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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
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:
- Concentrations: all
- Sampling method:
Soil samples (approximately 10 g soil d.w. per replicate) were taken of 0 (3 hours after application) and 28 days after application and the NO3-N-content was determined.
Soil was extracted by adding 50 mL 1 M KCl solution to the equivalent of 10 g soil d.w. and mixed on a rotator at 150 rpm for 60 minutes. Afterwards, the mixtures were centrifuged and analysed.

- Sample storage conditions before analysis:The analysis was performed on day of sampling.
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: lucerne meal (C/N ratio 13.2/1)


APPLICATION OF TEST SUBSTANCE TO SOIL
- Method:

The incubation of the soil samples was performed as a series of individual and equal sub-samples of each treatment group.
150 g soil dry weight (= one sub-sample) per test vessel was weighed. The soil was mixed with 0.5 % (i.e. 0.75 g/150 g soil d.w.) lucerne meal by means of a hand-stirrer (the C/N ratio of the lucerne meal was 13.2/1). One additional soil sample (without lucerne meal) was used for determination of the initial NO3-N-content. The NO3-N-content was 2.87 mg/100 g soil d.w.
The test item was mixed with deionised water and the test solution was then mixed with the soil by means of a hand-stirrer. Water was added to the soil to achieve a water content of approximately 45 % of WHC (see Appendix 2, page 19).
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 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. For details see Appendix 4, page 21.


VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): The test item was mixed with deionised water and the test solution was then mixed with the soil by means of a hand-stirrer.
- Concentration of vehicle in test medium (stock solution and final test solution): Concentration in stock solution: 12.5 and 37.5 mg/mL and 0.035 to 37.5 mg/mL in application solution.
- Evaporation of vehicle before use: not needed since deionized was was used.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.6 - 20.8 °C
Moisture:
41.89 - 44.21 % of WHC (WHC: 38.48 g/100 g dry soil)
Organic carbon content (% dry weight):
1.45
Nitrogen content (% dry weight):
0.14
Details on test conditions:
TEST SYSTEM
- Test container (type, material, size): wide-mouth glass flasks (500 mL)
- Amount of soil: 150 g soil dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: NA

SOIL INCUBATION
- Method: series of individual subsamples

The incubation of the soil samples was performed as a series of individual and equal sub-samples of each treatment group.
150 g soil dry weight (= one sub-sample) per test vessel was weighed. The soil was mixed with 0.5 % (i.e. 0.75 g/150 g soil d.w.) lucerne meal by means of a hand-stirrer (the C/N ratio of the lucerne meal was 13.2/1). One additional soil sample (without lucerne meal) was used for determination of the initial NO3-N-content. The NO3-N-content was 2.87 mg/100 g soil d.w.
The test item was mixed with deionised water and the test solution was then mixed with the soil by means of a hand-stirrer. Water was added to the soil to achieve a water content of approximately 45 % of WHC.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)

As stated in OECD Guideline 216, an agriculturally utilised soil was selected.
The supply, including the data of relevant soil analyses, was conducted by BioChem agrar GmbH.
The soil was removed to a depth of 20 cm as mixed sample. Afterwards, the soil was carefully dried at room temperature, passed through a 2 mm mesh sieve and then stored at a temperature of approximately 4 °C in containers under aerobic conditions in the dark.
Before application, the soil was adapted to test conditions.


- Geographical reference of sampling site (latitude, longitude): 12.694435960 degrees East; 51.403774567 degrees North
- History of site:
Cultivation:
at soil removal (2021) fallow ground
pre-cultivation (2020) fallow ground

- Treatments with pesticides or fertilizers:
Last application of plant protection products: 1990

Application of fertilisers:
organic fertilisers none since 2003
inorganic fertilisers none since 2003

pH (H2O): 6.0
Corg [%]: 1.45
Humus content [%]: 2.49
Carbon content of
microbial biomass [mg C/100 g soil d.w.] 28.09 = 1.94 % of Corg
Nmin [mg/100 g soil d.w.]: 1.99
Total-N [%]: 0.14

Particle size distribution [%]
According to USDA:
clay (< 0.002 mm): 11.2
silt (0.002 - 0.050 mm): 38.3
sand (0.050 - 2.0 mm): 50.5
soil class (USDA): loam


According to ISO 11277:
clay (< 0.002 mm): 8.9
silt (0.002 - 0.063 mm): 31.0
sand (0.063 - 2.0 mm): 60.2
soil class (DIN 4220): loamy sand

