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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:
March 2020 - October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Not Applicable
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Carbon transformation was determined by the glucose induced respiration method, based on the procedure described by Anderson and Domsch(6) 1978.
- Other:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: To achieve satisfactory incorporation of the test item into the test soil, Amines, tallow, alkyl, dodecylbenzene sulfonate was warmed in a water bath set at 60 °C until liquid and appropriate amounts then dissolved in acetone and applied at an appropriate concentration to sand, evaporated to dryness and then the treated sand (at a rate of 10 g sand per kg dry weight soil + test item) distributed evenly in several aliquots over the surface of each soil replicate (spread as a thin layer of approximately 2.5 cm). The treated test soil was thoroughly mixed after each aliquot addition to give the final nominal soil concentrations.

Following preparation, the soil group replicates were placed into individual 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange. The soil group replicates were incubated in the dark in a temperature controlled room set at 20 ± 2°C. The moisture content of the soil group replicates was checked weekly by weight and the moisture adjusted by adding sufficient distilled water to bring the soils back to their original moisture content following removal of samples

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone (organic solvent) + sand (carrier)
- Concentration of vehicle in test medium (stock solution and final test solution): To achieve satisfactory incorporation of Amines, tallow alkyl, dodecylbenzene sulfonate into the test soil, the test item was warmed in a water bath set at 60 °C until liquid and appropriate amounts to achieve each of the 5 treatment levels then dissolved in 20 ml acetone. The treated acetone containing test item was added to 64.32 g sand (equivalent to 8 replicates of 8.04 g sand*). The acetone was evaporated to dryness and three replicate 8.04 g portions of sand plus the calculated weight of test item (eg. Group 2: 8.04 g sand + 8.04 mg test item = 8.05 g treated sand) were used to treat the three 1 kg soil replicates per nitrogen treatment Group 2.
- Evaporation of vehicle before use: yes
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
temperatures between 18 to 22 °C
Moisture:
Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 40% of the soil water holding capacity (WHC).
Soil (Initial ) - Maximum WHC : 49.0 % w/w. Lead to = 19.6% moisture content.
Organic carbon content (% dry weight):
1.36
Nitrogen content (% dry weight):
0.3
Details on test conditions:
TEST SYSTEM
- Testing facility: Covance Laboratories
- Test container (type, material, size): 5 L polypropylene containers
- Amount of soil: 1 kg soil for each replicate
- No. of replicates per concentration: 3 replicates
- No. of replicates per control: 3 replicates
- No. of replicates per vehicle control: /

SOIL INCUBATION
- Method: bulk / series of individual subsamples : bulk (a single sandy loam soil was used).

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LandLook (Midlands) soil series.site near Rugby, Leamington Spa, Warwickshire, England (Ordinance Survey map reference SP3203469216)
- Source : Leamington Spa, Warwickshire. (agricultural land)
- History of site: The soil not have received treatment with plant protection products, organic and mineral fertilisers during the preceding year
- Vegetation cover: grass cover
- Treatments with pesticides or fertilizers: none
- Accidental contamination: none
- Other:
- Depth of sampling: top 5 - 20 cm of top soil
- Soil texture : Sandy loam
- % sand: 71 % w/w
- % silt: 16 % w/w
- % clay: 13 % w/w
- Soil taxonomic classification:
- Soil classification system:
- pH (in water): 6.3
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Soil 317: <0.003 g N/g dry weight soil (<0.3%)
- Maximum water holding capacity (in % dry weigth): 49.0 % w/w - Maximum WHC (0.001 bar [pF 0] disturbed soil)
- Cation exchange capacity (mmol/kg): 13.0 meq/100 g
- Pretreatment of soil: The test soil (Bromsgrove 317/23.01.20) was received at Covance on 27 January 2020 and stored at 2 - 8 °C and then prior to testing was sieved to 2mm and stored at 20 ± 2 °C for 14 days.
- Storage (condition, duration): The soil was stored at ambient temperature prior to delivery to Covance.
- Initial microbial biomass as % of total organic C: 371 mg C/kg soil (2.73 %)

