the losses were considered to be due to inconsistent apparatus used to incubate the bioitc and abiotic test systems. It is considered from the results of a subsequent study performed (Harlan Laboraotries Ltd., project number: 41200180), which utilized the same incubation apparatus for the abiotic and abiotic test systems, that the loss of the test item seen in this study in the abiotic vessels was due to the air flow being pulled over the soil, therefore causing losses possibly due to volatilization.

Executive summary:

Introduction.

The purpose of the study was to determine the rate of degradation of the test item in one soil incubated in the dark under aerobic conditions at 20 ± 2°C.

 

The work was designed to be compatible withOECD Guideline 307 for testing chemicals: Aerobic - Anaerobic Transformation in Soil (April 2002), SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation and Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.1 route of degradation, 7.1.1.2 rate of degradation, 7.1.1.2.1 laboratory studies - aerobic degradation.

 

It should be noted that this test was designed to evaluate the toxicity of single substances of low volatility and not complex substances such as the test item (GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-C50 –branched, cyclic and linear) which is considered a UVCB substance.[1]  This means that greater care is required in the interpretation of the data and the study design was altered by the inclusion of abiotic (sterile) controls as described below. In effect for the types of substances evaluated in this study the term degradation has been used for the aerobic systems although it should be noted that this is a combination of biological degradation and losses due to physical (abiotic) factors. The sterile controls were used to ascertain a disappearance rate to account for abiotic loss processes such as losses due to volatilisation or due to significant binding to soil (non extractable residues).

 

Method.

The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand.

The freshly collected soil was supplied sieved to 2 mm. The test item was then applied to soil samples (100 g dry weight) at a concentration of approximately 1000 mg/kg. The soil moisture, of biotic samples, content was adjusted with purified water. Abiotic sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilisation and/or bound residues. The treated soil samples were incubated at 20ºC in the dark. Prior to treatment and at the end of the incubation period, the microbial biomass was determined for each soil. The results show that the biotic soil remained viable during the study.

Samples were taken from each soil immediately after treatment with test item (Day 0) and after 7, 14, 27, 59, 90 and120 days. Each sample was exhaustively extracted bysolvent extraction at room temperature using tetrachloroethylene. The extracts were then subjected to spectrographic analysis.

Reference 4

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28-May-2010 to 11-Aug-2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.2 rate of degradation; 7.1.1.2.1 laboratory studies - aerobic degradation.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

aerobic

Remarks:

The purpose of this study was to determine the rate of degradation (DT50, DT75 and DT90) of the test item in one soil incubated in the dark under aerobic conditions at 20 ± 2 °C.

Soil classification:

other: The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand.

Year:

2010

Soil no.:

#1

Soil type:

loamy sand

% Clay:

8

% Silt:

10

% Sand:

82

% Org. C:

8.13

pH:

6.49

CEC:

4.08 other: (cmol+/k g soil)

Details on soil characteristics:

Soil Types

The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand.

Pesticide history: infrequent spraying to control broadleaf weeds but not normally on steep slope where samples are taken.

Soil Collection

The soil was freshly sampled by Land Research Associates, Lockington, Derby, from permanent pasture land in Ingleby, Derbyshire, UK (SK 346269 (SK 34636 26943, 52°51' 19.8” N, 1° 29' 13.9” W) in March 2010 and transported to Harlan Laboratories Ltd, Shardlow, UK shortly after sampling. The area had occasionally been treated, using MCPA, to control broadleaf weeds. It was sampled from shelf cut below the surface soil using a spade.


For all soils, the top plant cover was removed during sampling and soil was put in containers with free access to air. A unique sample identification label was placed inside the container and the same information written with indelible pen on the outside of the container.

All three soils had not been subjected to any pesticide, organic or inorganic fertiliser treatment for at least the last five years prior to sampling. There had also been no such treatment of the soils at Harlan Laboratories Ltd.

Soil Preparation

Prior to transportation to Harlan Laboratories Ltd, Shardlow, UK the soil had been sieved to 2mm by Land Research Associates, Lockington, Derby. The soil was stored at approximately 5 °C between sieving and delivery. Upon receipt, the soil was stored in a refrigerator until use and was watered if needed. The soil was characterized for particle size distribution , moisture content at water holding capacity , pH†, organic carbon content†, cation exchange capacity† and microbial biomass. The soil parameters are shown in Table 1.

The soil moisture content was determined for triplicate sub-samples by oven drying and weighing. An adequate water content was obtained by adding purified water to reach final water contents of 11.0 g water per 100 g soil. This value corresponds to a pF value of approximately 2.0.
Several days before the start of the study, the soil was conditioned to room temperature.

Soil Characteristics

Site location: Ingleby (Site B2)
Batch: EF52
Soil characteristics:
pH 6.49
Cation exchange capacity (cmol+/kg) 4.08
Organic matter carbon (%) 8.13
Soil type (according to USDA) Loamy Sand
Particle size analyses (% w/w) USDA :
2.00-0.050 mm (Sand) 82
0.050-0.002 mm (Silt) 10
> 0.002 (Clay) 8
MWHC (g water/100 g soil) at pF 2.0 11.0*
Microbial Biomass (µg organic C/g dry soil):
Start of incubation 55
End of incubation 76



Please also see attached Appendix 3 EF52 Soil Sampling Log

Soil No.:

#1

Duration:

51 d

Soil No.:

#1

Initial conc.:

1 000 other: mg a.i./kg dry soil

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Soil No.:

#1

Temp.:

20 ± 2°C

Microbial biomass:

Start 55 - End 76 µg organic C/g dry soil

Details on experimental conditions:

Determination of Soil Microbial Biomass

The microbial biomass of the soil was determined prior to treatment and at the end of the incubation period, by using the fumigation-extraction method as recommended by ISO Guideline 14240-2.

Experimental Conditions

Test System

Samples of 100 g of soil, based on dry weight, were incubated under aerobic conditions in all-glass flasks in the dark at 20 ± 2 °C. The flasks were fitted with a polyurethane bung which freely allowed air to pass into the systems.Each test system was uniquely identified.

Temperatures

The soil samples were incubated in air-conditioned rooms at a temperature of 20 ± 2 °C. The temperatures were continuously monitored.

Moisture Content

The soil moisture content was maintained at approximately pF2.0. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added where necessary.

Treatment and Sampling

Rationale for the Application Rate

The aim was to apply the test item, at two levels, to aliquots of fresh soil (100 g dry weight) at the target rates of 1000 mg/kg dry soil (100 mg/100 g dry soil) and 100 mg/kg dry soil (10 mg/100 g dry soil).
The analytical method was not validated for the lower test level (100 mg/kg dry soil) as the limit of detection was greater than 10% of the application rate. Therefore the study was not performed at the lower test level.

Preparation of the Application Solution

The test item was supplied as a liquid. A stock solution was prepared by diluting a nominal weight of test item (10 g) in 100 ml acetone. The nominal application solution concentration was 100000 mg/l.
An aliquot (1 ml) of the application solution was calculated to be applied to each sample to reach the target concentration (1000 mg/kg dry soil).

