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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
Study period:
20th of March 2019 to 1st of August 2019
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:
version 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: (untreated): 1.2; 3.7; 11; 33 and 100 mg/kg dry weight soil
- Sampling method:
Determination of NO3- ions to assess influences on the nitrification process:
At each sampling interval, 20 gram dry weight subsample was taken from each test sample and transferred to a Nalgene bottle. Thereafter, subsamples were extracted with 0.1 M potassium chloride solution for 60 minutes on a shaker table set at 150 rpm, followed by centrifugation for 10 minutes at 3800 rpm. The supernatant was decanted and each sample was additionally diluted with purified reagent water. An aliquot of the diluted extract was centrifuged at 14,000 rpm for 5 minutes and transferred to an HPLC vial. These vials were analysed using a validated HPLC-UV method.

- Sample storage conditions before analysis: The test vessels were covered with perforated parafilm to allow for air exchange while reducing water loss. The test samples were incubated in the dark in an environmental chamber. The temperature was maintained at 20 ± 2 °C and was monitored continuously with a temperature data logger. The soil moisture content of the test samples was checked and adjusted, if necessary, once a week to maintain the soil moisture content at 45 ± 5% of the soil’s maximum water holding capacity.
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Natural soil: The soil utilised for the test was Speyer 2.3 (sandy loam) collected on the 4th of January 2019, seived (2 mm) and characterised by Lufa-Speyer of Germany. No pesticides have been applied for at least four years. The soil was taken from the top 20 cm of the field and freshly-collected samples were shipped to Smithers Viscient in Wareham, Massachusetts, USA. Additional characterisation was conducted by Agvise Laboratories in Northwood, North Dakota and/or Smithers Viscient. The soil was initially stored refigerated (i.e. at approximately 4 °C) and in the dark. Prior to the start of the test, the soil was allowed to acclimate at test temperature for 2 to 28 days (2 days, actual).
The water holding capacity of the soil was 26.4 %. The initial moisture content of the soil was 7.89 % (wet weight basis), therefore, 33 mL of purified reagent water was added per kilogram of soil during dosing to bring the moisture content to 45 ± 5% of the water holding capacity.

The alfalfa (also known as Lucerne meal) , used to amend the test soil was obtained from Morrison's home and Garden in Pluymouth, Massachusetts on the 5th of December 2017. It was determined that the carbon content was 39.9 % and the nitrogen content was 3.0 % Resulting in a ratio between 12/1 and 16/1). The analysis was performed by Agvise Laboratories in Northwood, North Dakota. The alfalfa was amended to the soil at an amount of 5 grams of alfalfa per 1000 grams of soil.

- Other: The selection of nominal concentrations were intended to include both toxicant-effect and no-effect levels as well as an untreated control soil. Therefore, the concentrations were determined to 1.2; 3.7; 11; 33 and 100 mg a.i./kg dry weight soil.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: To determine the concentration, a 2.00 mL aliqout of the test substance was placed in a volumetric flask and the associated weight was 1.9252 g (1.9079 g as active ingredient). The concentration of this aliquot was determined to be 0.95 g/mL. The dosing occurred in the following way: Six 1.0 kg (dry weight equivalent) batches of soil were weighted out into beakers. Approximately half of the soil batch was transferred to a 4 L jar. The test substance was dropwise added to the soil surface followed by adding the remaining half of the soil batch. The soil was mixed by rolling the capped jar for approximately 3 hours at approximately 15 rpm on a rolling mill. The fortified soil was transferred to the bowl of a food mixer and mechanically mixed for 10 minutes at low speed. During this mechanical mixing, the soil was amended with 5.0 g of lucerne meal (i.e. alfalfa) and a purified reagent water was added to the soil in order to bring the moisture content to 45 ± 5% of the soil’s maximum water holding capacity. Subsequently, each batch of soil was divided equally into three replicate test samples in 1 L glass bottle test vessels and covered with perforated parafilm to allow for air exchange while reducing water loss. the test samples were incubated in the dark in an environmental chamber with a temperature maintained at 20 ± 2 °C (monitored with a temperature data logger). The soil moisture content of the test samples was checked and adjusted (if neccessary) once a week to maintain the soil moisture content at 45 ± 5% of the soil’s maximum water holding capacity.

VEHICLE: None
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C
Moisture:
Maintained at approximately 45% of the maximum water holding capacity.
Organic carbon content (% dry weight):
0.6
Nitrogen content (% dry weight):
3
Details on test conditions:
TEST SYSTEM
- Testing facility: Smithers Viscient
- Test container (type, material, size): 1 L glass bottles filled with dry weight soil. Test vessels were covered with perforated parafilm to allow for air exchange and to avoid loss of moisture.
- Amount of soil: 20 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: no vehicle control performed.

