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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:
supporting study
Study period:
15 May - 12 Jun 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatsministerium für Umwelt und Landwirtschaft, Freistaat Sachsen, Dresden Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Lucerne meal; source: Wassergut Canitz, Canitz, Germany, soil was mixed with 0.5% lucerne with a hand-stirrer. One additional soil sample (without lucerne meal) was used for determination of the initial NO3-N content. The initial NO3-N content was 1.06 mg/100 g soil d.w.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Sieved soil (2 mm) was treated with either 10 g ground quartz sand/kg dry weight soil or a mixture of quartz sand. The test item was thoroughly mixed with quartz sand. Subsequently the obtained mixture was added and mixed with the soil by means of a hand stirrer. For an optimum distribution of the test item in the soil, the test item quartz sand mixture was applied at a ratio of about 10 g per kilogram soil dry weight. Water was added to the soil to achieve a water content of approximately 45% of WHC. The incubation of the prepared soil was carried out in wide mouth glass flasks (500 mL) under the conditions mentioned above. The screw caps of the flasks used permitted an air exchange. The water content of the soil in each test vessel was determined at test start (after application) and adjusted once a week to the required range of 40-50% of WHC.

VEHICLE: no vehicle was used
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.4 - 21.2 °C in a climatic room
Moisture:
16.69 - 17.93 g/100 g soil d.w. (equivalent to 45.59 - 48.98% of WHC)
Details on test conditions:
TEST SYSTEM
- Testing facility: BioChem agrar, Gerichshain, Germany
- Test container (type, material, size): in wide mouth glass flasks (500 mL)
- Amount of soil: 200 g soil dry weight (= one sub-sample) per test vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: no vehicle was used

SOIL INCUBATION
- Method: incubation of the soil samples was performed as a series of individual and equally sized sub-samples of each treatment group

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Wassergut Canitz, Canitz, Germany, 12.694435960 degrees East, 51.403774567 degrees North
- History of site: last application of plant protection products: 1990
- Vegetation cover: fallow ground
- Treatments with pesticides or fertilizers: none since 2003
- Humus content [%]: 2.37
- Depth of sampling: 20 cm
Soil texture
- % sand: 54.3 (according to USDA) and 52.9 (according to DIN 11277)
- % silt: 35.4 (according to USDA); 36.8 (according to DIN 11277)
- % clay: 10.3 (according to USDA and DIN 11277)
- Soil taxonomic classification: sandy loam (according to USDA) or loamy sand (according to DIN 4220)
- pH (in water): 6.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 0.53 mg/100 g soil d.w.
- Maximum water holding capacity (in g/100 g soil d.w.): 36.61
- Cation exchange capacity: 8.9 cmol+/kg soil
- Pretreatment of soil: soil was dried at room temperature, passed through a 2 mm mesh sieve (05.03. - 13.03.2013); before application, the soil was adapted to test conditions (06.05. - 15.05.2013)
- Storage (condition, duration): at approx. 4 °C in containers under aerobic conditions in the dark (13.03. - 06.05.2013)
- Initial microbial biomass as % of total organic C: 35.10 mg C/100 g soil dry weight= 2.54% of Corg.

DETAILS OF PREINCUBATION OF SOIL (if any): no additional information

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The nitrogen transformation was determined on day 0 (after approximately 3 h) and at intervals of 7, 14, and 28 d after application.

VEHICLE CONTROL PERFORMED: no (no solvent was used)

RANGE-FINDING STUDY: no range-finding test was performed
Nominal and measured concentrations:
control, 0.233 kg/ha (corresponding to 0.269 mg a.s./kg soil d.w.) and 1.116 kg/ha (corresponding to 1.29 mg a.s./kg soil d.w.)
Reference substance (positive control):
yes
Remarks:
Dinoterb (2-tert-Butyl-4,6-dinitrophenol)
Duration:
28 d
Dose descriptor:
other: trigger value of 25% inhibition compared with the control
Effect conc.:
> 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Details on results:
The test item caused no adverse effects (difference to control < 25%, OECD 216) on the soil nitrogen transformation (measured as NO3-N production) at the end of the 28 d incubation period.
Results with reference substance (positive control):
In a separate study the reference item Dinoterb caused a stimulation of nitrogen transformation of +33.7% and +42.6% at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, determined 28 d after application.
Reported statistics and error estimates:
The % differences in the quantities of nitrogen formed between the control and the test item treatment groups were determined as follows: % difference to control = ((test item rate - control rate)/control rate) x 100%. A statistical evaluation of the test results was performed by means of a 2-sided Student-t-test (for homogeneous variances at 5% significance level).