Treatment of the Test System

Aliquots were applied evenly onto the soil surface. The treated soil was then mixed thoroughly. The total amount of organic solvent added to the samples was 1.0% v/w.
For determination of the microbial biomass, control samples were treated with 1 ml of acetone and incubated under the same conditions as the treated samples. After administration, the flasks were plugged with polyurethane bungs and were incubated in an air-conditioned room at 20 ± 2 °C.

Sampling

Soil Samples

Duplicate test samples (1000 mg/kg dry soil) and a single control sample were taken for extraction and analysis immediately after administration (Day 0), and after 1, 3, 7, 10, 14, 20, 35 and 51 days.

Extraction of test item from Soil

Each sample was subjected to the following extraction procedure: Each vessel was emptied into a 500 ml centrifuge tube and homogenised by placing on a flat bed shaker for 5 minutes at approximately 320 rpm.

An aliquot (approximately 5.0 g) of the homogenised sample was added to a 50 ml centrifuge tube. The soil was chemically dried by adding approximately 5.0 g of anhydrous sodium sulphate to the soil and shaking for 5 minutes, using a flat bed shaker at approximately 320 rpm.

The dried soil was extracted by adding acetone (4 ml) and sonicating for 2 minutes at approximately 20°C. Hexane (4 ml) was then added to the soil and the samples were ultrasonicated for a further 10 minutes at approximately 20 °C.

The soil was then centrifuged for 5 minutes at 750 rpm. The resulting liquid layer was removed via suction and passed though glass wool into a 50 ml measuring cylinder. The extraction step from the addition of acetone (4 ml) onwards was then repeated.

The soil was further extracted by adding acetone/hexane (1:1) (10 ml) to the soil cake and ultrasonicated for 15 minutes at approximately 20 °C. The soil was then centrifuged for 5 minutes at 750 rpm. The resulting liquid layer was removed via suction, passed through glass wool and combined in the same 50 ml measuring cylinder. The extraction step from the addition of acetone/hexane (1:1) (10 ml) onwards was then repeated.

The glass funnel and glass wool were rinsed with approximately 3 ml of acetone/hexane (1:1) and the final volume was adjusted to 40 ml. The combined extracts were quantified by chromatographic analysis.

Soil No.:

#1

% Recovery:

102

St. dev.:

7.4

Remarks on result:

other: Non-labelled test material. The total mean recoveries in mg/Kg

Soil No.:

#1

% Degr.:

5

Parameter:

test mat. analysis

Sampling time:

51 d

Soil No.:

#1

DT50:

11.4 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Soil No.:

#1

DT50:

23.2 d

Type:

other:

Remarks on result:

other: DT75 (d) at 20 °C

Soil No.:

#1

DT50:

38.7 d

Type:

other:

Remarks on result:

other: DT90 (d) at 20 °C

Transformation products:

no

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

RESULTS

Rate of Test Item Degradation

The results are summarised in Table 2 - (see in section any other information on results) in terms of percentage of fortification and in
table 3 - (see in section any other information on results) in mg equivalents per kg dry soil.

The rate of degradation is shown graphically in Figure 1 - see in attached section.

The percentage fortification was established for each sampling interval. On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as mean values.
The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) was 102%. By the end of the study the amount of test item recovered from the soil decreased rapidly to 5% fortification (Day 51).
The measured amounts of the test item in Table 2 were subjected to first-order reaction kinetics. The following DT50, DT75 and DT90 values were calculated for the test item, based on first-order kinetics:

Degradation Rate Time (Days)
DT50 11.4
DT75 23.2
DT90 38.7
r2 0.9908

The OECD Guideline 307 was designed to assess the degradation of pesticides and not complex substances such as the test substance where components of the substance may be lost due to volatilisation and/or irreversible binding to the soil (bound residues). Ideally killed control samples should have been incubated alongside the test samples to assess the loss of test item due to bound unextractable residues and volatilisation. This did not occur and the rate of loss of test item observed during the study therefore cannot be conclusively described as degradation of the test item and in strict scientific terms the rate of loss observed during this study should be described as the rate of disappearance of the test item from the soil. However, for this study the analytical data has shown that it is possible to recover the test substance components of soil indicating bound residues are not a specific concern. Furthermore, based on information from a previous non GLP study undertaken by the sponsor the rates of non-biological losses attributed to factors such as: volatilisation, irreversible binding of compounds to the soil particles, and inefficiencies in the soil extraction procedure for the test substance were only 10% after 10 weeks in a static soil study. As such it would appear that degradation is the dominant factor accounting for the disappearance of the test substance from soil.

Microbial Biomass

The microbial biomass of the soil was determined to be 55 µg organic C/g dry soil prior to the start of incubation. At the end of the incubation period, the microbial biomass was determined to be 76 µg organic C/g dry soil. These results demonstrate that the soil remained viable during the study.

Table 2 Pattern of Degradation of the Test Item

 

Soil	Replicate	Incubation Time (Days)
		0	1	3	7	10	14	20	35	51
Amount of Test Item                 (% Fortification)	A	102	100	80	59	50	55	32	6	5
	B	102	97	67	66	48	43	35	16	4
	Mean	102	98	74	62	49	49	34	11	5

 

Table 3      Pattern of Degradation of the Test Item in soil

 

Soil	Replicate	Incubation Time (Days)
		0	1	3	7	10	14	20	35	51
Amount of test item (mg/kg dry soil)	A	1019.499	1000.496	798.009	590.570	497.469	549.374	324.999	61.596	51.335
	B	1016.727	969.531	675.236	658.515	480.814	429.904	346.083	156.599	44.872
	Mean	1018.113	985.014	736.623	624.543	489.142	489.639	335.541	109.098	48.104

 

Conclusions:

The rate of degradation of the test item in a single soil type incubated in the dark under aerobic conditions at 20 ± 2°C was investigated. The test item has a DT50 degradation rate of 11.4 days at 20 ± 2°C.

Executive summary:

Results.

The percentage fortification recovered was established for each sampling interval. The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) was 102%. By the end of the study the amount of test item recovered from the soil decreased rapidly to 5% fortification (Day 51). The calculated DT₅₀, DT₇₅and DT₉₀ values for the test item based on first-order kinetics are presented below: Degradation Rate Time (Days) DT₅₀ 11.4 DT₇₅ 23.2 DT₉₀ 38.7 r²[1] 0.9908

The OECD Guideline 307 was designed to assess the degradation of pesticides and not complex substances such as the ‘Distillates (Fischer-Tropsch), C8-26 - branched and linear’ where components of the substance may be lost due to volatilisation and/or irreversible binding to the soil (bound residues). Ideally killed control samples should have been incubated alongside the test samples to assess the loss of test item due to bound unextractable residues and volatilisation. This did not occur and the rate of loss of test item observed during the study therefore cannot be conclusively described as degradation of the test item and in strict scientific terms the rate of loss observed during this study should be described as the rate of disappearance of the test item from the soil. However, for this study the analytical data has shown that it is possible to recover the substance´s components of soil indicating bound residues are not a specific concern. Furthermore, based on information from a previous non GLP study undertaken by the sponsor the rates of non-biological losses attributed to factors such as: volatilisation, irreversible binding of compounds to the soil particles, and inefficiencies in the soil extraction procedure for the test item were only 10% after 10 weeks in a static soil study. As such it would appear that degradation is the dominant factor accounting for the disappearance of the substance from soil.