SOIL INCUBATION
- Method: series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographical reference of sampling site: Offenbach, Rheinland-Pfalz, Germany "Rechts der Landauer Str."; No 826/7. (No coordinates provided).
- History of site: Not reported
- Vegetation cover: not reported
- Treatments with pesticides or fertilizers: None utilised within the last 4 years.
- Accidental contamination: None reported
- Other: n/a
- Depth of sampling: 20 cm
- Soil texture:
- % sand: 68
- % silt: 26
- % clay: 6
- Soil taxonomic classification: USDA
- Soil classification system: sandy loam
- pH (in water): 6.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in % dry weigth): 45 ± 5%
- Cation exchange capacity (mmol/kg): 57
- Pretreatment of soil: Not reported
- Storage (condition, duration): The soil was initially stored refigerated (i.e. at approximately 4 °C) and in the dark.
- Initial microbial biomass as % of total organic C: 2.2

DETAILS OF PREINCUBATION OF SOIL: n/a

EFFECT PARAMETERS MEASURED: Nitrate concentration at day 0 and day 28. Nitrogen transformation rate at day 28.

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY: None reported
Nominal and measured concentrations:
Nominal: Control, 1.2; 3.7; 11; 33 and 100 mg/kg dry weight soil.
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 100 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.:
> 100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Reported statistics and error estimates:
A relative standard deviation for the untreated control of ≤15% was used to establish validity of the test. The relative standard deviation of the nitrate concentration and formation rate for the untreated control samples ranged from 0.3 to 5.1%.

Table 1: The Concentration and Formation Rate of Nitrate.

Test System

Mean Day 0 Nitrate

Concentration

(mg/kg)

Mean Day 28 Nitrate

Concentration

(mg/kg)

Mean Nitrate

Formation Rate

(mg/kg/day)

Rate Difference from Untreated

Control

(%)

Untreated Control

84.3

253

6.01

NAa

1.2 mg/kg

84.1

243

5.67

-5.67

3.7 mg/kg

83.2

248

5.88

-2.10

11 mg/kg

84.1

255

6.12

1.84

33 mg/kg

84.7

248

5.85

-2.73

100 mg/kg

85.6

245

5.70

-5.09

a         NA = Not Applicable

The table shows the nitrate concentration as well as the formation rate of nitrate. None of the doses provided a significant effect.

Table 2 - Concentration and Formation Rate

System

Sample ID F0319-

Measured Conc. (μg/mL)

Dilution Ratio

Calculated Conc. (mg/kg)

Measured Conc. (μg/mL)

Dilution Ratio

Day 28 Calculated Conc. (mg/kg)

Formation Rate (mg/kg/day)

Untreated Control

148

8.46

10

84.6

26.47

10

265

6.432

Untreated Control

149

8.44

10

84.4

24.45

10

245

5.718

Untreated Control

150

8.4

10

84

24.86

10

249

5.879

Untreated Control

 

 

Mean:

84.3

 

Mean:

253

6.01

Untreated Control

 

 

SD:

0.25

 

SD:

8.72

0.306

Untreated Control

 

 

RSD (%):

0.3

 

RSD (%):

3.45

5.091

1.2 mg/kg

151

8.39

10

83.9

25.27

10

253

6.029

1.2 mg/kg

152

8.44

10

84.4

23.25

10

233

5.289

1.2 mg/kg

153

8.4

10

84

24.33

10

243

5.689

1.2 mg/kg

 

 

Mean:

84.1

 

Mean:

243

5.669

1.2 mg/kg

 

 

SD:

0.22

 

SD:

8.25

0.302

1.2 mg/kg

 

 

RSD (%):

0.26

 

RSD (%):

3.4

5.33

3.7 mg/kg

154

8.34

10

83.4

24.93

10

249

5.925

3.7 mg/kg

155

8.26

10

82.6

25.3

10

253

6.086

3.7 mg/kg

156

8.36

10

83.6

24.15

10

242

5.639

3.7 mg/kg

 

 

Mean:

83.2

 

Mean:

248

5.883

3.7 mg/kg

 

 

SD:

0.43

 

SD:

4.79

0.185

3.7 mg/kg

 

 

RSD (%):

0.52

 

RSD (%):

1.93

3.138

11 mg/kg

157

8.44

10

84.4

26.09

10

261

6.304

11 mg/kg

158

8.57

10

85.7

25.71

10

257

6.121

11 mg/kg

159

8.21

10

82.1

24.83

10

248

5.936

11 mg/kg

 

 

