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

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was performed between 01 October 2009 and 11 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling and analysis
On days 0, 7 and 28 triplicate control and six replicate test item vessels were sacrificed for nitrate analysis.
The contents of the test vessels were transferred to polyethylene bottles and an aliquot (250 ml) of potassium chloride (0.1M) added. The mixtures were then shaken (150 rpm, 60 minutes) using an INFORS TR-225 orbital platform shaker prior to removal of the aqueous phase by filtration (0.45 µm).
To an aliquot (35 ml) of acid mixture* was added 5 ml of the soil filtrate followed by 5 ml of 2,6-dimethylphenol solution**. The mixture was thoroughly mixed and allowed to stand for between 12 and 20 minutes prior to determination of the absorbance at 324 nm.

Vehicle:

no

Details on preparation and application of test substrate:

Test item preparation
To prepare the test series, an amount of test item (1770 µl equivalent to 1500 mg) was added to 15 g of quartz sand in a glass jar and mixed using an industrial mixer. After 24 hours the mixing was stopped and a final bulk soil weight of 1500 g* was added to each container prior to mixing for a further 3 hours. After this process, an amount of powdered Lucerne-green-grass-meal (7.5 g) and an aliquot (53.1 ml) of water was then separately added to the soil/sand/test item combination and thoroughly mixed (Kenwood Chef Mixer) for 5 minutes to give the required test concentration of 1000 mg/kg with a nominal moisture content of 40% of the Water Holding Capacity (WHC). The soil for the test concentration was then split into 18 replicates for incubation.
The control soil was prepared in an identical manner but not exposed to the test item. The soil for the control was then split into 9 replicates for incubation.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.  This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

GLP COMPLIANCE STATEMENT
With the exception noted below the work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception was considered not to affect the purpose or integrity of the study.

Exposure conditions
The soil samples were incubated in glass beakers, each containing 50 g* of soil. The test vessels were covered with loosely fitting lids in order to minimise moisture loss by evaporation and maintained in a temperature controlled room at approximately 21oC, in darkness.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

The test vessels were covered with loosely fitting lids in order to minimise moisture loss by evaporation and maintained in a temperature controlled room at approximately 21oC, in darkness.

Moisture:

The moisture content of the soil was determined prior to the start of the definitive test by drying an amount of soil (100.00 g) at approximately 110oC until a constant weight was obtained. The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 11%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.1 g/100g (see Appendix 1) and hence 17.7 ml of deionised reverse osmosis water per 0.5 kg* of soil was added. This gave a final water content of
14.04 g/100 g i.e. 40% of the WHC as recommended by the Test Guidelines.

Details on test conditions:

Soil Details
The soil for the range-finding and definitive tests was obtained on 8 April 2010 from LUFA Speyer, Obere Langgasse 40, 67346 Speyer, Germany (see attached Appendix 1). The sampling site had not been treated with crop protection products or organic fertiliser for at least 3 years prior to sampling (data from supplier).
The pH of the soil in water was 7.4, with an organic carbon content of 0.97 ± 0.07%. The sand content of the soil was measured to be 59.6 % (see attatched Appendix 1) and so satisfied the recommendations of the Test Guidelines.

On arrival of the soil at Harlan Laboratories Ltd., the soil was stored at approximately 4ºC prior to use. Prior to the start of the range-finding test, an amount of soil (approximately 2.5 kg) was removed and stored at approximately 21ºC for 3 days and shielded from light. Prior to the start of the definitive test, an amount of soil (approximately 3 kg) was removed and stored at approximately 21ºC for 4 days and shielded from light.

Preliminary Work
The test item was a poorly water soluble, non-viscous liquid. Therefore in order to ensure accurate addition of the test item, for the purpose of the test, the test item was added directly to the test vessels using an Eppendorf pipettor.
Preliminary work conducted showed that a volume of 5.9 µl of test item, measured using an Eppendorf pipetter gave a measured weight of 5.0 mg (mean of 15 separate weighings).
Additionally, preliminary work also showed that volumes of 59 and 590 µl of test item, measured using an Eppendorf pipetter gave measured weights of 50 and 500 mg respectively (mean of 15 separate weighings).
Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. In the range-finding test soil microorganisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg for a period of 28 days.