Conclusion.

The rate of degradation of the test item in a single soil type incubated in the dark under aerobic conditions at 20 ± 2°C was investigated. The test item has a DT₅₀ degradation rate of 11.4 days at 20 ± 2°C.

Reference 5

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The test was conducted between 17 August 2011 and 14 December 2011.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.2 rate of degradation; 7.1.1.2.1 laboratory studies - aerobic degradation.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

aerobic

Remarks:

The purpose of this study was to determine the rate of degradation (DT50 and, if the data permits, a DT75 and a DT90 will also be calculated) of the test item in one soil incubated in the dark under aerobic conditions at 20 ± 2 °C

Soil classification:

other: The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand

Year:

2011

Soil no.:

#1

Soil type:

silt loam

% Clay:

9

% Silt:

9

% Sand:

82

% Org. C:

1.6

pH:

5

CEC:

12.1 other: (mmol/100 g soil)

Details on soil characteristics:

Soil Collection
The soil was freshly sampled by Land Research Associates, Lockington, Derby, from a permanent pasture field in Ingleby, Derbyshire, UK (SK 346269 (SK 34636 26943, 52°51' 19.8” N, 1° 29' 13.9” W) on 21 July 2011 and transported to the relevant laboratory shortly after sampling.

Soil Preparation
Prior to transportation the soil had been sieved to 2mm by Land Research Associates, Lockington, Derby. The soil was stored at approximately 5 °C between sieving and delivery. Upon receipt, the soil was stored in a refrigerator until use and was watered if needed. The soil was characterized for particle size distribution*, moisture content at water holding capacity , pH, bulk density, organic carbon content*, cation exchange capacity* and microbial biomass. The soil parameters are shown in Table 1.
The soil moisture content was determined for triplicate sub-samples by oven drying and weighing. An adequate water content was obtained by adding purified water to reach final water contents of 16 g water per 100 g soil. This value corresponds to a pF value between pF2.0 and 2.5.
Sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilization and/or bound residues.
Several days before the start of the study, the soil was conditioned to room temperature.

Soil Characteristics

Site location: Ingleby (Site B2)
Batch: EF75
Soil characteristics:
pH 5.00
Cation exchange capacity (meq/100g) 12.1
Organic carbon (% w/w) 1.6
Bulk Density (g/ml) 1.38
† Soil type (according to USDA) Loamy Sand
Particle size analyses (% w/w) USDA :
2.00-0.050 mm (Sand) 82
0.050-0.002 mm (Silt) 9
< 0.002 (Clay) 9
*MWHC (% w/w) at pF 2.0
16.2
Microbial Biomass (mg C/100 g):
Start of incubation 14.80
End of incubation 17.80

*Parameters as determined by CEM Analytical Services, Glendale Park, Fernbank Road, North Ascot, Berkshire, UK (Study Number: CEMS-5196)
†According to USDA soil texture classification system
Moisture content at water holding capacity

Soil No.:

#1

Duration:

56 d

Soil No.:

#1

Initial conc.:

1 000 other: mg/kg dry soil

Based on:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear

Soil No.:

#1

Initial conc.:

10.5 other: mgkg dry soil

Based on:

other: Dodecane

Soil No.:

#1

Initial conc.:

32 other: mgkg dry soil

Based on:

other: Dodecane

Soil No.:

#1

Initial conc.:

26.6 other: mg/kg dry soil

Based on:

other: Eicosane

Soil No.:

#1

Temp.:

20+-2°C

Microbial biomass:

14.80 start

Soil No.:

#1

Temp.:

20+-2°C

Microbial biomass:

17.80 end

Details on experimental conditions:

Aerobic & Sterile Test System
Samples of 100 g of soil, based on dry weight, were incubated under aerobic conditions in all-glass flasks in the dark. The flasks were fitted with a polyurethane bung which freely allowed air to pass into the systems.
Each test system was uniquely identified.

Temperatures
The soil samples were incubated in air-conditioned rooms at a temperature of 20 ± 2 °C. The temperatures were continuously monitored.
Moisture Content

Aerobic systems
The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added where necessary.

Sterile Systems
The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of sterilization solution (an aqueous solution of cycloheximide and sodium azide) equal to that lost by evaporation was added where necessary.

Treatment and Sampling

Rationale for the Application Rate
The aim was to apply the test item and reference items to aliquots of fresh soil (100 g dry weight) at the target rates specified in the table below:
Test/Reference Item Target Rate System Identification
Test Item - Distillates (Fischer-Tropsch), C8-26 - branched and linear) 1000 mg/kg [System 1]
Reference Item 1 - Dodecane 10.5 mg/kg [System 2]
Refernce Item 2 - Hexadecane 32 mg/kg [System 3]
Reference Item 3 - Eicosane 26.6 mg/kg [System 4]

Preparation of the Application Solution

Test Item – System 1
The test item was supplied as a liquid. An application solution was prepared by diluting a nominal weight of test item (5 g) in 50 ml acetone. The nominal application solution concentration was 100000 mg/l.
An aliquot (1 ml) of the application solution was calculated to be applied to each sample to reach the target concentration (1000 mg/kg dry soil).

Reference Item 2 – System 3
The reference item was supplied as a liquid. An application solution was prepared by diluting a nominal weight of reference item 2 (0.32 g) in 100 ml hexane. The nominal application solution concentration was 3200 mg/l.
An aliquot (1 ml) of the application solution was calculated to be applied to each sample to reach the target concentration (32 mg/kg dry soil).

Reference Item 3 – System 4
The reference item was supplied as a liquid. An application solution was prepared by diluting a nominal weight of reference item 3 (0.27 g) in 100 ml acetone. The nominal application solution concentration was 2700 mg/l.
An aliquot (1 ml) of the application solution was calculated to be applied to each sample to reach the target concentration (26.6 mg/kg dry soil).

Treatment of the Test Systems
Aliquots of test and reference item application solutions were applied evenly onto the soil surface. The total amount of organic solvent added to the samples was approximately 1 % v/w.
Synthetic control samples and samples for the determination of the microbial biomass were treated with 1 ml of acetone and incubated under the same conditions as the treated samples.
After treatment polyurethane bungs were placed in to the neck of the flasks and were incubated in an air-conditioned room.

Soil Sampling
Duplicate test samples from Systems 1 to 4, aerobic, sterile and a single synthetic control sample were taken for extraction and analysis immediately after administration (Day 0), and after 1, 3, 7, 14, 28 and 56 days.