DETAILS OF PREINCUBATION OF SOIL (if any): To achieve satisfactory incorporation of the test item into the test soil, Amines, tallow, alkyl, dodecylbenzene sulfonate was warmed in a water bath set at 60 °C until liquid and appropriate amounts then dissolved in acetone and applied at an appropriate concentration to sand (see Attachment 12.3), evaporated to dryness and then the treated sand (at a rate of 10 g sand per kg dry weight soil + test item) distributed evenly in several aliquots over the surface of each soil replicate (spread as a thin layer of approximately 2.5 cm). The treated test soil was thoroughly mixed after each aliquot addition to give the final nominal soil concentrations. Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 40% of the soil water holding capacity (WHC). The control soil mixture was similarly treated with the same volume of acetone on the same amount of sand, evaporated, mixed, and water added to bring moisture content to 40% of WHC

- Preparation and Incubation of Soil Treatment Groups
Following preparation, the soil group replicates were placed into individual 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange. The soil group replicates were incubated in the dark in a temperature controlled room set at 20 ± 2°C. The moisture content of the soil group replicates was checked weekly by weight and the moisture adjusted by adding sufficient distilled water to bring the soils back to their original moisture content following removal of samples.
Nominal and measured concentrations:
Soil microorganisms were exposed to the test item, Amines, tallow alkyl, dodecylbenzene sulfonate, at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil).
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
320 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Details on results:
Carbon transformation in treated soil Groups 2 – 5 was broadly comparable to the non-treated control soil (Group 1) over the 28 day study period. Statistically significant differences in rates of carbon transformation were noted at a small number of hourly intervals for treated soil Groups 2 – 5, however the percent deviation in carbon transformation activity between non-treated control soil (Group 1) and treated Groups 2 – 5 at the majority of hourly interval measurements over the study period was below the level of 25% deviation that can be applied as an indication of biological significance in this study type. Carbon transformation in treated soil Group 6 was broadly comparable to the non-treated control soil (Group 1) at Day 0 and 7 days. However at 28 days the rates of carbon transformation at the majority of hourly time intervals showed statistically significant increases for treated soil Group 6 compared to the non-treated control soil with the percent deviation in carbon transformation activity greater than 25% different for the majority of hourly interval measurements.

For all time points on Days 0, 7 and 28, the EC10, EC25 and EC50 values and 95% confidence intervals could not be reliably estimated for carbon transformation due to an insufficient dose-response relationship across the treatment groups. Accounting for the statistically significant increases in carbon transformation observed for treated soil Group 6 (1000 mg/kg dry weight soil), the NOEC was considered to be 320 mg/kg dry weight soil.
Results with reference substance (positive control):
None
Reported statistics and error estimates:
For more details, see statistical analysis and tables (document attached).

The mean results presented below are also attached in a document in the background justification part to get access to a more friendly presentation. For more details, the individual results by replicate are also attached in the same document in the background justification part.

Table10.3: Carbon transformation in soil - Day 0 group mean values

Day 0

 

Group

 

Gp 2 : Gp 1
Deviation (%)

Gp 3 : Gp 1
Deviation (%)

Hourly interval after treatment

1

Non-treated control soil

2

10 mg/kg
Test item

3

32 mg/kg
Test item

1

5.44

5.48

5.44

0.7

0.0

2

5.06

5.10

5.07

0.8

0.2

3

5.01

5.05

5.07

0.8

1.2

4

5.26

5.33

5.35

1.3

1.7

5

5.59

5.63

5.66

0.8

1.4

6

5.84

5.89

5.92

0.8

1.5

7

6.12

6.20

6.20

1.3

1.3

8

6.20

6.34

6.37

2.1

2.6

9

6.54

6.66

6.66

1.9

1.8

10

6.99

7.17

7.16

2.6

2.4

11

7.48

7.73

7.70

3.3

3.0

12

8.12

8.43

8.35

3.8

2.8

 

 

 

Day 0

 

Group

 

Gp 4 : Gp 1
Deviation (%)

Gp 5 : Gp 1
Deviation (%)

Gp 6 : Gp 1
Deviation (%)

Hourly interval after treatment

4

100 mg/kg
Test item

5

320 mg/kg
Test item

6

1000 mg/kg
Test item

 