To prepare the test series, amounts of test item (5.9, 59 and 590 µl equivalent to 5.0, 50 and 500 mg) were each added separately to 5 g of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours the mixing was stopped and a final bulk soil weight of 500 g* was added to each container prior to mixing for a further 3 hours. After this process, an amount of powdered Lucerne-grass-green-meal (2.5 g) and an aliquot (19.8 ml) of water was then separately added to each soil/sand/test item combination and thoroughly mixed (Kenwood Chef Mixer) for 5 minutes to give the required test concentrations of 10, 100 and 1000 mg/kg with a nominal moisture content of 40% of the Water Holding Capacity (WHC). Lucerne-grass-green meal was added to act as respirometry substrate.
The control soil was prepared in an identical manner but not exposed to the test item.
The range-finding test was conducted in glass beakers, each containing 50 g* of soil, and sealed with loosely fitting lids in order to minimise moisture loss by evaporation. Four replicate vessels were prepared for each control and test concentration.
At the start of the range-finding test two replicate control and test item vessels for each concentration were sacrificed for nitrate analyses. The remainder of the vessels were then sealed with loosely fitting lids and maintained in a temperature controlled room at approximately 21ºC, in darkness, for 28 days.
After 28 days the nitrate concentration in each control and test vessel was determined.
Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/kg to confirm that at this concentration no effect on nitrification activity was observed.

* Dry Weight

Nominal and measured concentrations:

Following a preliminary range-finding test soil microorganisms were exposed to the test item at a single concentration of 1000 mg/kg for 28 days

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil ww

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95 % Cl not reported

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil ww

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95 % CL limits not recorded

Details on results:

RESULTS
Range-finding Test
Absorbance values, nitrate concentrations and percentage inhibition of nitrogen transformation activity determined during the range-finding test are given in Table 1.
The results showed no significant effect on nitrogen transformation activity.
Based on this information a single test concentration of 1000 mg/kg soil was selected for the definitive test. This experimental design conforms to a “limit test” to confirm that at the maximum recommended concentration given in the Test Guidelines no effect on nitrogen transformation was observed.
Definitive Test
Absorbance values and nitrate concentrations determined on each sampling day are given in Table 2. The nitrate formation rates and percentage inhibition of nitrogen transformation activity on days 7 and 28 are given in Table 3.
The microbial biomass of the soil was shown to be 134 µg C/g (ISO 14240-2: 1997). This was equivalent to 1.4% of the total soil organic carbon content.
The variation between replicate control nitrification rates was less than 15% and therefore satisfied the validation criterion given in the Test Guidelines.
Accordingly the following results were determined from the data:
EC10 (28 days): >1000 mg/kg
EC25 (28 days): >1000 mg/kg
EC50 (28 days): >1000 mg/kg

where ECx is the test concentration that reduced soil nitrogen transformation activity by x %.
Statistical analysis of the nitrate concentration values was carried out for the control and test item group and showed no statistically significant decreases between the control and 1000 mg/kg test concentration (P≥0.05). Therefore the ‘No Observed Effect Concentration’ (NOEC) was 1000 mg/kg.
Temperature was maintained at approximately 21ºC throughout the test and the moisture content of each control and test vessel was checked on days 7, 14, 21 and 28 to ensure that the moisture content of the soil was maintained to 40% ± 5% of the WHC of the soil.
The mean nitrate formation rate in the range-finding test was significantly higher in the 1000 mg/kg test concentration than in the control; however inthe definitive test the mean nitrate formation rate in the control and 1000 mg/kg test concentration were similar.
The results in Tables 1 and 2 also show a significant increase in the nitrate formation rate between the control and 1000 mg/kg test vessels prepared in the range-finding and definitive test.  This increase may be due to the soil having been stored between the range-finding and definitive tests. The difference in nitrogen transformation rates between the range-finding and definitive tests is considered to have had no adverse effect on the study given that in both tests no inhibition of the nitrogen transformation rate between the control and test group was observed.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical analysis
A Students t-test was carried out on the nitrate concentration values on Day 28 for the control and the 1000 mg/kg test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table 1: Absorbance Readings, Nitrate Concentrations and Inhibition of Nitrogen Transformation Activity from the Range-finding Test