Extraction of test item from Soil
Each sample was subjected to the following extraction procedure: Each vessel was emptied into a vessel and homogenised by placing on a flat bed shaker for 10 minutes at approximately 320 rpm.
An aliquot (approximately 5 g) of the homogenised soil sample was added to a 50 ml glass centrifuge tube. The soil was chemically dried by adding approximately 5 g of anhydrous sodium sulphate to the soil and shaking for 5 minutes, using a flat bed shaker at approximately 320 rpm.
The dried soil was extracted by adding acetone (4 ml) and ultrasonicated for 2 minutes at approximately 20°C. Hexane (4 ml) was then added to the soil and the samples were shaken and ultrasonicated for a further 10 minutes at approximately 20 °C.
The soil was then centrifuged for 5 minutes at 1000 rpm. The resulting liquid layer was removed via pipette and passed though glass wool into a 50 ml measuring cylinder. The extraction step from the addition of acetone (4 ml) onwards was then repeated.
The soil was further extracted by adding acetone/hexane (1:1) (10 ml) to the soil cake shaken and ultrasonicated for 15 minutes at approximately 20 °C. The soil was then centrifuged for 5 minutes at 1000 rpm. The resulting liquid layer was removed via suction, passed through glass wool and combined in the same 50 ml measuring cylinder. The extraction step from the addition of acetone/hexane (1:1) (10 ml) onwards was then repeated.
The glass funnel and glass wool were rinsed with approximately 3 ml of acetone/hexane (1:1) and the final volume was adjusted to 40 ml (except on one occasion where a control sample was diluted to 50 ml in error, this was considered to have no adverse impact on the integrity of the results). The combined extracts were shaken manually for 30 seconds and were then quantified by chromatographic analysis.

Soil No.:

#1

% Recovery:

91.2

St. dev.:

3.7

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.7%

Soil No.:

#1

DT50:

22.4 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear (Aerobic)

Soil No.:

#1

DT50:

82.6 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear (Sterile)

Soil No.:

#1

DT50:

43.4 d

Type:

other: DT 75

Remarks on result:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear (Aerobic)

Soil No.:

#1

DT50:

169.3 d

Type:

other: DT 75

Remarks on result:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear (Sterile)

Soil No.:

#1

DT50:

71.2 d

Type:

other: DT90

Remarks on result:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear (Aerobic)

Soil No.:

#1

DT50:

283.8 d

Type:

other: DT90

Remarks on result:

other: Distillates (Fischer-Tropsch), C8-26 - branched and linear (Sterile)

Soil No.:

#1

DT50:

1.5 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Hexadecane (Aerobic)

Soil No.:

#1

DT50:

32.9 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Hexadecane (Sterile)

Soil No.:

#1

DT50:

5.4 d

Type:

other: DT75

Remarks on result:

other: Hexadecane (Aerobic)

Soil No.:

#1

DT50:

86.2 d

Type:

other: DT75

Remarks on result:

other: Hexadecane (Sterile)

Soil No.:

#1

DT50:

10.6 d

Type:

other: DT90

Remarks on result:

other: Hexadecane (Aerobic)

Soil No.:

#1

DT50:

156.7 d

Type:

other: DT90

Remarks on result:

other: Hexadecane (Sterile)

Soil No.:

#1

DT50:

11.4 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Eicosane (Aerobic)

Soil No.:

#1

DT50:

122.4 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: Eicosane (Sterile)

Soil No.:

#1

DT50:

26.5 d

Type:

other: DT75

Remarks on result:

other: Eicosane (Aerobic)

Soil No.:

#1

DT50:

261 d

Type:

other: DT75

Remarks on result:

other: Eicosane (Sterile)

Soil No.:

#1

DT50:

46.4 d

Type:

other: DT90

Remarks on result:

other: Eicosane (Aerobic)

Soil No.:

#1

DT50:

444.3 d

Type:

other: DT90

Remarks on result:

other: Eicosane (Sterile)

Transformation products:

no

Evaporation of parent compound:

no

Volatile metabolites:

no

Residues:

no

Details on results:

Rate of Test and Reference Item Degradation Rates
The results are summarized in Tables 2 to 9 in terms of percentage of fortification and in Appendix 2 to 9 in mg equivalents per kg dry soil. The rate of degradation is shown graphically in Figure 1 to 6.
The percentage fortification was established for each sampling interval. On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as mean values.
The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) and Day 56 is shown in the table below:
Recovery at Day 0 (% Fortification) Recovery at Day 56 (% Fortification)
Aerobic Sterile Aerobic Sterile
System 1 91 112 13 70
System 2 39 25 System 3 84 87 na 37
System 4 108 112 7 80
The measured amounts of the test and reference were used to plot degradation curves. The following DT50, DT75 and DT90 values were calculated:
System 1 System 2 System 3 System 4
Disappearance
Rate (days) Aerobic Sterile Aerobic Sterile Aerobic Sterile Aerobic Sterile
DT50 22.4 82.6 - - 1.5 32.9 11.4 122.4
DT75 43.4 169.3 - - 5.4 86.2 26.5 261.0
DT90 71.2 283.8 - - 10.6 156.7 46.4 444.3
r2 0.955 0.940 - - 0.807 0.673 0.921 0.741
The amount of reference item 1 observed at Day 0 in System 2 aerobic and sterile was 39% and 25% fortification, respectively. Reference item 1 disappeared from System 2 rapidly and could only be quantified in 3 aerobic test systems and 4 sterile test systems. Therefore not enough time points generated to produce a degradation curve.

Microbial Biomass
The microbial biomass of the aerobic soil was determined to be 14.80 mg C/ 100 g dry soil prior to the start of incubation. At the end of the incubation period, the microbial biomass of the aerobic soil was determined to be 17.80 mg C/ 100 g dry soil (Table 1). These results demonstrate that the aerobic soil remained viable during the study.
The results for the sterile soil at the beginning and end of the study showed no microbial activity.

Table 2         System 1, Distillates (Fischer-Tropsch), C8-26 - branched and linear - Pattern of Degradation (Aerobic)

Test item	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	88	79	89	69	81	45	10
	B	93	80	82	73	65	47	16
	Mean	91	80	86	71	73	46	13

Table 3         System 1, Distillates (Fischer-Tropsch), C8-26 - branched and linear - Pattern of Degradation (Sterile)

Test item	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	113	86	109	97	96	76	73
	B	111	87	111	94	96	95	67
	Mean	112	87	110	96	96	86	70

 

Table 4         System 2, Dodecane - Pattern of Degradation (Aerobic)

Reference item 1	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	40	13	13	<LOQ	<LOQ[1]	ND	<LOQ
	B	38	23	20	<LOQ	ND	ND	<LOQ
	Mean	39	18	17	na	na	na	na

Table 5         System 2, Dodecane - Pattern of Degradation (Sterile)

Reference item 1	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	31	21	30	16	<LOQ	ND	<LOQ
	B	18	21	29	13	<LOQ	ND	<LOQ
	Mean	25	21	30	15	na	na	na

 

[1]<LOQ: Less than limit of quantitation

ND:     Not detected

na:       Not applicable

Table 6         System 3, Hexadecane - Pattern of Degradation (Aerobic)