Table 1: Effects on nitrogen transformation in soil after treatment with test item

Time Interval (days)

Control

0.265 mg a.s./kg soil dry weight equivalent to 0.233 kg test item/ha

1.31 mg a.s./kg soil dry weight equivalent to 1.116 kg test item/ha

Nitrate-N1)

Nitrate-N1)

% difference to control

Nitrate-N1)

% difference to control

0-7

3.80

±

0.04

4.08

±

0.20

-7.4 n.s.

4.10

±

0.06

+8.0 *s

7-14

1.81

±

0.04

1.27

±

0.11

-30.2 *s

0.97

±

0.11

-46.7 *s

14-28

1.03

±

0.05

1.15

±

0.26

+11.3 n.s.

1.25

±

0.25

+21.3 n.s.

The calculations were performed with unrounded values

1) Rate: Nitrate-N in mg/kg soil dry weight/time interval/day, mean of 3 replicates and standard deviation

n.s. = No statistically significant difference to the control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

*s. = statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05

Validity criteria fulfilled:
yes
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov - 11 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatsministerium für Umwelt und Landwirtschaft, Freistaat Sachsen, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: glucose concentration was determined as 0.6%

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: For this purpose soil samples (100 g soil d.w.) were taken, mixed with glucose by means of a hand-stirrer and filled into reaction flasks (500 mL). Afterwards, small vessels containing 18 mL 1 M NaOH solution were placed in the reaction flasks, which were tightly closed and then connected with a respirometer.

VEHICLE: no vehicle was used
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
18.9 - 20.6 °C in a climatic room
Moisture:
43.63 [g/100 g soil d.w.] water holding capacity
Details on test conditions:
TEST SYSTEM
- Testing facility: BioChem agrar; Labor für biologische und chemische Analytik GmbH, Gerichshain, Germany
- Test container (type, material, size): 4 L steel vessel, used lids allowed air exchange
- Amount of soil: 100 g soil dry weight (= one sub-sample) per vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: no vehicle was used

SOIL INCUBATION
- Method: Temperature: incubation of the soil samples was performed as series of individual and equally sized sub-samples (1000 g soil dry weight) of each treatment group, Temperature: 18.9 - 20.6 °C in a climatic room; incubated in the dark; Water content: 21.08 - 21.56 g/100 g soil d.w. (equivalent to 48.31 - 49.41% of WHC); duration: 28 d

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Wyler, North Rhine-Westphalia, Germany
- History of site: agriculturally utilised soil
- Vegetation cover: fallow ground
- Treatments with pesticides or fertilizers: none since 2009
- Depth of sampling: 20 cm
Soil texture
- % sand: 28.3 (according to USDA), 27.5 (according to DIN 11277)
- % silt: 33.1 (according to USDA), 33.9 (according to DIN 11277)
- % clay: 38.9 (according to USDA and DIN 11277)
- Soil taxonomic classification: clay loam
- Soil classification system: USDA and DIN 4220
- pH (in water): 7.0
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 3.15 mg/100 g soil d.w.
- Maximum water holding capacity (in % dry weigth): 43.63 g/100 g soil d.w.
- Cation exchange capacity: 22.0 cmol+/kg soil
- Pretreatment of soil: dried at room temperature, passed through a 2 mm mesh sieve (17.08. - 21.08.2015). Before application, the soil was adapted to test conditions (conditioning phase: 06.11. - 13.11.2015)
- Storage (condition, duration): at approx. 4 °C, under aerobic conditions in the dark 21.08 - 06.11.2015
- Initial microbial biomass as % of total organic C: 2.00% of Corg Carbon content of microbial biomass

DETAILS OF PREINCUBATION OF SOIL (if any): no additional information

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Effects on non-target soil microorganisms on day 0 (3 hours), and after 7,14 and 28 days of incubation based to the glucosestimulatined soil respiration