Nominal Concentration (mg/kg soil)		Day 0		Nominal Concentration (mg/kg soil)		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)			Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Mean Nitrate Formation Rate (mgNO3/kg/day)	% Inhibition
Control	R1	0.245	88.35	Control	R3	0.359	128.50	1.33	1
	R2	0.244	88.00		R4	0.342	122.50
10	R1	0.242	87.30	10	R3	0.359	128.50	1.52	[14]
	R2	0.235	84.85		R4	0.360	128.85
100	R1	0.251	90.50	100	R3	0.367	131.30	1.59	[20]
	R2	0.248	89.40		R4	0.385	137.65
1000	R1	0.245	88.35	1000	R3	0.615	218.55	5.01	[277]
	R2	0.248	89.40		R4	0.675	239.70

 

*Corrected for blank absorbance value

[Increase in nitrate formation rate as compared to controls]

 R₁-R₄= Replicates R₁to R₄

Table 2: Absorbance and Nitrate Concentrations from the Definitive Test

Nominal Concentration (mg/kg soil)		Day 0
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.304	109.15
	R2	0.295	105.95
	R3	0.291	104.55
	Mean	-	106.55
	CV%	-	2
1000	R1	0.313	112.30
	R2	0.306	109.85
	R3	0.310	111.25
	R4	0.295	105.95
	R5	0.306	109.85
	R6	0.307	110.20
	Mean	-	109.90
	CV%	-	2

 

Nominal Concentration (mg/kg soil)		Day 7
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R4	0.543	193.25
	R5	0.530	188.65
	R6	0.510	181.60
	Mean	-	187.33
	CV%	-	3
1000	R7	0.524	186.55
	R8	0.554	197.10
	R9	0.505	179.85
	R10	0.519	184.80
	R11	0.534	190.05
	R12	0.515	183.40
	Mean	-	186.96
	CV%	-	18

*Corrected for blank absorbance value

R₁-R₁₂= Replicates 1 to 12

CV = Coefficient of Variation

Table 2 (continued): Absorbance Readings and Nitrate Concentrations from the Definitive Test

Nominal Concentration (mg/kg soil)		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R7	1.194	422.30
	R8	1.188	420.20
	R9	1.192	421.60
	Mean	-	421.37
	CV%	-	<1
1000	R13	1.192	421.60
	R14	1.198	423.75
	R15	1.193	421.95
	R16	1.175	415.65
	R17	1.232	435.70
	R18	1.199	424.10
	Mean	-	423.79
	CV%	-	2

*Corrected for blank absorbance value

 R₇-R₉= Replicates 7 to 9

 R₁₃-R₁₈= Replicates 13 to 18

CV = Coefficient of Variation

Table 3: Inhibition of Nitrogen Transformation Activity

Nominal Concentration (mg/kg)	Day 7		Day 28
	Nitrate Formation Rate (mg NO3/kg/day)	% Inhibition	Nitrate Formation Rate (mg NO3/kg/day)	% Inhibition
Control	11.61	-	11.24	-
1000	11.01	5	11.21	0

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the nitrogen transformation activity of soil microorganisms has been investigated over a 28-Day period and gave an EC50 value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the long-term effect of the test item ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' after a single exposure, on the nitrogen transformation activity of soil microorganisms. The method followed that described in the OECD Guidelines for Testing of Chemicals (2000) No 216 “Soil Microorganisms: Nitrogen Transformation Test” referenced as Method C.21 of Commission Regulation (EC) No. 440/2008; EPPO (1994) Decision Making Scheme for the Environmental Risk Assessment of Plant Protection Products, Chapter 7: Soil Microflora, EPPO Bulletin 24 and SETAC-Europe (1995) Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides.

Methods.

Following a preliminary range-finding test soil microorganisms were exposed to the test item at a single concentration of 1000 mg/kg for 28 days at a temperature of approximately 21 ºC, in the dark with the addition of powdered Lucerne-green-grass meal to act as a respiratory substrate.

The inhibitory effect of the test item on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples on Days 0, 7 and 28 and compared to data obtained from control soil samples.

Results.

The effect of the test item on the nitrogen transformation activity of soil microorganisms has been investigated over a 28-Day period and gave an EC₅₀ value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

CONCLUSION

The effect of the test item on the nitrogen transformation activity of soil microorganisms has been investigated over a 28-Day period and gave an EC₅₀ value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Reference 2

Endpoint:

toxicity to soil microorganisms

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) is 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 substances contain 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 7.3 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 toxicity profiles. There is no evidence of toxicity in the studies on the source substances, and no evidence of toxicity in studies on hydrocarbons with shorter carbon chain length and a narrower range of carbon number, therefore there is no evidence for mixture effects or for increased toxicity with increased concentration.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

320 mg/kg soil ww

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

180 mg/kg soil ww

Basis for effect:

nitrate formation rate

Reference 3

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 01 October 2009 and 24 March 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with one minor deviations from standard test guidelines and/or minor methodological deficiencies, which does 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. The deviation was as follows: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception was considered not to affect the purpose or integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

On days 0, 7 and 28 triplicate control and test item vessels were sacrificed for nitrate analysis.