Reference item 2	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	82	59	16	10	6	ND[1]	ND
	B	85	64	17	11	6	ND	ND
	Mean	84	62	17	11	6	na	na

Table 7         System 3, Hexadecane - Pattern of Degradation (Sterile)

Reference item 2	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	88	66	61	58	57	35	39
	B	86	59	58	61	60	32	35
	Mean	87	63	60	60	59	34	37

[1]ND:     Not detected

na:       Not applicable

Table 8         System 4, Eicosane - Pattern of Degradation (Aerobic)

Eicosane	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	107	93	101	16	<LOQ[1]	<LOQ*	7
	B	108	93	106	59	4	<LOQ*	7
	Mean	108	93	104	38	4*	na*	7

Table9         System 4, Eicosane- Pattern of Disappearance (Sterile)

Eicosane	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
(% Fortification)	A	113	100	105	95	84	86	93
	B	110	107	106	89	89	91	66
	Mean	112	104	106	92	87	89	80

[*]:        Considered to be anomalous therefore not included in mean calculation or degradation curve

<LOQ: Less than limit of quantitation

Appendix 2      System 1, Distillates (Fischer-Tropsch), C8-26 - branched and linear - Pattern of Degradation– (Aerobic)

Test item	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification (mg/kg dry soil)	A	884.718	792.976	893.221	686.755	807.845	446.954	95.367
	B	933.029	796.573	823.765	726.105	652.355	470.603	159.118
	Mean	908.874	794.775	858.493	706.430	730.100	458.799	127.243

 

Appendix 3      System 1 - Pattern of Degradation – (Sterile)

Test item	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification (mg/kg dry soil)	A	1125.971	868.957	1094.172	966.820	955.129	768.790	735.327
	B	1106.448	873.027	1117.416	937.134	959.559	954.696	678.683
	Mean	1116.210	870.992	1105.794	951.977	957.344	861.743	707.005

 

Appendix 4      System 2 - Pattern of Degradation – (Aerobic)

Reference Item 1	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification   (mg/kg dry soil)	A	4.535	1.440	1.453	<LOQ	<LOQ	ND[1]	<LOQ
	B	4.345	2.577	2.298	<LOQ	ND	ND	<LOQ
	Mean	4.440	2.009	1.876	na	na	na	na

 

Appendix 5      System 2 - Pattern of Degradation – (Sterile)

Reference Item 1	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification  (mg/kg dry soil)	A	3.521	2.406	3.460	1.770	<LOQ[2]	ND	<LOQ
	B	2.040	2.372	3.238	1.473	<LOQ	ND	<LOQ
	Mean	2.781	2.389	3.349	1.622	na	na	na

 

Appendix 6      System 3 - Pattern of Degradation – (Aerobic)

Reference Item 2	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification  (mg/kg dry soil)	A	26.229	18.820	5.141	3.332	2.064	ND[3]	ND
	B	27.186	20.453	5.417	3.518	1.973	ND	ND
	Mean	26.708	19.637	5.279	3.425	2.019	na	na

 

Appendix7      System 3 - Pattern ofDegradation – (Sterile)

Reference Item 2	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification  (mg/kg dry soil)	A	28.113	21.113	19.512	18.468	18.368	11.130	12.532
	B	27.626	18.795	18.504	19.598	19.360	10.171	11.173
	Mean	27.870	19.954	19.008	19.033	18.864	10.651	11.853

 

Appendix8      System 4, Eicosane - Pattern ofDegradation – (Aerobic)

Eicosane	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification  (mg/kg dry soil)	A	29.2	25.2	27.5	4.4	<LOQ[1]	<LOQ*	2.0
	B	29.3	25.2	28.9	16.1	1.0	<LOQ*	1.9
	Mean	29.2	25.2	28.2	10.2	1.0	na	1.9

[*]:        Considered to be anomalous therefore not included in mean calculation

<LOQ: Less than limit of quantitation

Appendix9      System 4 - Pattern ofDegradation – (Sterile)

Reference Item 3	Replicate	Incubation Time (Days)
		0	1	3	7	14	28	56
Fortification  (mg/kg dry soil)	A	30.603	27.294	28.599	25.730	22.786	23.298	25.076
	B	29.925	29.171	28.667	24.294	24.387	24.628	17.893
	Mean	30.264	28.233	28.633	25.012	23.587	23.963	21.485

  

[1]<LOQ: Less than limit of quantitation

ND:     Not detected

na:       Not applicable

<LOQ: Less than limit of quantitation

[2]ND:     Not detected

na:       Not applicable

[3]ND:     Not detected

na:       Not applicable

Conclusions:

The test item (System 1) has a DT50 degradation rate, at 1000 mg/kg, of 22.4 days in aerobic soil, when incubated in the dark at 20 ± 2°C. In sterilized soil (System 1) the test item has a degradation rate, at 1000 mg/kg, of 82.6 days, when incubated in the dark at 20 ± 2°C
The reference item 1 (System 2) degradation rate could not be calculated due to the low recovery at Day 0 and the lack of time point data generated. The low recovery could be attributed to the reference item’s high volatility.
Reference item 2 (System 3) has a DT50 degradation rate, at 32 mg/kg, of 1.5 days in aerobic soil, when incubated in the dark at 20 ± 2°C. In sterilized soil System 3, reference item 2 has a degradation rate, at 32 mg/kg, of 32.9 days, when incubated in the dark at 20 ± 2°C
Reference item 3 (System 4) has a DT50 degradation rate, at 26.6 mg/kg, of 11.4 days in aerobic soil, when incubated in the dark at 20 ± 2°C. In sterilized soil System 4, reference item 3 has a degradation rate, at 26.6 mg/kg, of 122.4 days, when incubated in the dark at 20 ± 2°C
The rate of disappearance of the test item from aerobic System 1 (22.4 days) was quicker than the rate of disappearance from sterile System 1 (82.6 days). The losses from sterile System 1 is considered to be mainly via volatilization due to the chemical nature of many of the components of the test item but may also be due to losses due to bound residues. Therefore a significant amount of loss of the test item in aerobic System 1 can be considered to be attributed to microbial degradation.
Reference item 2 is more volatile than reference item 3. Losses from sterile System 3 were observed to be quicker than System 4. This data is consistent with the reference item’s relative volatility, supporting the theory that the losses of the test item (sterile System 1) observed are mainly due to volatilization.
The rate of disappearance from aerobic System 4 is quicker than aerobic System 1, however the rate of disappearance from sterile System 4 is much slower than System 1. This may be due to the test item having a toxic/ inhibiting effect on the microbes in the soil due to the relative high concentration of the test item in System 1 (1000 mg/kg) compared to the concentration of reference item 3 (26.6 mg/kg).
The rate of degradation of the test item in a single soil type incubated in the dark under aerobic conditions at 20 ± 2°C was investigated. The disappearance of the test item under aerobic conditions is rapid, with a DT50 of 22.4 days. Significant microbial degradation of the test item has been determined to occur along with losses of components of the test item via volatilization.

