Paraffin waxes (Fischer-Tropsch), isomerization

Biodegradation in soil

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-20 - 2012-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

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 (incl. QA statement)

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:

2012

Details on soil characteristics:

The soil was freshly sampled 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 13 February 2012 and transported to the laboratory shortly after sampling.

Prior to transportation to the laboratory the soil had been sieved to < 2mm. 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 characteristics are shown in Table 1 (see "Any other information on results incl. tables").

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 approximately 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 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:

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 recovery samples were inconsistently poor. Therefore the study was not performed at the lower test level. This was considered to have no adverse effect on the integrity of the study.

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 (10 g) in 100 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 were treated with 1 ml of hexane 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 of aerobic, sterile and a single synthetic control sample were taken for extraction and analysis immediately after administration (Day 0), and after 7, 14, 27, 59, 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, approximately 57.5g anhydrous sodium sulphate was added and the entire sample was homogenised by placing on a flat bed shaker for 10 minutes at approximately 300 rpm.

An aliquot of soil (7.5 g) was weighed into glass vessels. Tetrachloroethylene (10 mL) was then added to the soil and the samples were shaken for a 30 minutes at approximately 200 rpm. The soil was allowed to settle and the supernatant was removed and passed though glass wool into a 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 a 30 minutes at approximately 200 rpm. The soil was allowed to settle and the supernatant was removed and filtered though glass wool into a volumetric.

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

Values 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

% Degr.:

ca. 85

Parameter:

test mat. analysis

Remarks:

Biotic

Sampling time:

120 d

Soil No.:

#1

% Degr.:

ca. 46

Parameter:

test mat. analysis

Remarks:

Sterile

Sampling time:

120 d

Soil No.:

#1

DT50:

ca. 35.6 d

Type:

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

Remarks on result:

other: Biotic

Soil No.:

#1

DT50:

ca. 101.9 d

Type:

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

Remarks on result:

other: Sterile

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 Disappearance
The results are summarised in Tables 2 and 3 in terms of percentage of fortification (see "Any other information on results incl. tables"). The rate of disappearance is shown graphically in Figure 1 and 2 (see "Attached background material").

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, with the exception of sterile day 0.

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)
Biotic Sterile Biotic Sterile
98 105 15 54

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 35.6 101.9
DT75 72.1 217.4
DT90 120.3 > 1 Yr
r2 0.893 0.889

Microbial Biomass
The microbial biomass of the biotic soil was determined to be 8.12 mg C/ 100 g dry soil prior to the start of incubation. At the end of the incubation period, the microbial biomass of the biotic soil was determined to be 10.98 mg C/ 100 g dry soil (Table 1, see "Any other information on results incl. tables"). 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.

Tables

Table1     Soil Characteristics

Parameters	Soil
Site location:	Ingleby (Site B2)
Batch:	EF76
Soil characteristics:
pH	5.21
*Cation exchange capacity (meq/100g)	8.5
*Organic carbon (% w/w)	1.1
Bulk Density (g/ml)	1.32
*Soil type (according to USDA)	Loamy Sand
[1]*Particle size analyses (% w/w)USDA[2]**:
2.00-0.050 mm (Sand)	82
0.050-0.002 mm (Silt)	9
< 0.002 (Clay)	10
*5MWHC[3](% w/w) at pF 2.0	15.7
Microbial Biomass (mg C/100 g):
Start of incubation	8.12*
End of incubation	10.98*

 

Table2     Pattern of Disappearance of the Test Item (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

 

Table3     Pattern of Disappearance of the Test Item (Sterile)

1000 mg/kg Sterile	Replicate	Incubation Time (Days)
		0	7	14	27	59	90	120
Amount of Test Item                 (% Fortification)	A	115[4]	90	92	82	78	47	48
	B	105	85	91	75	69	52	60
	Mean	105	88	92	79	74	50	54

 

^*      Parameters as determined by CEM Analytical Services, Glendale Park, Fernbank Road, North Ascot,  Berkshire, UK (Study Number: CEMS-5391)

^**[2]       According to USDA soil texture classification system

MWHC[3]= Moisture content at water holding capacity

[4]Result not included in mean calculation as considered to be anomalous

Conclusions:

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

Recovery at Day 0 (% Fortification) Recovery at Day 120 (% Fortification)
Biotic Sterile Biotic Sterile
1000 mg/Kg 98 105 15 54

The measured amounts of the test item were subjected to first-order reaction kinetics. The following DT50, DT75, DT90 and r2 values were calculated for the test item:

1000 mg/kg
Disappearance
Rate (days) Aerobic Sterile
DT50 35.6 101.9
DT75 72.1 217.4
DT90 120.3 > 1 Yr
r2 0.893 0.889

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

As the microbial activity measurements confirmed that sterility was maintained, the losses from the sterile system were due to abiotic factors. These were considered to be mainly via binding to the soil since, due to the physical chemical nature of the test item, these losses were unlikely to be via volatilization. The results indicate that microbial degradation plays a significant role in removing the test item in the biotic soil.