The contents of the test vessels were transferred to 500 ml polyethylene bottles and an aliquot (250 ml) of potassium chloride (0.1M) added. The mixtures were then shaken (150 rpm, 60 minutes) using an INFORS TR-225 orbital platform shaker prior to removal of the aqueous phase by filtration (0.45 µm).

To an aliquot (35 ml) of acid mixture* was added 5 ml of the particle free extract followed by 5 ml of 2,6-dimethylphenol solution**. The mixture was thoroughly mixed and allowed to stand for between 10 and 19 minutes prior to determination of the absorbance at 324 nm.

A calibration curve was prepared by measuring absorbance values at 324 nm of standard solutions of potassium nitrate at the following concentrations: 1.0, 5.0, 10, 15, 20 and 25 mg NO3- N/l. The standard solutions were treated in the same manner as the test samples. Linear regression analysis of the standard curve data produced an equation for the best-fit line into which the test sample extract absorbance values were substituted to determine the nitrate nitrogen concentration.

The concentration of nitrate (mg/l) in the test sample extracts was obtained by multiplying the nitrate nitrogen concentration (mg/l) by a factor of 4.427.

* 250 ml 98% sulphuric acid, 250 ml 85% orthophosphoric acid and 20 mg amidosulfonic acid

** 300 mg 2,6-dimethylphenol dissolved in 250 ml glacial acetic acid (99.79%)

Vehicle:

no

Details on preparation and application of test substrate:

Soil Details
The soil for the range-finding and definitive tests was obtained on 4 January 2010 from LUFA Speyer, Obere Langgasse 40, 67346 Speyer, Germany
(see Appendix 1 attached). The sampling site had not been treated with crop protection products or organic fertiliser for at least 3 years prior to sampling (data from supplier).

The pH of the soil in water was 7.0, with an organic carbon content of 0.97 ± 0.07% and the sand content of the soil was measured to be 59.6%
(see Appendix 1 attached) and so satisfied the recommendations of the Test Guidelines.


Preliminary Work
The test item was a poorly water soluble, non-viscous liquid. Therefore in order to ensure accurate addition of the test item, for the purpose of the test, the test item was added directly to the test vessels using a high precision volumetric syringe or Eppendorf pipettor.

Preliminary work conducted showed that a volume of 6.4 µl of test item, measured using a gas tight micro-syringe gave a measured weight of 5.0 mg, mean of 15 separate weighings.

Additionally, preliminary work also showed that volumes of 66 and 650 µl of test item, measured using an Eppendorf pipetter gave measured weights of 50 and 500 mg respectively, mean of 15 separate weighings.

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. In the range-finding test soil microorganisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/kg for a period of 28 days.

To prepare the test series, amounts of test item (6.4, 66 and 650 µl equivalent to 5.0, 50 and 500 mg) were each added separately to 5g of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours the mixing was stopped and a final bulk soil weight of 500 g was added to each container prior to mixing for a further 3 hours. After this process, an amount of powdered Lucerne-grass-green-meal (2.5 g) and an aliquot (26.1 ml) of water was then separately added to each soil/sand/test item combination and thoroughly mixed (Kenwood Chef Mixer) for 5 minutes to give the required test concentrations of 10, 100 and 1000 mg/kg with a nominal moisture content of 40% of the Water Holding Capacity (WHC).
Lucerne-grass-green-meal was added to act as a respiratory substrate.

The control soil was prepared in an identical manner but not exposed to the test item.

The range-finding test was conducted in glass beakers, each containing 50 g* of soil, and sealed with loosely fitting lids in order to minimise moisture loss by evaporation. Four replicate vessels were prepared for each control and test concentration.

At the start of the range-finding test two replicate control and test item vessels for each concentration were sacrificed for nitrate analyses. The remainder of the vessels were then sealed with loosely fitting lids and maintained in a temperature controlled room at approximately 21oC, in darkness, for 28 days.