Executive summary:

Introduction

The purpose of this study was to determine the rate of degradation (DT₅₀and, if the data permits, a DT₇₅and a DT₉₀will also be calculated) of 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' in one soil incubated in the dark under aerobic conditions at 20 ± 2 °C. The test item was applied to a single soil to monitor the rate of degradation. Three alkanes were applied separately to a single soil to monitor the rates of degradation of single compounds compared to the test item. The degradation rates have been compared to see if they differ. Sterile soil was also treated with the test item and individual alkanes to assess any loss due to volatilization and/or bound residues. The information will be used to predict the likelihood of the substances persisting in the environment.

The work was designed to be compatible with OECD Guideline 307 for testing chemicals: Aerobic - Anaerobic Transformation in Soil (April 2002), SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation and Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.1 route of degradation, 7.1.1.2 rate of degradation, 7.1.1.2.1 laboratory studies - aerobic degradation.

Method

The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand. The freshly collected soil was supplied sieved to 2 mm. The test and reference items 1, 2 and 3 (Individual alkanes: Dodecane, Hexadecane and Eicosane respectively) were then applied to soil samples (100 g dry weight), designated System 1 to 4. The soil moisture content, of aerobic samples, was adjusted with purified water. Sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilization and/or bound residues. The treated soil samples were incubated at 20± 2 ºC in the dark. Prior to treatment and at the end of the incubation period, the microbial biomass was determined. The results show that the aerobic soil remained viable during the study and that the sterile sample remained sterile over the duration of the study.

Samples were taken from Systems 1 to 4 immediately after treatment (Day 0) and after 1, 3, 7, 14, 28 and 56 days. Each sample was extracted by solvent extraction using acetone and hexane. The extracts were then subjected to chromatographic analysis.

Results

The percentage fortification recovered was established for each sampling interval.

The total mean recovery in terms of percentage fortification immediately after treatment   (Day 0) is shown in the table below:

	Recovery at Day 0 (% Fortification)		Recovery at Day 56 (% Fortification)
	Aerobic	Sterile	Aerobic	Sterile
System 1	91	112	13	70
System 2	39	25	<LOQ	<LOQ
System 3	84	87	ND	37
System 4	108	112	7	80

The calculated DT₅₀, DT₇₅and DT₉₀values for the test and reference items are presented below:

	System 1		System 2		System 3		System 4
Disappearance Rate (days)	Aerobic	Sterile	Aerobic	Sterile	Aerobic	Sterile	Aerobic	Sterile
DT50	22.4	82.6	-	-	1.5	32.9	11.4	122.4
DT75	43.4	169.3	-	-	5.4	86.2	26.5	261.0
DT90	71.2	283.8	-	-	10.6	156.7	46.4	444.3
r^2[1]	0.955	0.940	-	-	0.807	0.673	0.921	0.741

Conclusion

The test item 'Distillates (Fischer-Tropsch), C8-26 - branched and linear' (System 1), has a DT₅₀degradation rate, at 1000 mg/kg, 22.4 days in aerobic soil, when incubated in the dark at 20 ± 2°C. In sterilized soil (System 1) after 56 days of incubation a loss of 30% was observed, when incubated in the dark at 20 ± 2°C.

Dodecane (System 2) degradation rate could not be calculated due to the low recovery at Day 0 and the lack of time point data generated. The low recovery could be attributed to the high volatility of Dodecane. However, the results indicated that the removal of the Dodecane present after soil preparation on Day 0 was quicker in the biotic treatments in comparison to the sterile controls. For example, after 7 days Dodecane in the aerobic system was below the LOQ whereas 15% of the initial nominal dose remained in the sterile treatments.

Hexadecane (System 3) has a DT₅₀degradation rate, at 32 mg/kg, of 1.5 days in aerobic soil, when incubated in the dark at 20 ± 2°C. In sterilized soil System 3, after 56 days of incubation a loss of 63% was observed, when incubated in the dark at 20 ± 2°C.

 Eicosane (System 4) has a DT₅₀degradation rate, at 26.6 mg/kg, of 11.4 days in aerobic soil, when incubated in the dark at 20 ± 2°C. In sterilized soil System 4, after 56 days of incubation a loss of 20% was observed, when incubated in the dark at 20 ± 2°C.

The rate of disappearance of the test item 'Distillates (Fischer-Tropsch), C8 -26 - branched and linear' from aerobic System 1 (22.4 days) was quicker than the extrapolated rate of disappearance from sterile System 1 (82.6 days). The losses from sterile System 1 is considered to be mainly via volatilization due to the chemical nature of many of the components of the test item but may also be due to losses due to non-extractable residues. Therefore as only 30% of the test item was lost after 56 days in the sterile system in comparison to 87% in the aerobic system a significant amount of loss of the test item in aerobic System 1 can be considered to be attributed to microbial degradation.

The results of the studies with the n-alkanes show the significance that abiotic factors have in determining the fate of such substances in OECD 307 studies. For example, even during soil dosing there were significant losses of the most volatile n-alkane (Dodecane) which continued to occur in the sterile system. Hexadecane is more volatile than Eicosane and this was also manifested in the losses from their respective sterile systems.

 

Reference 6

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-08-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Read-across is considered to be reliability 2.

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.2 rate of degradation; 7.1.1.2.1 laboratory studies - aerobic degradation.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

aerobic

Soil classification:

other: The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand.

Year:

2011

Soil no.:

#1

Soil type:

loamy sand

% Clay:

9

% Silt:

9

% Sand:

82

% Org. C:

1.6

pH:

5

CEC:

12.1 other: (meq/100 g soil)

Bulk density (g/cm³):

1.38

Details on soil characteristics:

Soil Collection
The soil was freshly sampled by Land Research Associates, Lockington, Derby, from a permanent pasture field in Ingleby, Derbyshire, UK (SK 346269 (SK 34636 26943, 52°51' 19.8” N, 1° 29' 13.9” W) on 21 July 2011 and transported to Harlan Laboratories Ltd, Shardlow, UK shortly after sampling.

Soil Preparation
Prior to transportation to Harlan Laboratories Ltd, Shardlow, UK the soil had been sieved to 2mm by Land Research Associates, Lockington, Derby. The soil was stored at approximately 5 °C between sieving and delivery. Upon receipt, the soil was stored in a refrigerator until use and was watered if needed. The soil was characterized for particle size distribution*, moisture content at water holding capacity , pH†, bulk density†, organic carbon content*, cation exchange capacity* and microbial biomass†. The soil parameters are shown in Table 1.

The soil moisture content was determined for triplicate sub-samples by oven drying and weighing. An adequate water content was obtained by adding purified water to reach final water contents of 16 g water per 100 g soil. This value corresponds to a pF value between pF2.0 and 2.5.

Sterile samples were prepared by the addition of an aliquot (1 ml) of an aqueous solution containing cycloheximide (0.02 M) and sodium azide (0.8 M) to assess any loss due to volatilization and/or bound residues.

Several days before the start of the study, the soil was conditioned to room temperature.