Water holding capacity WHC [g/100 g soil d.w.]: 38.48
Water content [g/100 g soil d.w.]: 13.21
Cation exchange capacity [cmol+/kg soil]: 6.1

soil d.w. = soil dry weight


DETAILS OF PREINCUBATION OF SOIL (if any):
Soil removal: 17.02.2021
Drying and sieving (at room temperature): 17.02. - 22.02.2021
Storage (at approximately 4 °C): 22.02. - 08.03.2021
Soil conditioning (under test conditions): 08.03. - 22.03.2021
Application: 22.03.2021


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
A sample of each replicate of each treatment was taken on 0 (about 3 hours after application) and on day 28 after application and the mineral nitrogen content of the soil was determined.

RANGE-FINDING STUDYNo range-finding experiment was performed.
Nominal and measured concentrations:
Control, 1.000, 3.333, 10.00, 33.33, 100.0, 333.3, 1000 mg test item/kg soil dry weight.
Reference substance (positive control):
yes
Remarks:
Dicyandiamide was tested as reference item in a separate study to verify the sensitivity of the test system
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
262.58 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
492.45 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
923.54 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
In a separate study the reference item Dicyandiamide caused an effect of nitrogen transformation of -44.9 %, -53.9 % and -59.3 % at 50 mg, 100 mg and 200 mg Dicyandiamide per kg soil dry weight, respectively, 28 days after application
Reported statistics and error estimates:
The statistical analysis was performed with the software ToxRat Professional 3.3.0 (RATTE, 2018).
The Williams-t-test (one-sided, smaller) was used to compare the control with the independent test item groups. The Logit analysis using linear maximum likelihood regression used for EC10, 25, 50 determination.

Effects on nitrogen transformation in soil after treatment with Trichloroacetic acid

Days after application

Treatment group

 

Mean NO3-N in mg/kg soil dry weight

% deviation from control

0

Control

29.73

-

 

1.000 mg test item/kg soil d.w.

27.93

-6.1

 

3.333 mg test item/kg soil d.w

30.40

+2.2

 

10.00 mg test item/kg soil d.w

30.50

+2.6

 

33.33 mg test item/kg soil d.w

30.07

+1.1

 

100.0 mg test item/kg soil d.w

28.87

-2.9

 

333.3 mg test item/kg soil d.w

29.23

-1.7

 

1000 mg test item/kg soil d.w

30.13

+1.3

28

Control

86.43

-

 

1.000 mg test item/kg soil d.w.

86.40

±0.0

 

3.333 mg test item/kg soil d.w

87.47

+1.2

 

10.00 mg test item/kg soil d.w

91.00

+5.3

 

33.33 mg test item/kg soil d.w

90.77

+5.0

 

100.0 mg test item/kg soil d.w

94.67

+9.5

 

333.3 mg test item/kg soil d.w

73.40

-15.1*

 

1000 mg test item/kg soil d.w

40.37

-53.3*

- = inhibition; + = stimulation

*    statistically significant compared to control on day 28 after application (Williams-t-test,a= 0.05, one-sided smaller)

 The calculations were performed with unrounded values.

Validity criteria fulfilled:
yes
Remarks:
The coefficients of variation in the control were maximum 7.0 % and thus fulfilled the demanded range (<= 15 %).
Conclusions:
EC10: 262.58 mg/kg dw
EC25: 492.45 mg/kg dw
EC50: 923.54 mg/kg dw
Executive summary:

In a GLP 28 -day nitrogen transformation test (OECD 216, Klimisch 1) with Trichloroacetic acid, the EC10, EC25and EC50values for nitrogen transformation (measured as NO3-N-production) were calculated to be 262.58, 492.45 and 923.54 mg test item/kg soil dry weight, respectively.The NOEC for nitrogen transformation was determined to be 100.0 mg test item/kg soil dry. The test fulfilled the validity criteria.

The results of this test are considered relevant and reliable for the risk assessment.

Description of key information








In a GLP 28 -day nitrogen transformation test (OECD 216, Klimisch 1) with Trichloroacetic acid, the EC10, EC25and EC50values for nitrogen transformation (measured as NO3-N-production) were calculated to be 262.58, 492.45 and 923.54 mg test item/kg soil dry weight, respectively.The NOEC for nitrogen transformation was determined to be 100.0 mg test item/kg soil dry. The test fulfilled the validity criteria.








Key value for chemical safety assessment

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

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