After 28 days the nitrate concentration in each control and test vessel was determined.


Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 100, 180, 320, 560, and 1000 mg/kg

Preparation of soil

The moisture content of the soil was determined prior to the start of the definitive test by drying an amount of soil (100.38 g) at approximately 110oC until a constant weight was obtained. The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 9%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.1 g/100g (see Appendix 1 in attached section) and hence 27.1 ml of deionised reverse osmosis water per 0.5 kg* of soil was added.

This gave a final water content of 14 g/100 g i.e. 40% of the WHC as recommended by the Test Guidelines.

* Dry Weight

Test item preparation
To prepare the test series, amounts of test item (66, 114, 202, 356 and 650 µl equivalent to 50, 90, 160, 280 and 500 mg) were each added separately to 5g of quartz sand in an amber glass jar and mixed using an industrial mixer. After 24 hours the mixing was stopped and a final bulk soil weight of 500 g* was added to each container prior to mixing for a further 3 hours. After this process, an amount of powdered Lucerne-green-grass-meal (2.5 g) and an aliquot (27.1 ml) of water was then separately added to each soil/sand/test item combination and thoroughly mixed (Kenwood Chef Mixer) for 5 minutes to give the required test concentrations of 10, 100 and 1000 mg/kg with a nominal moisture content of 40% of the Water Holding Capacity (WHC). The soil for each test concentration was then split into 9 replicates for incubation.

The control soil was prepared in an identical manner but not exposed to the test item.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

The test vessels were covered with loosely fitting lids in order to minimise moisture loss by evaporation and maintained in a temperature controlled room at approximately 21 Deg C, in darkness.

Moisture:

The moisture content of the soil was determined prior to the start of the definitive test by drying an amount of soil (100.38 g) at approximately 110 dgree C until a constant weight was obtained.

The moisture content of the soil, expressed as a percentage of the dry weight, was determined to be 9%. The Water Holding Capacity (WHC) of the soil supplied by LUFA Speyer was 35.1 g/100g (see Appendix 1 in attached section) and hence 27.1 ml of deionised reverse osmosis water per 0.5 kg (dry weight) of soil was added.

This gave a final water content of 14 g/100 g i.e. 40% of the WHC as recommended by the Test Guidelines.

Details on test conditions:

Exposure conditions
The soil samples were incubated in glass beakers, each containing 50 g* of soil. The test vessels were covered with loosely fitting lids in order to minimise moisture loss by evaporation and maintained in a temperature controlled room at approximately 21 degree C, in darkness.
* Dry Weight

Physico-chemical measurements
Room temperature was recorded daily throughout the test. Each individual test vessel was weighed on Day 0 and deionised reverse osmosis water added on a weight basis on days 7, 14, 21 and 28 where necessary in order to maintain the moisture content within +/-5% of the initial moisture
content.

Nominal and measured concentrations:

A significant effect on nitrogen transformation activity was observed in the range-finding test at 1000 mg/kg, however, there were no significant effects at 10 and 100 mg/kg. Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/kg were selected for the definitive test.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

320 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 290 -360 mg/l

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

180 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95 % CL limits not recorded

Details on results:

Range-finding Test
Absorbance values, nitrate concentrations and percentage inhibition of nitrogen transformation activity determined during the range-finding test are given in Table 1 in section any other information on results incl. tables.


Definitive Test
Absorbance values and nitrate concentrations determined on each sampling day are given in Table 2 in section any other information on results incl. tables. The nitrate formation rates and percentage inhibition of nitrogen transformation activity on days 7 and 28 are given in Table 3 in section any other information on results incl. tables.

Inhibition of nitrogen transformation activity is presented graphically in Figure 1 - see in attached section

The microbial biomass of the soil was shown to be 121 µg C/g (ISO 14240-2: 1997). This was equivalent to 1.2% of the total soil organic carbon content.

The variation between replicate control nitrification rates was less than 15% and therefore satisfied the validation criterion given in the Test Guidelines.
Accordingly the following results were determined from the data:
EC10 (28 days): 130 mg/kg
EC25 (28 days): 210 mg/kg
EC50 (28 days): 320 mg/kg, 95% confidence limits 290 - 360 mg/kg

where ECx is the test concentration that reduced soil nitrogen transformation activity by x%.