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:
nominal concentrations: 0.30 mg test item/kg dry weight soil (0.223 kg test item/ha) and 1.49 mg test item/kg dry weight soil (1.116 kg test item/ha)
Reference substance (positive control):
yes
Remarks:
Dinoterb
Duration:
28 d
Dose descriptor:
other: trigger value of 25% inhibition compared with the control
Effect conc.:
> 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
respiration rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
respiration rate
Remarks on result:
other: Endpoint was not part of the report but derivable from the reported results
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.265 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
respiration rate
Remarks on result:
other: Endpoint was not part of the report but derivable from the reported results
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: Test item did not caused any adverse effects (deviation from control < 25 %, OECD 217) on the soil carbon transformation (measured as O2-consumption) at the end of the 28-day incubation period.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: reference item Dinoterb caused inhibitions of carbon transformation of -33.1 %, -47.6 % and -44.7 % at 13.00 mg, 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, determined 28 days after application
Reported statistics and error estimates:
A statistical evaluation of the test results was performed by means of a 2-sided Student-t-test (for homogeneous variances at 5% significance level).

Validity criteria:

The coefficients of variation in the control were maximum 9.6% and thus fulfilled the demanded range (≤ 15%).

The NOEC was determined to be at 0.265 mg test item/kg soil d.w. since no statistically significant effects were observed at this test item concentration. The EC10 was determined to be >1.31 mg test item/kg soil d.w. since the inhibition at this highest test item concentration was below 10%.

Table 1: Effects on carbon transformation in soil after treatment

Days after application

Control

0.265 mg a.s./kg soil d.w. equivalent to 0.223 kg test item/ha

1.31 mg a.s./kg soil d.w. equivalent 1.116 kg test item/ha

O2-consumption [mg/kg soil d.w./h]

CV [%]

O2-consumption [mg/kg soil d.w./h]

CV [%]

% difference to control

O2-consumption [mg/kg soil d.w./h]

CV [%]

% difference to control

0

16.81

5.1

15.89

2.4

-5.5 n.s.

15.74

5.7

-6.4 n.s.

7

15.82

2.2

15.21

1.5

-3.8 n.s.

15.26

6.9

-3.5 n.s.

14

15.15

9.6

14.17

8.7

-6.5 n.s.

15.27

6.5

+0.8 n.s.

28

13.62

1.9

13.65

3.8

+0.2 n.s.

12.81

2.1

-6.0 *s.

The calculations were performed with unrounded values.

CV [%] = Coefficient of Variation

n.s. = No statistically significant difference to the control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

*s. = statistically significantly different to control (Student-t test for homogeneous variances, 2-sided, p ≤ 0.05)

Validity criteria fulfilled:
yes
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov - 11 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatsministerium für Umwelt und Landwirtschaft, Freistaat Sachsen, Dresden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: soil was enriched with lucerne meal (concentration in soil 0.5%, Wassergut Canitz, 42.4% carbon content, 3.20% nitrogen content, 13.2/1 C/N relation, particle size: <0.25 mm). One additional soil sample (without lucerne meal) was used for
determination of the initial NO3-N-content. The initial NO3-N-content was 4.13 mg/100 g soil d.w.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: test item was mixed with quartz sand and was mixed with the soil by means of a hand stirrer. For an optimum distribution of the test item in the soil, the test item quartz sand mixture was applied at a ratio of about 10 g/kg soil dry weight. Water was added to the soil to achieve a water content of approximately 45 % of WHC. The water content of the soil in each test vessel was determined at test start (after application) and adjusted once a week to the required range of 40 - 50% of WHC.