1

6.20

6.08

5.91

14.0

11.7

8.6

2

5.58

5.46

5.41

10.3

8.0

7.0

3

5.26

5.17

5.12

5.0

3.2

2.2

4

5.30

5.15

5.12

0.7

-2.1

-2.6

5

5.61

5.44

5.45

0.4

-2.6

-2.5

6

5.84

5.67

5.68

0.0

-2.9

-2.7

7

5.99

5.87

5.82

-2.1

-4.2

-4.9

8

6.15

5.97

5.95

-0.9

-3.8

-4.0

9

6.35

6.16

6.17

-3.0

-5.9

-5.7

10

6.69

6.47

6.50

-4.3

-7.5

-7.1

11

7.04

6.73

6.84

-5.8

-10.0

-8.5

12

7.54

7.19

7.22

-7.2

-11.5

-11.1

      Values expressed as mg C as CO2per kg dry weight soil/hour; each value is a mean figure derived from 3 replicate analyses

        Test item – Amines, tallow alkyl, dodecylbenzene sulfonate

 


 

Table10.4: Carbon transformation in soil - Day 7 group mean values

 

Day 7

 

Group

 

Gp 2 : Gp 1
Deviation (%)

Gp 3 : Gp 1
Deviation (%)

Hourly interval after treatment

1

Non-treated control soil

2

10 mg/kg
Test item

3

32 mg/kg
Test item

1

7.24

7.01

5.42

-3.2

-25.2

2

6.20

6.02

4.98

-2.9

-19.7

3

5.78

5.63

4.78

-2.5

-17.3

4

5.69

5.60

4.79

-1.7

-15.8

5

5.67

5.61

4.87

-1.2

-14.1

6

5.76

5.67

4.96

-1.6

-14.0

7

5.78

5.70

4.97

-1.4

-14.1

8

5.87

5.80

5.06

-1.3

-13.8

9

6.09

6.04

5.29

-0.9

-13.2

10

6.24

6.21

5.43

-0.5

-12.9

11

6.43

6.41

5.60

-0.4

-12.9

12

6.82

6.78

5.98

-0.6

-12.3

 

 

 

Day 7

 

Group

 

Gp 4 : Gp 1
Deviation (%)

Gp 5 : Gp 1
Deviation (%)

Gp 6 : Gp 1
Deviation (%)

Hourly interval after treatment

4

100 mg/kg
Test item

5

320 mg/kg
Test item

6

1000 mg/kg
Test item

 

1

5.63

5.18

5.10

-22.3

-28.5

-29.5

2

5.18

4.85

4.82

-16.5

-21.7

-22.2

3

5.03

4.72

4.72

-12.8

-18.2

-18.2

4

4.95

4.72

4.68

-13.0

-17.1

-17.8

5

4.96

4.75

4.76

-12.5

-16.2

-16.1

6

4.89

4.67

4.67

-15.1

-19.0

-19.0

7

4.90

4.69

4.69

-15.1

-18.8

-18.9

8

5.02

4.78

4.79

-14.6

-18.6

-18.4

9

5.16

4.94

4.97

-15.4

-19.0

-18.4

10

5.20

4.97

5.03

-16.6

-20.2

-19.3

11

5.30

5.05

5.10

-17.6

-21.5

-20.7

12

5.54

5.24

5.28

-18.8

-23.2

-22.6

      Values expressed as mg C as CO2per kg dry weight soil/hour; each value is a mean figure derived from 3 replicate analyses

        Test item – Amines, tallow alkyl, dodecylbenzene sulfonate

           

 


 

Table10.5: Carbon transformation in soil - Day 28 group mean values

 

Day 28

 

Group

 

Gp 2 : Gp 1
Deviation (%)

Gp 3 : Gp 1
Deviation (%)

Hourly interval after treatment

1

Non-treated control soil

2

10 mg/kg
Test item

3

32 mg/kg
Test item

1

5.68

5.75

5.00

1.4

-12.0

2

4.85

4.97

4.46

2.3

-8.1

3

4.53

4.67

4.28

3.0

-5.6

4

4.62

4.74

4.40

2.6

-4.8

5

4.56

4.72

4.41

3.5

-3.3

6

4.18

4.39

4.10

4.9

-2.0

7

4.14

4.35

4.02

5.0

-2.9

8

4.45

4.66

4.30

4.6

-3.4

9

4.64

4.83

4.56

4.1

-1.8

10

4.82

5.07

4.76

5.2

-1.2

11

5.11

5.35

4.97

4.8

-2.6

12

5.58

5.85

5.49

4.7

-1.7

 