Determination of Soil Microbial Biomass
The microbial biomass of the soil was determined prior to treatment and at the end of the incubation period, by using a modification of the respiratory method described in ISO Guideline 14240-1 Soil quality – Determination of soil microbial biomass Part 1: Substrate induced respiration method.
Sub-samples of soil were amended with glucose and incubated at 22 ± 1oC in a CES Multi-channel Aerobic Respirometer. The total volume of carbon dioxide evolved per hour was calculated and from this the microbial biomass was determined.

Soil No.:

#1

Duration:

56 d

Soil No.:

#1

Initial conc.:

100 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#1

Temp.:

20

Details on experimental conditions:

Experimental Conditions

Aerobic & Sterile Test System
Samples of 100 g of soil, based on dry weight, were incubated under aerobic conditions in all-glass flasks in the dark. The flasks were fitted with a polyurethane bung which freely allowed air to pass into the systems.

Each test system was uniquely identified.

Temperatures
The soil samples were incubated in air-conditioned rooms at a temperature of 20 ± 2 °C. The temperatures were continuously monitored.

Moisture Content
Aerobic systems
The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added where necessary.

Sterile Systems
The soil moisture content was maintained at between pF2.0 and 2.5. Samples were weighed periodically during the incubation period to determine the amount of water lost by evaporation. To compensate for this loss, an amount of sterilization solution (an aqueous solution of cycloheximide and sodium azide) equal to that lost by evaporation was added where necessary.

Treatment and Sampling
Rationale for the Application Rate
The aim was to apply the test item to aliquots of fresh soil (100 g dry weight) at the target rates of 1000 mg/kg dry soil.

Preparation of the Application Solution
The test item was supplied as a liquid. An application solution was prepared by diluting a nominal weight of test item (5 g) in 50 ml hexane. The nominal application solution concentration was 100000 mg/l.

An aliquot (1 ml) of the application solution was calculated to be applied to each sample to reach the target concentration (1000 mg/kg dry soil).

Treatment of the Test System
Aliquots were applied evenly onto the soil surface. The total amount of organic solvent added to the samples was approximately 1 % v/w.
Synthetic control samples and samples for the determination of the microbial biomass, shared with Harlan Laboratories Ltd. project number 41101425, were treated with 1 ml of acetone and incubated under the same conditions as the treated samples. The solvent used for the synthetic control was not the same as the test systems as the controls were shared with Harlan Laboratories Ltd. project number 41101425. The test item was found to be insoluble in acetone. The use of a differing solvent in the synthetic controls was considered to have no significant adverse effect on the integrity of the results.

After treatment polyurethane bungs were placed in to the neck of the flasks and were incubated in an air-conditioned room.

Soil Sampling
Duplicate test samples of aerobic, sterile and a single synthetic control sample were taken for extraction and analysis immediately after administration (Day 0), and after 1, 3, 7, 14, 28 and 56 days.

Extraction of test item from Soil
Each sample was subjected to the following extraction procedure: Each vessel was emptied into a vessel and homogenised by placing on a flat bed shaker for 10 minutes at approximately 320 rpm.

An aliquot (approximately 5.0 g) of the homogenised soil sample was added to a 50 ml glass centrifuge tube. The soil was chemically dried by adding approximately 5 g of anhydrous sodium sulphate to the soil and shaking for 5 minutes, using a flat bed shaker at approximately 320 rpm.
The dried soil was extracted by adding acetone (4 ml) and ultrasonicated for 2 minutes at approximately 20°C. Hexane (4 ml) was then added to the soil and the samples were ultrasonicated for a further 10 minutes at approximately 20 °C.

The soil was then centrifuged for 5 minutes at 1000 rpm. The resulting liquid layer was removed via pipette and passed through glass wool into a 50 ml measuring cylinder. The extraction step from the addition of acetone (4 ml) onwards was then repeated.

The soil was further extracted by adding acetone/hexane (1:1) (10 ml) to the soil cake and ultrasonicated for 15 minutes at approximately 20 °C. The soil was then centrifuged for 5 minutes at 1000 rpm. The resulting liquid layer was removed via pipette, passed through glass wool and combined in the same 50 ml measuring cylinder. The extraction step from the addition of acetone/hexane (1:1) (10 ml) onwards was then repeated.

The glass wool were rinsed with approximately 3 ml of acetone/hexane (1:1) and the final volume was adjusted to 40 ml. The combined extracts, shaken manually for 30 seconds, were quantified by chromatographic analysis.

Calculations
The calculations were performed by means of a commercially available computer program (Microsoft Excel, 2007). The results given in the tables are rounded numbers. Thus, hand calculations may differ slightly from those presented.

The amount of test item found in the soil samples was expressed in mg/kg dry soil. The amount of test item was also expressed as percentage of the application rate (% Fortification) used to calculate the rate of disappearance of the test item. The following two equations were employed:

% Fortification = (mg in extract) / (mg applied) x 100
mg/kg = (mg in extract) / (weight of dry soil g ) x 1000

Calculation of DT50, DT75 and DT90 Values
The rate of disappearance of the test item under aerobic conditions was calculated by nonlinear regression assuming Single First-Order kinetics.

Determination of the Microbial Biomass
The microbial biomass was determined prior to treatment and at the end of the incubation period, by using a modification of the respiratory method described in ISO Guideline 14240-1 Soil quality – Determination of soil microbial biomass Part 1: Substrate induced respiration method.

Soil samples amended with glucose were placed in a CES Multi-channel Aerobic Respirometer. The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current are proportional to the amount of oxygen supplied to the micro-organisms.

Using the oxygen data generated, the volume of carbon dioxide evolved per hour per 100 g of soil was calculated and the microbial biomass determined.

Soil No.:

#1

% Recovery:

33

Remarks on result:

other: Aerobic

Soil No.:

#1

% Recovery:

99

Remarks on result:

other: Sterile

Soil No.:

#1

% Degr.:

67

Parameter:

test mat. analysis

Remarks:

Aerobic

Sampling time:

56 d

Soil No.:

#1

% Degr.:

1

Parameter:

test mat. analysis

Remarks:

Sterile

Sampling time:

56 d

Soil No.:

#1

DT50:

35.8 d

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C Aerobic

Soil No.:

#1

DT50:

231.4 d

Type:

other: DT90 (d)

Remarks on result:

other: DT90 (d) at 20 °C Aerobic

Soil No.:

#1

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C Sterile

Soil No.:

#1

DT50:

> 1 yr

Type:

other: DT90 (d)

Remarks on result:

other: DT90 (d) at 20 °C Sterile

Transformation products:

no

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

Details on results:

RESULTS
Rate of Test Item Degradation
The results are summarised in Tables 2 and 3 in terms of percentage of fortification and in Appendix 2 and 3 in mg equivalents per kg dry soil. The rate of degradation is shown graphically in Figure 1 and 2.

The percentage fortification was established for each sampling interval. On the whole, duplicate samples gave similar results; therefore the results in the following sections are expressed as mean values.