VEHICLE: no vehicle was used
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
18.9 - 20.6 °C in a climatic room
Moisture:
21.08 - 21.72 g/100 g soil d.w. (equivalent to 48.32 - 49.79% of WHC)
Details on test conditions:
TEST SYSTEM
- Testing facility: BioChem agrar GmbH, Gerichshain, Germany
- Test container (type, material, size): wide mouth glass flasks (500 mL). The screw caps of the flasks used permitted an air exchange.
- Amount of soil: 200 g soil dry weight (= one sub-sample) per test vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: incubation of the soil samples was performed as a series of individual and equally sized sub-samples of each treatment group

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Arnheimscher Deich, Wyler, Germany, sampling date: 17 Aug 2015
History of site:
- Vegetation cover: fallow ground
- Treatments with pesticides or fertilizers: none since 2009, 2009 (last application of plant protection product)
- Depth of sampling: 20 cm
Soil texture
- % sand: 28.3% (according to USDA) and 27.5% (according to DIN 11277)
- % silt: 33.1% (according to USDA) and 33.9% (according to DIN 11277)
- % clay: 38.6% (according to USDA and DIN 11277)
- Soil taxonomic classification: clay loam
- Soil classification system: according to USDA and DIN 4220
- pH (in water): 7.0
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 3.15 mg/100 g soil dry weight
- Maximum water holding capacity (in % dry weight): 43.63 g/100 g soil dry weight
- Cation exchange capacity: 22.0 cmol+/kg soil
- Pretreatment of soil: soil was dried at room temperature, passed through a 2 mm mesh sieve (17.08. - 21.08.2015); before application, the soil was adapted to test conditions (06.11. - 13.11.2015)
- Storage (condition, duration): at approx. 4 °C in containers under aerobic conditions in the dark (21.08. - 06.11.2015)
- Initial microbial biomass as % of total organic C: 48.30 mg C/100 g soil dry weight = 2.00% of Corg.

DETAILS OF PREINCUBATION OF SOIL (if any): no additional information

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The nitrogen transformation was determined on day 0 (after approximately 3 hours) and at intervals of 7, 14, and 28 days after application.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY: no range-finding test was performed
Nominal and measured concentrations:
control, 0.2 kg a.s./ha (corresponding to 0.223 kg test item/ha) and 1.0 kg a.s./ha (corresponding to 1.116 kg test item/ha)
Reference substance (positive control):
yes
Remarks:
Dinoterb
Duration:
28 d
Dose descriptor:
other: trigger value of 25% inhibition compared with the control
Effect conc.:
> 1.116 kg/ha
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Remarks on result:
other: Endpoint was not part of the report but derivable from the reported results
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Remarks on result:
other: Endpoint was not part of the report but derivable from the reported results
Details on results:
The substance caused no adverse effects (difference to control <25%, OECD 216) on the soil nitrogen transformation (expressed as NO3-N-production) at the end of the 28 d incubation period. The study was performed in a field soil at concentrations up to 1.31 mg a.s./kg soil dry weight, which are equivalent to application rates up to 1.116 test item/ha.
Results with reference substance (positive control):
In a separate study the reference item Dinoterb caused stimulations of nitrogen transformation of +109.5%, +212.8% and +155.6% at 13.00 mg, 27.00 mg and 40.00 mg Dinoterb per kg soil dry weight, respectively, determined 28 d after application (time interval 14-28).
Reported statistics and error estimates:
A statistical evaluation of the test results was performed by means of a 2-sided Student-t-test (for homogeneous variances at 5% significance level).

Table 1: Water content during the test [g/100 g soil d.w.]

Treatment group

days after application

0

7

14

21

28

Control

21.44

21.20

21.16

21.08

21.10

21.67

21.40

21.33

21.33

21.33

21.57

21.31

21.34

21.23

21.31

0.30 mg test item/kg soil d.w.

21.43

21.14

21.15

21.09

21.11

21.69

21.41

21.42

21.38

21.39

21.66

21.38

21.32

21.45

21.31

1.49 mg test item/kg soil d.w.

21.57

21.36

21.27

21.25

21.17

21.51

21.24

21.20

21.20

21.20

21.72

21.46

21.38

21.41

21.39

 

Table 2: Effects on nitrogen transformation in soil after treatment

Time Interval (days)

Control

0.265 mg a.s./kg soil dry weight equivalent to 0.223 kg test item/ha

1.31 mg a.s./kg soil dry weight equivalent to 1.116 kg test item/ha

Nitrate-N1)

Nitrate-N1)

% difference to control

Nitrate-N1)

% difference to control

0-7

4.00

±

0.14

3.59

±

0.24

-10.1 n.s.

3.92

±

0.22

-1.9 n.s.

7-14

1.07

±

0.28

1.27

±

0.66

+18.8 n.s.