Statistical analysis of the nitrate concentration values was carried out for the control and test item groups and showed no statistically significant decreases between the control and 100 and 180 mg/kg test concentrations (P> or equal to 0.05), however the 320, 560 and 1000 mg/kg test concentrations were significantly lower than the control (P<0.05). Therefore the ‘No Observed Effect Concentration’ (NOEC) was 180 mg/kg.
Temperature was maintained at 21 ± 1ºC throughout the test and the moisture content of each control and test vessel was adjusted to 40% of the WHC (± 5%) of the soil on days 7, 14, 21 and 28.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the nitrate concentration values on Day 28 to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Absorbance, Nitrate Concentrations and Inhibition of Nitrogen Transformation Activity from the Range-finding Test

Nominal Concentration (mg/kg soil)		Day 0		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Mean Nitrate Formation Rate (mgNO3/kg/day)	% Inhibition
Control	R1	0.201	69.40	0.686	202.85	4.71	-
	R2	0.202	69.80	0.678	200.00
10	R1	0.245	85.20	0.665	195.35	4.15	12
	R2	0.247	85.90	0.700	207.90
100	R1	0.229	79.45	0.705	209.70	4.84	[3]
	R2	0.207	71.55	0.713	212.55
1000	R1	0.261	90.95	0.423	108.50	0.72	85
	R2	0.254	88.45	0.430	111.05

  *Corrected for blank absorbance value

[Increase in nitrate formation rate as compared to controls]

 R₁-R₂= Replicates R₁and R₂

Table 2              Absorbanceand Nitrate Concentrations from the Definitive Test

Nominal Concentration (mg/kg)		Day 0		Day 7		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
Control	R1	0.205	140.00	0.373	258.20	0.473	328.60
	R2	0.213	145.60	0.366	253.30	0.509	353.90
	R3	0.211	144.20	0.360	249.10	0.508	353.20
	Mean	-	143.27	-	253.53	-	345.23
	CV%	-	2	-	1	-	3
100	R1	0.209	142.80	0.379	262.50	0.489	339.90
	R2	0.212	144.90	0.396	274.40	0.493	342.70
	R3	0.210	143.50	0.393	272.30	0.553	384.90
	Mean	-	143.73	-	269.73	-	355.83
	CV%	-	1	-	2	-	6
180	R1	0.208	142.10	0.336	232.20	0.474	329.30
	R2	0.215	147.00	0.363	251.20	0.463	321.60
	R3	0.217	148.40	0.368	254.70	0.437	303.30
	Mean	-	145.83	-	246.03	-	318.07
	CV%	-	2	-	4	-	3

*Corrected for blank absorbance value

R₁-R₃= Replicates 1 to 3

CV = Coefficient of Variation

Table 2 (continued)         Absorbanceand Nitrate Concentrations from the Definitive Test

Nominal Concentration (mg/kg)		Day 0		Day 7		Day 28
		Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)	Absorbance* (324 nm)	Nitrate Concentration (mg NO3/kg)
320	R1	0.206	140.70	0.306	211.10	0.338	233.60
	R2	0.220	150.50	0.279	192.10	0.321	221.60
	R3	0.211	144.20	0.306	211.10	0.311	214.60
	Mean	-	145.13	-	204.77	-	223.27
	CV%	-	3	-	4	-	4
560	R1	0.218	149.10	0.273	187.80	0.285	196.30
	R2	0.221	151.20	0.264	181.50	0.283	194.90
	R3	0.222	152.00	0.269	185.00	0.325	224.40
	Mean	-	150.77	-	184.77	-	205.20
	CV%	-	1	-	1	-	7
1000	R1	0.228	156.20	0.261	179.40	0.255	175.20
	R2	0.242	166.00	0.263	180.80	0.255	175.20
	R3	0.229	156.90	0.261	179.40	0.267	183.60
	Mean	-	159.70		179.87	-	178.00
	CV%	-	3		0	-	2

*Corrected for blank absorbance value

R₁-R₃= Replicates 1 to 3

CV = Coefficient of Variation

Table 3              Inhibition of Nitrogen Transformation Activity

Nominal Concentration (mg/kg)	Day 7		Day 28
	Nitrate Formation Rate (mg NO3/kg/day)	% Inhibition	Nitrate Formation Rate (mg NO3/kg/day)	% Inhibition
Control	15.75	-	7.21	-
100	18.00	[14]	7.58	[5]
180	14.31	9	6.15	15
320	8.52	46	2.79	61
560	4.86	69	1.94	73
1000	2.88	82	0.65	91

*Corrected for blank absorbance value

[Increase in nitrate formation rate as compared to controls]

Validity criteria fulfilled:

yes

Remarks:

This study is classified as acceptable and satisfies the guideline requirements for this study end point.