Mean:

84.1

 

Mean:

255

6.12

11 mg/kg

 

 

SD:

1.49

 

SD:

5.28

0.15

11 mg/kg

 

 

RSD (%):

1.77

 

RSD (%):

2.07

2.454

33 mg/kg

160

8.4

10

84

25.25

10

253

6.018

33 mg/kg

161

8.49

10

84.9

25.37

10

254

6.029

33 mg/kg

162

8.53

10

85.3

23.9

10

239

5.489

33 mg/kg

 

 

Mean:

84.7

 

Mean:

248

5.845

33 mg/kg

 

 

SD:

0.54

 

SD:

6.66

0.252

33 mg/kg

 

 

RSD (%):

0.64

 

RSD (%):

2.68

4.307

100 mg/kg

163

8.55

10

85.5

23.17

10

232

5.221

100 mg/kg

164

8.46

10

84.6

26.18

10

262

6.329

100 mg/kg

165

8.66

10

86.6

24.23

10

242

5.561

100 mg/kg

 

 

Mean:

85.6

 

Mean:

245

5.704

100 mg/kg

 

 

SD:

0.82

 

SD:

12.5

0.463

100 mg/kg

 

 

RSD (%):

0.96

 

RSD (%):

5.08

8.12

Table 3 - Gained Validation Values

Criterion  Acceptable Limits  Result 
Specificity  Interfering peaks in blank control samples should be less than 30% of the lowest calibration standard.  Average area of 6.5%, maximum area of 7.6%. 
Linearity  At least 80% of calibration standards yield recovery of 80 to 120% of nominal.  All calibration standards within 80 to 120% 
  The linear regression’s coefficient of determination (r2) should be greater than or equal to 0.990.  r2>0.999 
Accuracy  Mean recoveries of each set of recovery samples should be 70.0 to 110% of their nominal dosed concentration, and ideally 80 to 100%.  LOQ: 97.3% High: 99.7% 
Precision  The relative standard deviation (RSD) for each set of samples should be ≤20%.  Blank Control: 14.1% 
Precision  The relative standard deviation (RSD) for each set of samples should be ≤20%.  LOQ: 1.2% 
Precision  The relative standard deviation (RSD) for each set of samples should be ≤20%.  Untreated Control: 1.3% High: 1.1% 
Limit of Quantification (LOQ) The limit of quantification is the nominal concentration of the LOQ recovery samples.  LOQ = 7.5 μg/g 
Limit Of Detection (LOD)  The LOD is equal to mean + 3 standard deviations (SD) of the blank control samples’ area.  LOD = 2.9 mAU×s 
Method Detection Limit (MDL)  The MDL will be the product of the concentration of the lowest calibration standard and the lowest dilution factor of the samples.  MDL = 5.0 µg/g 
Confirmation of Analyte Identification  A secondary HPLC-UV method will be used to confirm proper identification of the analyte peak.  The second HPLC-UV method confirmed the primary results. 
Validity criteria fulfilled:
yes
Conclusions:
A study investigating the toxicity to soil microorganism was performed during 28 days, conducted according to OECD 216 and in compliance with GLP. A nominal EC10 value of >100 mg/kg soil dw was determined which was the highest concentration tested. The result was based on no long-term effects on the nitrogen transformation rate of soil microflora at environmentally-relevant concentrations.
Executive summary:

A study investigating the toxicity to soil microorganism was performed during 28 days, conducted according to OECD 216 and in compliance with GLP. A nominal EC10 value of >100 mg/kg soil dw was determined which was the highest concentration tested. The result was based on no long-term effects on the nitrogen transformation rate of soil microflora at environmentally-relevant concentrations.

Description of key information

Soil microflora: 28-day EC10: >100 mg/kg dry weight (nominal concentration) (highest concentration tested), nitrate formation rate.

Key value for chemical safety assessment

Additional information

A 28-day toxicity to soil microorganisms test for effects of D6 on nitrate formation rate of soil microflora, tested in a series of concentrations up to 100 mg/kg soil dry weight, has been conducted in accordance with OECD TG 216 (Soil Microorganisms: Nitrogen Transformation Test) and in compliance with GLP. No effects on nitrate formation were observed:

A 28-day EC10 value of >100 mg/kg dry weight (highest concentration tested) has been determined for the effects of the test substance on nitrate formation rate, based on nominal concentrations (Smithers 2019).

 

Analytical verification of test substance concentrations during the earthworm toxicity test indicate that the test substance is relatively stable in the soil. The soil was prepared using a similar method in both the earthworm and microorganisms tests, therefore it is likely that the soil microorganisms were exposed to the test substance throughout the test.