 

 

Day 28

 

Group

 

Gp 4 : Gp 1
Deviation (%)

Gp 5 : Gp 1
Deviation (%)

Gp 6 : Gp 1
Deviation (%)

Hourly interval after treatment

4

100 mg/kg
Test item

5

320 mg/kg
Test item

6

1000 mg/kg
Test item

 

1

4.99

5.22

6.00

-12.1

-8.0

5.7

2

4.65

4.88

5.75

-4.3

0.5

18.5

3

4.62

4.88

5.75

1.9

7.8

26.9

4

4.49

4.83

5.76

-3.0

4.5

24.6

5

4.55

4.91

6.01

-0.1

7.6

31.9

6

5.23

5.61

6.81

25.1

34.2

62.9

7

4.38

4.70

5.82

5.6

13.4

40.4

8

4.35

4.75

5.98

-2.4

6.6

34.2

9

4.55

4.99

6.37

-2.0

7.4

37.3

10

4.72

5.22

6.78

-2.0

8.5

40.9

11

4.91

5.41

7.11

-3.8

5.9

39.3

12

5.22

5.81

7.66

-6.5

4.0

37.1

      Values expressed as mg C as CO2per kg dry weight soil/hour; each value is a mean figure derived from 3 replicate analyses

        Test item – Amines, tallow alkyl, dodecylbenzene sulfonate

 

 

 

 


 

Validity criteria fulfilled:
yes
Remarks:
The variation between triplicate control soil samples (Group 1) for carbon transformation was less than ± 15% at the beginning of the study on Day 0 and at the end of the study on Day 28 and was in compliance with the OECD 217 validity criteria.
Conclusions:
For all time points on Days 0, 7 and 28, the EC10, EC25 and EC50 values and 95% confidence intervals could not be reliably estimated for carbon transformation due to an insufficient dose-response relationship across the treatment groups. Accounting for the statistically significant increases in carbon transformation observed for treated soil Group 6 (1000 mg/kg dry weight soil), the NOEC was considered to be 320 mg/kg dry weight soil.
Executive summary:

This study was performed to assess the long-term effect of the test item after a single exposure, on carbon transformation activities of soil micro-organisms. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2000) No. 217, "Soil Microorganisms: Carbon Transformation Test".


Soil microorganisms were exposed to the test item at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil) for 28 days at temperatures of between 18 to 22 °C in the dark. The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling. 


Following preparation, the soil group replicates were placed into individual 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange. The moisture content of the soil group replicates was checked weekly by weight and the moisture adjusted by adding sufficient distilled water to bring the soils back to their original moisture content following removal of samples.


Carbon transformation was assessed in non-treated control soil and soils treated with the test item, prepared as three replicate samples per treatment group. Triplicate portions of each soil group replicate were sampled on Day 0, 7 and 28 days after treatment and glucose-induced respiration rates were determined on each sampling day by mixing sufficient glucose into the soil of each replicate to elicit an “immediate maximum respiratory response”. The glucose amended soil samples (100 g) were distributed into respirometer flasks and incubated in a temperature controlled room set to maintain 20°C ± 2°C. Carbon dioxide formation was determined hourly for 12 hours using an Infrared Gas Analyser.


The variation between triplicate control soil samples (Group 1) for carbon transformation was less than ± 15% at the beginning of the study on Day 0 and at the end of the study on Day 28 and was therefore in compliance with the OECD Guideline 217 validity criteria.


For carbon transformation, an insufficient dose-response relationship across the treatment groups prevented calculation of EC values. Whilst the NOEC level was considered to be 320 mg/kg dry weight soil, it was considered that Amines, tallow alkyl, dodecylbenzene sulfonate showedno long-term inhibitory effect on carbon transformation in soil at the concentrations tested in this study.

Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2020 - October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
Sampling and Analysis of Soils for Nitrogen Transformation:
- Concentrations: 10, 32, 100, 320 and 1000 mg/kg dry weight soil
- Sampling method: Triplicate samples of 20 g soil were withdrawn from each of the non-treated control soil and treated soil groups within 6 hours of treatment on Day 0, 7 and 28 days after preparation. In addition, on Day 0 a further set of triplicate 20 g soil samples was removed from the non-treated control (Group 1) and stored frozen at ca. -20 °C until used for preparing spike recoveries. Soil replicates were thoroughly mixed prior to sub-sampling at each time point. The soil samples were extracted on the same day of sampling using 100 mL 0.1M potassium chloride solution by shaking on a mechanical shaker at approximately 150 rpm for 60 minutes.
- Sample storage conditions before analysis: The supernatant solutions were filtered through filter paper (Fisherbrand 11784263, Range QL115) and then the sample extracts were transferred to a deep-freezer at approximately -20°C to await analysis. On the day of analysis the frozen sample extracts were allowed to thaw and then analysed for their concentrations of nitrate spectrophotometrically. The absorbance was then measured using a Shimadzu UV-1800 spectrophotometer.

The measured absorbance values for soil extract filtrates were plotted against the calibration curve to derive the sample nitrate nitrogen (N) concentration (µg N/mL). The concentration of nitrate in the test sample (µg/mL) was obtained by multiplying by the conversion constant 4.427.

Nitrate concentration in soil (µg NO3/Kg) = µg NO3/ml from analysis × KCl used (mL) / Dry weight of soil (g)

The means of the triplicate analyses were calculated and used to determine the amount of nitrate formed per gram dry-weight of soil for each soil group and the nitrate per gram dry-weight of soil against time was plotted graphically.
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Lucerne meal (sieved to 2 mm maximum) having a carbon:nitrogen ratio of 12.43 : 1 was used and 4.02g was added to 1 kg test soil, having a dry weight of 804 g, to give a final concentration of lucerne meal of 0.5% on a soil dry weight basis. Analysis of the carbon:nitrogen content of the lucerne meal was performed in a separate non-GLP study by Butterworth Laboratories Ltd. Lucerne meal batch 205: Carbon 42.38%, Hydrogen 6.08%, Nitrogen 3.41%

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: To achieve satisfactory incorporation of the test item into the test soil, Amines, tallow, alkyl, dodecylbenzene sulfonate was warmed in a water bath set at 60 °C until liquid and appropriate amounts then dissolved in acetone and applied at an appropriate concentration to sand, evaporated to dryness and then the treated sand (at a rate of 10 g sand per kg dry weight soil + test item) distributed evenly in several aliquots over the surface of each soil replicate (spread as a thin layer of approximately 2.5 cm). The treated test soil was thoroughly mixed after each aliquot addition to give the final nominal soil concentrations. Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 40% of the soil water holding capacity (WHC). The control soil mixture was similarly treated with the same volume of acetone on the same amount of sand, evaporated, mixed, and water added to bring moisture content to 40% of WHC.

Following preparation, the soil group replicates were placed into individual 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange. The soil group replicates were incubated in the dark in a temperature controlled room set at 20 ± 2°C. The moisture content of the soil group replicates was checked weekly by weight and the moisture adjusted by adding sufficient distilled water to bring the soils back to their original moisture content following removal of samples.


VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone (organic solvant) + Sand (carrier)
- Concentration of vehicle in test medium (stock solution and final test solution): To achieve satisfactory incorporation of Amines, tallow alkyl, dodecylbenzene sulfonate into the test soil, the test item was warmed in a water bath set at 60 °C until liquid and appropriate amounts to achieve each of the 5 treatment levels then dissolved in 20 ml acetone. The treated acetone containing test item was added to 64.32 g sand (equivalent to 8 replicates of 8.04 g sand*). The acetone was evaporated to dryness and three replicate 8.04 g portions of sand plus the calculated weight of test item (eg. Group 2: 8.04 g sand + 8.04 mg test item = 8.05 g treated sand) were used to treat the three 1 kg soil replicates per nitrogen treatment Group 2.
- Evaporation of vehicle before use: yes for Acetone (organic solvant)
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
temperatures between 18 to 22 °C
Moisture:
Sufficient distilled water was sprinkled onto the test soil to increase the soil moisture content to 40% of the soil water holding capacity (WHC).
Soil (Initial ) - Maximum WHC : 49.0 % w/w, lead to 19.6% moisture content.
Organic carbon content (% dry weight):
1.36
Nitrogen content (% dry weight):
0.3
Details on test conditions:
TEST SYSTEM
- Testing facility: Covance Laboratories
- Test container (type, material, size): 5 L polypropylene containers
- Amount of soil: 1 kg wet soil
- No. of replicates per concentration: triplicate
- No. of replicates per control: triplicate
- No. of replicates per vehicle control: /