0.61

±

0.25

-42.4 n.s.

14-28

1.08

±

0.24

1.10

±

0.18

+1.8 n.s.

1.09

±

0.05

+0.9 n.s.

The calculations were performed with unrounded values

1) Rate: Nitrate-N in mg/kg soil dry weight/time interval/day, mean of 3 replicates and standard deviation

n.s. = No statistically significant difference to the control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

Validity criteria fulfilled:
yes
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 May - 12 Jun 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatsministerium für Umwelt und Landwirtschaft, Freistaat Sachsen, Dresden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: optimal glucose concentration was 0.4%

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: 1000 g soil dry weight (= one sub-sample) per vessel was weighed in the mixing vessel of a laboratory mixer. Test item was mixed with quartz sand and soil in the laboratory mixer. The tested test item concentrations were 0.233 kg/ha resulted in 0.30 mg/kg soil d.w. and a fivefold application rate of 1.116 kg/ha resulted in 1.49 mg/kg soil d.w.. The test item quartz sand mixture was applied at a ratio of about 10 g per kilogram soil dry weight.

VEHICLE: no vehicle was used
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.4 - 21.2 °C in a climatic room
Moisture:
36.61 g/100 g soil d.w. water holding capacity
Details on test conditions:
TEST SYSTEM
- Testing facility: BioChem agrar; Labor für biologische und chemische Analytik GmbH, Gerichshain, Germany
- Test container (type, material, size): 4 L steel vessel, used lids allowed air exchange
- Amount of soil: 1000 g soil dry weight (= one sub-sample) per vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: no vehicle was used

SOIL INCUBATION
- Method: Temperature: 19.4 - 21.2 °C in a climatic room; darkness; Water content: 17.59 - 18.27 g/100 g soil d.w. (equivalent to 48.04 - 49.92% of WHC); Duration: 28 d

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Wassergut Canitz, Saxony, Germany, 12.694435960 degrees East and 51.403774567 degrees North
- History of site: agriculturally utilised soil was selected
- Vegetation cover: fallow ground
- Treatments with pesticides or fertilizers: last application of plant protection products: 1990 organic fertiliser: none since 2003, inorganic fertiliser: none since 2003
- Depth of sampling: a depth of 20 cm
- Soil texture
- % sand: 52.9 (according to DIN 12277), 54.3 (according to USDA)
- % silt: 36.8 (according to DIN 12277), 35.4 (according to USDA)
- % clay: 10.3 (according to DIN 12277), 10.3 (according to USDA)
- Soil taxonomic classification: loamy sand (according to DIN 4220), sandy loam (according to USDA)
- Soil classification system: DIN 12277 and USDA
- pH (in water): 6.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 0.53 Nmin [mg/100 g soil d.w.]
- Maximum water holding capacity (g/100 g soil dry weight): 36.61
- Cation exchange capacity: 8.9 cmol+/kg soil
- Pretreatment of soil: dried at room temperature, passed through a 2 mm mesh sieve (05 Mar to 13 Mar 2013). Before application, the soil was adapted to test conditions (conditioning phase: 06 May - 15 May 2013)
- Storage (condition, duration): at a temperature of approx. 4 °C in containers under aerobic conditions in the dark from 13. Mar to 06 May 2013
- Initial microbial biomass as % of total organic C: 2.54% of Corg Carbon content of microbial biomass

DETAILS OF PREINCUBATION OF SOIL (if any): no additional information

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Effects on non-target soil microorganisms on day 0, and after 7, 14 and 28 d of incubation based to the glucosestimulatined soil respiration