The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) and Day 56 is shown in the table below:

Recovery at Day 0 (% Fortification) Recovery at Day 56 (% Fortification)
Aerobic Sterile Aerobic Sterile
102 96 33 99

The measured amounts of the test item were subjected to first-order reaction kinetics. The following DT50, DT75 and DT90 values were calculated for the test item:

1000 mg/kg
Disappearance
Rate (days) Aerobic Sterile
DT50 35.8 > 1 Yr
DT75 70.4 > 1 Yr
DT90 231.4 > 1 Yr
r2 0.987 0.072

Microbial Biomass
The microbial biomass of the aerobic soil was determined to be 14.80 mg C/ 100 g dry soil prior to the start of incubation. At the end of the incubation period, the microbial biomass of the aerobic soil was determined to be 17.80 mg C/ 100 g dry soil (Table 1). These results demonstrate that the aerobic soil remained viable during the study.

The results for the sterile soil at the beginning and end of the study showed no microbial activity.

Table1              Soil Characteristics

 

Parameters	Soil
Site location:	Ingleby (Site B2)
Batch:	EF75
Soil characteristics:
pH	5.00[1]
Cation exchange capacity (meq/100g)	12.1^†
Organic carbon (% w/w)	1.6^†
Bulk Density (g/ml)	1.38*
Soil type (according to USDA)	Loamy Sand
^[2]Particle size analyses (% w/w)USDA^[3]:
2.00-0.050 mm (Sand)	82
0.050-0.002 mm (Silt)	9
< 0.002 (Clay)	9
†MWHC^[4](% w/w) at pF 2.0	16.2
Microbial Biomass (mg C/100 g):
Start of incubation	14.80*
End of incubation	17.80*

 

Table2         Pattern ofDegradation of the GTL base oil 3 (Aerobic)

1000 mg/kg Aerobic		Replicate		Incubation Time (Days)
			0		1		3		7		14		28		56
Amount of Test Item                 (% Fortification)		A		37[5]		106		100		90		80		64		33
		B		102		97		101		89		75		68		ND*
		Mean		102		102		101		90		78		66		33

Table3         Pattern ofDegradation of the GTL base oil 3 (Sterile)

1000 mg/kg Sterile		Replicate		Incubation Time (Days)
			0		1		3		7		14		28		56
Amount of Test Item                 (% Fortification)		A		98		101		96		103		100		91		99
		B		93		104		101		100		102		97		98
		Mean		96		103		99		102		101		94		99

 

[1]Data shared with Harlan Laboratories Ltd project number 41101425

^[2]Parameters as determined by CEM Analytical Services, Glendale Park, Fernbank Road, North Ascot,  Berkshire, UK (Study Number: CEMS-5196)

^[3]According to USDA soil texture classification system

^[4]Moisture content at water holding capacity

[5]Considered to be anomalous therefore excluded from mean.

Conclusions:

CONCLUSION
The rate of degradation of the test item in a single soil type incubated in the dark under aerobic conditions at 20 ± 2°C was investigated. The sterile soil showed minimal loss of test item via volatilization or binding to the soil. Therefore the losses observed in the aerobic soil can be considered to be attributed to microbial degradation.

The test item has a DT50 degradation rate, at 1000 mg/kg, of 35.8 days in aerobic soil, incubated in the dark at 20 ± 2°C. In sterilized soil the test item showed no significant degradation and the degradation rate at 1000 mg/kg was predicted to be greater than 1 year when incubated in the dark at 20 ± 2°C.

Executive summary:

Introduction.The purpose of the study was to determine the rate of degradation of the test item in one soil incubated in the dark under aerobic conditions at20 ± 2°C.The test item was treated to a single soil and the rate of degradation monitored. Sterile soil was also treated with test item to assess any loss due to volatilization and/or bound residues. The information will be used to predict the likelihood of the substance persisting in the environment.

The work was designed to be compatible withOECD Guideline 307 for testing chemicals: Aerobic - Anaerobic Transformation in Soil (April 2002), SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation and Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.1 route of degradation, 7.1.1.2 rate of degradation, 7.1.1.2.1 laboratory studies - aerobic degradation.

Method.The following soil was used for the study: Site B2 Ingleby, Derbyshire; Loamy Sand.

The freshly collected soil was supplied sieved to 2 mm.The test item was then applied to soil samples (100 g dry weight) at a concentration of approximately 1000 mg/kg.The soil moisture, of aerobic samples, content was adjusted with purified water. Sterile samples were prepared by the addition of an aqueous solution of cycloheximide and sodium azide to assess any loss due to volatilization and/or bound residues.The treated soil samples were incubated at 20± 2 ºC in the dark. Prior to treatment and at the end of the incubation period, the microbial biomass was determined. The results show that the aerobic soil remained viable during the study and that the sterile sample remained sterile over the duration of the study.

Samples were taken from each soil immediately after treatment with test item (Day 0) and after 1, 3, 7, 14, 28 and 56 days. Each sample was extracted by solvent extraction using acetone and hexane. The extracts were then subjected to chromatographic analysis.

Results.The total mean recovery in terms of percentage fortification immediately after treatment (Day 0) and Day 56 is shown in the following table:

Recovery at Day 0 (% Fortification)		Recovery at Day 56 (% Fortification)
Aerobic	Sterile	Aerobic	Sterile
102	96	33	99

The measured amounts of the test item were subjected to first-order reaction kinetics. The followingDT₅₀, DT₇₅and DT₉₀values were calculated for the test item:

	1000 mg/kg
Disappearance Rate (days)	Aerobic	Sterile
DT50	35.8	> 1 Yr
DT75	70.4	> 1 Yr
DT90	231.4	> 1 Yr
r^2[1]	0.987	0.072

Conclusion.The rate of degradation of the test item in a single soil type incubated in the dark under aerobic conditions at 20 ± 2°C was investigated. The sterile soil showed minimal loss of test item via volatilization or binding to the soil. Therefore the losses observed in the aerobic soil can be considered to be attributed to microbial degradation.

The test item has a DT₅₀degradation rate, at 1000 mg/kg, of 35.8 days in aerobic soil, incubated in the dark at 20 ± 2°C. In sterilized soil the test item showed no significant degradation and the degradation rate at 1000 mg/kg was predicted to be greater than 1 year when incubated in the dark at 20 ± 2°C.

[1]        :Coefficient of determination

> 1 Yr  :Greater than 1 year based on extrapolation

 

Additional information

The available measured data for degradation in soil indicate that after 51 days contact time, constituents of GTL Gasoil were not detectable. It was not firmly established whether this is due to biodegradation, loss by evaporation or that the constituents were irreversibly bound to the soil matrix. Scientific judgment would suggest that it is probably a combination of all three. Similarly, studies with full-range GTL Base Oil Distillates and GTL Base Oil 3 indicated a significant degree of test substance removal, most of which can be attributed to biodegradation processes.

Since the constituents of Hydrocarbons, C18-C24, isoalkanes, <2% aromatics are within the carbon number range covered by the read across substances, it can therefore be concluded, based on the available weight of evidence, that a significant degree of removal, including biodegradation, will occur in soil.