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LandLook (Midlands) soil series.site near Rugby, Leamington Spa, Warwickshire, England
- Source : Leamington Spa, Warwickshire. (agricultural land)
- History of site: The soil not have received treatment with plant protection products, organic and mineral fertilisers during the preceding year
- Vegetation cover: grass cover
- Treatments with pesticides or fertilizers: none
- Accidental contamination: none
- Other:
- Depth of sampling: top 5 - 20 cm of top soil
- Soil texture : Sandy loam
- % sand: 71 % w/w
- % silt: 16 % w/w
- % clay: 13 % w/w
- Soil taxonomic classification: /
- Soil classification system: /
- pH (in water): 6.3
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Soil 317: <0.003 g N/g dry weight soil (<0.3%)
- Maximum water holding capacity (in % dry weigth):49.0 % w/w - Maximum WHC (0.001 bar [pF 0] disturbed soil)
- Cation exchange capacity (mmol/kg): 13.0 meq/100 g
- Pretreatment of soil: The test soil (Bromsgrove 317/23.01.20) was received at Covance on 27 January 2020 and stored at 2 - 8 °C and then prior to testing was sieved to 2mm and stored at 20 ± 2 °C for 14 days.
- Storage (condition, duration): The soil was stored at ambient temperature prior to delivery to Covance.
- Initial microbial biomass as % of total organic C: 371 mg C/kg soil (2.73 %)



Nominal and measured concentrations:
The test soil was treated with test item at five test concentrations (10, 32, 100, 320 and 1000 mg per kg dry weight soil).
For nitrogen transformation, the following mixtures of soil, nitrogen source (lucerne meal), distilled water and sand containing test item were prepared:
Group N1 Soil + nitrogen source (non-treated control)
Group N2 Soil + nitrogen source + 10 mg test item/kg dry weight soil
Group N3 Soil + nitrogen source + 32 mg test item /kg dry weight soil
Group N4 Soil + nitrogen source + 100 mg test item /kg dry weight soil
Group N5 Soil + nitrogen source + 320 mg test item /kg dry weight soil
Group N6 Soil + nitrogen source + 1000 mg test item /kg dry weight soil
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
Nitrogen transformation in non-treated control soil (Group 1) and in Groups 2 – 6 treated with the test item was evident from the increase in nitrate concentrations over the study period (Figure 11.1). On Day 28 the 32 and 100 mg/kg dry weight soil treated groups (Group 3 and Group 4 respectively) had significantly higher nitrate concentrations than the control group (p=0.029, Dunnett’s test). Over the 28 day study period, the percent deviation in soil nitrogen transformation activity between non-treated control soil (Group 1) and treated Groups 2 – 6 ranged from -10.10% to 9.03% and was therefore below the level of 25% deviation that can be applied as an indication of biological significance in this study type.

On Day 0, 7 and 28 the EC10, EC25 and EC50 and 95% confidence intervals could not be reliably estimated for nitrogen transformation due to an insufficient dose-response relationship across the treatment groups. The No Observed Effect Concentration (NOEC) was considered to be 1000 mg/kg dry weight soil.
Results with reference substance (positive control):
no
Reported statistics and error estimates:
For more details, see statistical analysis and tables (document attached in background justification part).

The mean results presented below are also attached in a document in the background justification part to get access to a more friendly presentation. For more details, the individual results by replicate are also attached in the same document in the background justification part.

Table 10.1: Nitrate concentrations in soil

Day

NitrateConcentration
(mg NO3/kg dry weight soil)

Group 2: Group 1

% Deviation

Group 3: Group 1
% Deviation

Group

1

Non-treated

control soil

2

10 mg/kg
Test item

3

32 mg/kg
Test item

0

136.28

131.04

134.95

-3.85

-0.98

7

135.90

129.41

122.83

-4.78

-9.62

28

335.98

332.93

366.31

-0.91

9.03

 

 

Day

NitrateConcentration
(mg NO3/kg dry weight soil)

Group 4: Group 1

% Deviation

Group 5: Group 1

% Deviation

Group 6: Group 1

% Deviation

Group

4

100 mg/kg
Test item

5

320 mg/kg
Test item

6

1000 mg/kg
Test item

0

135.61

139.81

138.86

-0.49

2.59

1.89

7

122.17

131.99

135.71

-10.10

-2.88

-0.14

28

363.45

337.6

318.24

8.18

0.48

-5.28

Nitrate concentrations in soil, expressed asmg NO3/kgdry weight soil, are mean values derived from three replicate analyses.

