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Toxicity to soil microorganisms

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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 11 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C18 to C50
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.

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:
other: guideline study on Gas-to-liquids (GTL) substance covering the carbon range from C8 to C26
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 ww
Nominal / measured:
meas. (initial)
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 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:
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

- EC50 (28d): >1000 mg/kg soil dw, test mat. 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (nominal) based on: nitrate formation rate (OECD 216)

- NOEC (28d): ≥1000 mg/kg soil dw, test mat. 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (nominal) based on: nitrate formation rate (OECD 216)

- EC50 (28d): 320 mg/kg soil dw, test mat. 'Distillates (Fischer-Tropsch), C8-26 - branched and linear'(nominal) based on: nitrate formation rate (OECD 216)

- NOEC (28d): 100 mg/kg soil dw, test mat. 'Distillates (Fischer-Tropsch), C8-26 - branched and linear'(nominal) based on: nitrate formation rate (OECD 216)

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

No toxicity studies to soil microorganisms are available for the substance 'Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear':

A nitrogen transformation test is available for the closely related substance GTL Base Oil Distillates covering the carbon range from C18 to C50 (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 soild dw 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 soil dw.

A similar nitrogen transformation test is also available for the supporting substance GTL Gasoil covering the carbon range from C8 to C26 (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 soil dw 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.

Conclusion:

Soil microorganism toxicity data are available for two GTL-derived substances in the relevant carbon number range for 'Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear'. For GTL Base Oil Distillates (C18-C50) there were no effects up to the highest dose level of 1000 mg/kg soil dw. For GTL Gasoil (C8-C26), the EC50 (28-d) was determined to be 320 mg/kg soil dw, and the corresponding NOEC was 100 mg/kg soil dw. 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.