The test item has a DT50 disappearance rate, at 1000 mg/kg, of 35.6 days in aerobic soil, incubated in the dark at 20 ± 2 °C. In sterilized soil the test item has a disappearance rate, at 1000 mg/kg, of 101.9 days, 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 GTL base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'] in one soil incubated in the dark under aerobic conditions at 20 ± 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 OECD 307 test was designed to assess the degradation rate of single chemicals in soil. However, for complex substances the test will in effect measure the rate of disappearance of the test item. This is because some components may be lost by abiotic factors and not biodegradation per se.

 

Methods

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 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 each soil immediately after treatment with test item (Day 0) and after 7, 14, 27, 59, 90 and 120 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 120 is shown in the following table:

 

	Recovery at Day 0 (% Fortification)		Recovery at Day 120 (% Fortification)
	Biotic	Sterile	Biotic	Sterile
1000 mg/Kg	98	105	15	54

 

The measured amounts of the test item were subjected to first-order reaction kinetics. The following DT50, DT75, DT90 and r² values were calculated for the test item:

	1000 mg/kg
Disappearance Rate (days)	Aerobic	Sterile
DT50	35.6	101.9
DT75	72.1	217.4
DT90	120.3	> 1 Yr
r^2[1]	0.893	0.889

[1] r²=Coefficient of determination

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

As the microbial activity measurements confirmed that sterility was maintained, the losses from the sterile system were due to abiotic factors. These were considered to be mainly via binding to the soil since, due to the physical chemical nature of the test item, these losses were unlikely to be via volatilization. The results indicate that microbial degradation plays a significant role in removing the test item in the biotic soil.

 The test item has a DT50 disappearance rate, at 1000 mg/kg, of 35.6 days in aerobic soil, incubated in the dark at 20 ± 2 °C. In sterilized soil the test item has a disappearance rate, at 1000 mg/kg, of 101.9 days, incubated in the dark at 20 ± 2 °C.

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

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

Remarks:

read-across from closely related Gas-to-liquids (GTL) product [covering the low molecular weight fraction of the registration substance, C18-30]

Adequacy of study:

supporting study

Study period:

2011-08-23 - 2011-12-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study on supporting 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

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 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 laboratory shortly after sampling.

Soil Preparation
Prior to transportation to the laboratory the soil had been sieved to 2mm. 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 characteristics are shown in Table 1 (see "Any other information on results incl. tables").

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 (see "Any other information on results incl. tables", 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^[2]
Organic carbon (% w/w)	1.6 ^[2]
Bulk Density (g/ml)	1.38^[1]
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
^[2] MWHC^[4](% w/w) at pF 2.0	16.2
Microbial Biomass (mg C/100 g):
Start of incubation	14.80 ^[1]
End of incubation	17.80 ^[1]

[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.

Table2         Pattern of Degradation of the test material (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

* considered to be anomalous therefore excluded from mean

Table3         Pattern of Degradation of the test material (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

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 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 at 20 ± 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 following DT₅₀, 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

Description of key information

- test material GTL Base oil ['Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'; CAS 848301-69-9, EC 482-220-0] [OECD 307]: indicative half-life DT₅₀ in biotic system at 1000 mg/kg, of 35.6 days (at 20 ± 2°C) compared to predicted DT₅₀ of 101.9 days in a sterile system (one soil type only);

- longer biodegradation half-lives in soil can be expected for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' containing higher molecular weight constituents (C25-150, about 30-55 % >C50). 

Additional information

Soil biodegradability data are not available for the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' itself.

The available measured data on degradation in soil indicate that after 51 days contact time, constituents of the closely related substance GTL base oil (C18-C50; CAS 848301-69-9, EC 482-220-0) 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.

The results indicate that a significant degree of test substance removal occurs, most of which can be attributed to biodegradation processes.

The results should also apply to the substance 'Paraffin waxes (Fischer-Tropsch), isomerization' containing higher molecular weight constituents (C25 - C150, about 30-55 % >C50), only longer biodegradation half-lives in soil are to be expected for it. 

It is safe to assume that biodegradability in soil - that is the metabolic ability of microorganisms to transform or mineralize hydrocarbons into less harmful, non-hazardous substances - is influenced by complex arrays of factors, such as nutrients, oxygen, pH value, concentration and bioavailability of the individual costituents, and the soil’s chemical and physical characteristics. A published review is available that summarises the major metabolic pathways for biodegradation of alkanes by microorganisms (see IUCLID section 5.6) and is relevant and useful for consideration of the primary degradation step for constituents of the registration substance. Under aerobic conditions, which are of most significance for conventional understanding of persistence and exposure, oxidation is understood to be the major breakdown mechanism and various microbial alkane hydroxylase enzymes are of particular significance. In general, the initial activation to form a primary alcohol is the first step, followed by oxidation to the aldehyde and subsequently the carboxylic acid. The carboxylic acids undergo β-oxidation. Oxidation may occur at both ends of longer chains, creating a ω-hydroxy fatty acid. Degradation of very long chains and branched structures is a more specialised process, but several bacterial strains are known to degrade such structures.