Conclusions:

The effect of the test item on the nitrogen transformation activity of soil microorganisms has been investigated over a 28-Day period and gave an
EC50 value of 320 mg/kg, 95% confidence limits 290 to 360 mg/kg. The No Observed Effect Concentration was 180 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the long-term effect of the test item, after a single exposure, on nitrogen transformation activity of soil microorganisms. The method followed that described in the OECD Guidelines for Testing of Chemicals (2000) No 216 “Soil Microorganisms: Nitrogen Transformation Test” referenced as Method C.21 of Commission Regulation (EC) No. 440/2008; EPPO (1994) Decision Making Scheme for the Environmental Risk Assessnt of Plant Protection Products, Chapter 7: Soil Microflora, EPPO Bulletin 24 and SETAC-Europe (1995) Procedures for Assessing the Environntal Fate and Ecotoxicity of Pesticides.

Methods.

Following a preliminary range-finding test soil microorganisms were exposed to the test item at concentrations of 100, 180, 320, 560 and 1000 mg/kg for 28 days at a temperature of approximately 21°C, in the dark with the addition of powdered Lucerne-green-grassal to act as a respiratory substrate.

The inhibitory effect of the test item on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples on Days 0, 7 and 28 and compared to data obtained from control soil samples.

Results and Conclusion.

The effect of the test item on the nitrogen transformation activity of the soil microorganisms gave an EC₅₀of 320 mg/kg, 95% confidence limits 290 - 360 mg/kg. The No Observed Effect Concentration (NOEC) was180 mg/kg.

Description of key information

(28-day) EC50 320 mg/kg dw soil micro-organisms (read-across, reliability 1)
(28-day) NOEC 100 mg/kg dw soil micro-organisms (read-across, reliability 1)

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

320 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

100 mg/kg soil dw

Additional information

A nitrogen transformation test is available for GTL Gasoil (Clarke, 2010a). The test was conducted in accordance with OECD 216 and GLP.

Soil microorganisms were exposed to GTL Gasoil at concentrations of 100, 180, 320, 560 and 1000 mg/kg (dry weight) for 28 days at a temperature of 21°C, in the dark, with addition of Lucerne-green-grass meal as a respiratory substrate. The inhibitory effect of the test substance on nitrogen transformation was determined by measurement of nitrate concentrations in soil samples taken on Days 7, 14, 21 and 28, in comparison to control samples.

The effect of the test substance on nitrogen transformation gave an EC₅₀of 320 mg/kg dw and a NOEC of 180 mg/kg dw.

A nitrogen transformation test is available for GTL Base Oil Distillates (Clarke, 2010b). The test was conducted in accordance with OECD 216 and GLP.

Soil microorganisms were exposed to GTL Base Oil Distillates at concentrations of 1000 mg/kg (dry weight) for 28 days at a temperature of 21°C, in the dark, on natural soil with an organic carbon content of 0.97 ± 0.07% with addition of Lucerne-green-grass meal as a respiratory substrate. The inhibitory effect of the test substance on nitrogen transformation was determined by measurement of nitrate concentrations in soil samples taken on Days 7, 14, 21 and 28, in comparison to control samples.

The effect of the test substance on nitrogen transformation gave an EC₅₀ greater than 1000 mg/kg dw.

Conclusion

Soil microorganism toxicity data are available for two GTL-derived substances in the relevant carbon number range for Hydrocarbons, C18 -C24, isoalkanes, <2% aromatics. For GTL Base Oil Distillates (C18 -C50) there were no effects up to the highest dose level of 1000 mg/kg dw soil. For GTL Gasoil (C8-C26), the EC50 (28-d) was determined to be 320 mg/kg dw soil, and the corresponding NOEC was 100 mg/kg dw soil. It is likely that the effects observed in the study with GTL Gasoil are attributable to the lower carbon number constituents but in the absence of individual constituent data for the terrestrial compartment to support this, the worst-case EC₅₀ and NOEC are read across to the registration substance. It is not possible to derive a meaningful PNEC for the terrestrial compartment from these whole-substance data.