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY: no range-finding test was performed
Nominal and measured concentrations:
control, 0.30 (application rate in the test: 0.2 kg a.s./ha corresponding to 0.223 kg test item/ha) and 1.49 mg test item/kg soil dry weight (application rate in the test 1.0 kg a.s./ha corresponding to 1.116 kg test item/ha
Reference substance (positive control):
yes
Remarks:
Dinoterb (2-tert-Butyl-4,6-dinitrophenol)
Duration:
28 d
Dose descriptor:
other: trigger value of 25% inhibition compared with the control
Effect conc.:
> 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
respiration rate
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 1.31 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Remarks on result:
other: Endpoint was not part of the report but derivable from the reported results
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.265 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Remarks on result:
other: Endpoint was not part of the report but derivable from the reported results
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: During the 28 d tests, the recommended field rate of test item (0.233 kg test item/ha) and the 5-fold overdose of the test item had no influence on carbon transformation after addition of glucose to a loamy sand (DIN 11277).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: reference item Dinoterb caused an inhibition of carbon transformation of -39.3% and -45.2% at 16.00 mg and 27.00 mg Dinoterb per kg soil dry weight, respectively, 28 d after application
Reported statistics and error estimates:
A statistical evaluation of the test results was performed by means of a 2-sided Student-t-test (for homogeneous variances at 5% significance level).

Biological Results:

The NOEC was determined to be at 0.265 mg test item/kg soil d.w. since no statistically significant effects were observed at this test item cocnentration.

The EC10 was determined to be >1.31 mg a.i./kg soil d.w. since the inhibition at this highest test item concentration was below 10%.

Table 1: Effects on carbon transformation in soil after treatment with the test item.

Days after application

Control

0.265 mg a.i./kg soil d.w. equivalent to 0.233 kg test item/ha

1.31 mg a.i./kg soil d.w. equivalent to 1.116 kg test item/ha

O2 consumption [mg/kg soil d.w./h]

CV [%]

O2 consumption [mg/kg soil d.w./h]

CV [%]

Deviation from control [%]1

O2 consumption [mg/kg soil d.w./h]

CV [%]

Deviation from control [%]1

0

13.68

1.7

13.89

0.5

+1.51 n.s.

13.17

1.5

 -3.76 *s.

7

12.25

1.0

12.22

3.2

-0.31 n.s.

12.05

0.5

-1.70 n.s.

14

10.98

2.0

10.82

1.8

-1.47 n.s.

10.74

1.2

-2.23 n.s.

28

11.41

1.0

11.42

1.2

+0.05 n.s.

10.95

0.4

-4.04 *s.

The calculations were performed with unrounded values.

CV [%] = Coefficient of Variation

1) based on O2 consumption; - = inhibition; + = stimulation

n.s. = No statistically significant difference to the control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

*s. = statistically significantly different to control (Student-t-test for homogeneous variances, 2-sided, p ≤ 0.05)

Validity criteria fulfilled:
yes

Description of key information

NOEC (28 d): ≥ 1.31 mg a.i./kg soil dw (OECD 216)
EC10 (28 d): > 1.31 mg a.i./kg soil dw (OECD 216)

NOEC (28 d): 0.265 mg a.i./kg soil dw (OECD 217)
EC10 (28 d): > 1.31 mg a.i./kg soil dw (OECD 217)

Key value for chemical safety assessment

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

Additional information

The potential effects of the test item on nitrogen (M-460650-01-1 and M-546745-01-1) and carbon transformation (M-460648-01-1 and M-546743-01-1) of soil microorganisms was investigated using natural soil from an uncultivated site. The studies were performed according to OECD 216 (nitrogen transformation) and 217 (carbon transformation) under GLP conditions.
In all studies two application rates were tested (0.233 kg/ha and 1.116 kg/ha corresponding to test concentrations of 0.265 and 1.31 mg a.s./kg soil dw). The test design targets the requirements for plant protection products and they were not initially planned for the derivation of an EC10/NOEC. However, both test designs only differ in the selected concentrations but not in the number of replicates. For the assessment the NOEC/EC10 values were derived based on raw data and the available statistical analysis within the study reports.
In both studies a natural soil was mixed with appropriate amounts of the test item in order to achieve the desired concentrations. Test vessels were incubated under controlled conditions for 28 d. In the studies on nitrogen transformation no inhibition was recorded after 28 d. Thus, the EC10 and NOEC (28 d) values were derived to be > 1.29 mg/kg soil dw and ≥ 1.29 mg/kg soil dw, respectively. 
For carbon transformation a significant inhibition was recorded after 28 d at the concentration of 1.31 mg/kg soil dw (maximum of 6%). Thus, the NOEC (28 d) was set to 0.265 mg/kg soil dw and the EC10 (28 d) was derived to be > 1.31 mg/kg soil dw. In all studies the validity criteria were met and the studies are valid.