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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:
30 June - 28 July 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)
Deviations:
no
GLP compliance:
yes
Remarks:
Test substance and nitrate calibration standard were not characterised in accordance with GLP standards, however the nitrate standard was characterised under stringent ISO guideline standards.
Analytical monitoring:
no
Details on sampling:
- Concentrations: In addition to the nitrate analysis, a subsample from each test sample was analyzed on day 0 using gas chromatography with mass selective detection and electron impact ionization (GC-MSD/EI) for confirmation of test substance identity and concentration, after extraction with acetonitrile/water (80/20, v/v) and partitioning with cyclohexane. Samples were analyzed using a validated GC-MSD/EI method.
- Sampling method: Recovery samples were extracted three times with 80/20 acetonitrile/purified reagent water (v/v) and cyclohexane. The sample extracts were subsequently diluted into the calibration standard range with an internal standard diluent to a final composition of 0.1% peanut oil in cyclohexane.
- Sample storage conditions before analysis: All primary and secondary stock solutions for the test substance were stored refrigerated (2 to 8 °C) in amber glass bottles fitted with Teflon-lined caps. All primary and secondary stock solutions for the internal standard were stored in a freezer (-25 to -10 °C) in amber glass bottles fitted with Teflon-lined caps. Diluent solutions were prepared fresh on the day of use and discarded after use.
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Natural soil
- Other: Lufa Speyer 2.3

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The control sample was treated the same way as the dosed samples but without being dosed. Six 1.0-kg (dry weight equivalent) batches of soil were weighed out into beakers. Starting with the control, each batch of soil was dosed and mixed as follows. Approximately half of the soil batch was transferred to a 4-L jar. An appropriate amount of the test substance was dosed dropwise across the soil surface using a Hamilton gas tight syringe. The remaining half of the soil batch was then added to the 4-L jar. 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. After dosing and mixing, each batch of soil was divided equally into three replicate test samples in 1-L glass bottle test vessels.

VEHICLE: no vehicle used
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 +/- 2 degrees C
Moisture:
45 +/- 5%
Details on test conditions:
TEST SYSTEM
- Testing facility: Smithers, 790 Main Street, Wareham, Massachusetts 02571-1037
- Test container (type, material, size): 1 L glass bottle test vessels covered with perforated parafilm to allow for air exchange while reducing water loss.
- Amount of soil: approximately 333g
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: not applicable

SOIL INCUBATION
- Method: bulk - The test samples were incubated in the dark in an environmental chamber.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Rheinland-Pfalz, Germany, "rechts der Landauer Str." No. 826/7
- History of site: No pesticides or organic fertilizers used within the last four years.
- Vegetation cover: Not reported
- Treatments with pesticides or fertilizers: No pesticides or organic fertilizers used within the last four years.
- Accidental contamination: Not reported
- Other:
- Depth of sampling: 0 - 20cm
- Soil texture
- % sand: 63
- % silt: 28
- % clay: 9
- Soil taxonomic classification: Sandy loam
- Soil classification system: USDA classification system
- pH (in water): 6.1
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): mean = 55.1 across all treatments and replicates
- Maximum water holding capacity: 26.5g/100g dry soil
- Cation exchange capacity: 5.9 meq/100g
- Pretreatment of soil: Lucerne meal (alfalfa) was used to amend the test soil
- Storage (condition, duration): not reported
- Initial microbial biomass as % of total organic C: 1.59

DETAILS OF PREINCUBATION OF SOIL (if any): Before use, the soil was transferred to an environmental chamber set to maintain a temperature of 20 degrees C and allowed to equilibrate to incubation conditions for 2 - 28 days (11 days actual)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : nitrate concentrations were determined by HPLC-UV on days 0 and 28.

VEHICLE CONTROL PERFORMED: not necessary as no vehicle used
Nominal and measured concentrations:
Nominal: control, 1.2, 3.7, 11, 33, 100 mg a.i./kg dwt
Reference substance (positive control):
no
Key result
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
Key result
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
Key result
Duration:
28 d
Dose descriptor:
NOEC
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:
Difference between the control and treatment soils were tested for significance using the ANOVA and Kruskal-Wallis test statistical programs. The nitrate formation rates were analyzed using the linear interpolation statistical program in CETIS to determine ECx values.

Table 1. Nitrate concentrations and formation rates of soil microorganisms after 28-days' exposure to the test substance

Nominal concentration (mg/kg)

Replicate

Day 0 nitrate concentration (mg/kg)

Day 28 nitrate concentration (mg/kg)

Nitrate formation rate (mg/kg/day)

Control

1

53.3

176

4.38

2

54.4

187

4.73

3

55.0

187

4.71

Mean (RSD)

54.3 (1.30)

183 (2.83)

4.61 (3.51)

1.2

1

55.4

198

5.10

2

52.6

198

5.19

3

54.6

198

5.14

Mean (RSD)

54.2 (2.14)

198 (0.10)

5.14 (0.76)

3.7

1

53.8

181

4.55

2

55.0

181

4.51

3

56.0

181

4.45

Mean (RSD)

54.9 (1.58)

181 (0.18)

4.50 (0.88)

11

1

56.8

189

4.73

2

56.3

188

4.72

3

56.6

190

4.78

Mean (RSD)

56.6 (0.39)

189 (0.43)

4.74 (0.54)

33

1

54.3

169

4.08

2

54.7

178

4.42

3

55.2

176

4.32

Mean (RSD)

54.7 (0.67)

174 (2.41)

4.27 (3.33)

100

1

56.1

169

4.02

2

55.9

206

5.36

3

55.1

179

4.44

Mean (RSD)

55.7 (0.74)

185 (8.50)

4.61 (12.16)

Validity criteria fulfilled:
yes
Remarks:
Relative standard deviation in nitrate concentration and formation rate for the untreated control samples ranged from 1.30 to 3.51% meeting the less than 15% validity criteria.
Conclusions:
28-day EC50, EC10 and NOEC values of >100, >100 and ≥100 mg/kg dry weight have been determined for the effects of the test substance on the nitrogen formation rate of soil microorganisms, based on nominal concentrations. These values are equivalent to >357, >357 and ≥357 mg/kg dry weight when normalised to 2% organic carbon content.

Description of key information

28-day EC50 >100 mg/kg soil dry weight, EC10 >100 mg/kg soil dry weight and NOEC ≥100 mg/kg soil dry weight, effects of the test substance on nitrogen formation rate of soil microflora.

Key value for chemical safety assessment

Additional information

An OECD TG 216 soil microorganism test has been conducted with the registered substance in accordance with ECHA final decision TPE-D-2114497761-36-01/F.

A 28-day EC50 value of >100 mg/kg soil dry weight, an EC10 value of >100 mg/kg soil dry weight and a NOEC value of ≥100 mg/kg soil dry weight have been determined for the effects of the test substance on nitrogen formation rate of soil microflora, in accordance with test guideline OECD 216 and in compliance with GLP (Smithers, 2020). 

These values are equivalent to >357, >357 and ≥357 mg/kg dry weight when normalised to 2% organic carbon content.

Although analytical verification of test substance concentrations was not carried out during this test, test concentrations have been measured in earthworm and plant studies conducted with the registered substance, where similar methods for application of the test substance to soil were used. The measured concentrations from the plant and earthworm studies indicate that the substance is relatively stable in soil throughout the test duration. The test organisms are therefore likely to have been exposed to concentrations of the test substance that are close to the nominal concentration.