Test item – Amines, tallow alkyl, dodecylbenzene sulfonate

Validity criteria fulfilled:
yes
Remarks:
The variation between triplicate control soil samples (Group 1) for nitrate analysis was less than ± 15% on Day 0 and on Day 28 and was therefore in compliance with the OECD Guideline 216 validity criteria.
Conclusions:
In conclusion the lack of treatment related effects of the test item on nitrogen transformation activity in soil at each of the treatment levels prevented calculation of EC values and demonstrated the NOEC to be 1000 mg/kg dry weight soil. Therefore, it was considered that the test item showed no long-term effect on nitrogen transformation in soil at the concentrations tested in this study.
Executive summary:

This study was performed to assess the long-term effect of the test item after a single exposure, on the nitrogen transformation activities of soil micro-organisms. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2000) No. 216, "Soil Microorganisms: Nitrogen Transformation Test".


 


Soil microorganisms were exposed to the test item at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil) for 28 days at temperatures of between 18 to 22 °C in the dark. The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling. 


 


Following preparation, the soil group replicates were placed into individual 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange. The moisture content of the soil group replicates was checked weekly by weight and the moisture adjusted by adding sufficient distilled water to bring the soils back to their original moisture content following removal of samples.


 


Nitrogen transformation was assessed in non-treated control soil and soils treated with the test item, prepared as 3 replicate samples per group. Each soil group replicate was supplemented with lucerne meal as a nitrogen source. Triplicate portions of each soil group replicate were analysed for soil nitrate concentrations in samples on Day 0, 7 and 28 days after treatment.


 


On Day 0, 7 and 28 the EC10, EC25 and EC50 and 95% confidence intervals could not be reliably estimated for nitrogen transformation due to an insufficient dose-response relationship across the treatment groups. The No Observed Effect Concentration (NOEC) was considered to be 1000 mg/kg dry weight soil for nitrogen transformation.


 


The variation between triplicate control soil samples (Group 1) for nitrate analysis was less than ± 15% at the beginning of the study on Day 0 and at the end of the study on Day 28 and was therefore in compliance with the OECD Guideline 216 validity criteria.


 


In conclusion the lack of treatment related effects of the test item on nitrogen transformation activity in soil at each of the treatment levels prevented calculation of EC values and demonstrated the NOEC to be 1000 mg/kg dry weight soil. Therefore, it was considered that the test item showed no long-term effect on nitrogen transformation in soil at the concentrations tested in this study.

Description of key information

According the OECD 216 & 217 and GLP studies, the chronic toxicity of the registered substance (EC 701-164-2) on terrestrial micro-organisms is considered to be :


- OECD 217 : NOEC - 28d = 320 mg a.i/kg dry soil.


- OECD 216 : NOEC - 28d > 1000 mg a.i/kg dry soil.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
320 mg/kg soil dw

Additional information

Toxicity on micro-organisms (OECD 216 & 217, GLP):


This study was performed to assess the long-term effect of the test item (EC 701-164-2), after a single exposure, on the nitrogen transformation and carbon transformation activities of soil micro-organisms.


 


Soil microorganisms were exposed to the test item at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil) for 28 days at temperatures of between 18 to 22 °C in the dark. The test soil was a sandy loam soil obtained from a site in Rugby, Warwickshire, England that had received no pesticides or fertilisers for 5 years prior to sampling.


 


To summarize, the lack of treatment related effects of the test item on nitrogen transformation activity in soil at each of the treatment levels prevented calculation of EC values and demonstrated the NOEC to be 1000 mg/kg dry weight soil. Whilst the NOEC level was considered to be 320 mg/kg dry weight soil in carbon transformation, it was considered that the test item showed no long-term inhibitory effect on carbon transformation in soil at the concentrations tested in this study.