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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:
9 June 2020 - 7 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:
Details on the test substance including identity, strength, purity, stability were not determined under GLP standards. Calibration standard (nitrate) was characterised prior to use in this study according to stringent ISO guidelines, not GLP standards.
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Speyer 2.3 soil
- Other:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Due to the limited water solubility of the test substance and because the test substance is a liquid at ambient conditions, the test substance was applied directly to soil without a carrier. The control test soil was treated the same way as the dosed test soils 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. A volume of 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. An appropriate amount of the test substance was dosed dropwise across the soil surface using a pipette. 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 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). After dosing and mixing, each batch of soil was divided equally into three replicate test samples in 1-L glass bottle test vessels.

VEHICLE: not used
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 +/- 2 degrees C
Moisture:
45 +/- 5%
Organic carbon content (% dry weight):
0.56
Details on test conditions:
TEST SYSTEM
- Testing facility: Smithers, 790 Main Street, Wareham, Massachusetts 02571-1037
- Test container (type, material, size): 1L glass bottles 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. 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): Offenback, Rheinland-Pfalz, Germany, 'Rechts de Landauer' No. 826
- History of site: No pesticides or organic fertilizers have been applied to the collection site over the past 4 years.
- Vegetation cover: Not reported
- Treatments with pesticides or fertilizers: No pesticides or organic fertilizers have been applied to the collection site over the past 4 years.
- Accidental contamination: Not reported
- Other: Date collected: 19 May 2020
- Depth of sampling: top 20 centimeters
- Soil texture
- % sand: 63
- % silt: 28
- % clay: 9
- Soil taxonomic classification: Speyer 2.3
- Soil classification system: Not reported
- pH (in water): 6.1
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Mean across all vessels (including control and all treatments) 59.1 mg/kg
- Maximum water holding capacity (in % dry weight): 26.5g/100g dry soil
- Cation exchange capacity (mmol/kg): 5.9meq/100g
- Pretreatment of soil: Not reported
- Storage (condition, duration): 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 (4 days actual)
- 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 (4 days actual)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : A subsample from each test sample was analysed for the determination of the concentration of nitrate after 0 and 28 days of incubation

VEHICLE CONTROL PERFORMED: not necessary as no vehicle was used

RANGE-FINDING STUDY not reported
Nominal and measured concentrations:
Nominal: 1.2, 3.7, 11, 33, 100 mg a.i./kg dry weight soil and control
Reference substance (positive control):
no
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
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:
NOEC
Effect conc.:
>= 100 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: None reported. Test material was supplied as a liquid
Reported statistics and error estimates:
Inhibition between the untreated control and the treated soils were tested for significance using the ANOVA and Dunnett’s Multiple Comparison Test statistical programs. The nitrate formation rates were analyzed using the linear interpolation (ICPIN) statistical program in CETIS (Ives 2020) to determine EC10, EC25, and EC50 values. The results were investigated for outliers using the Grubbs Extreme Value Test statistical program in CETIS.

Table 1. The effect of the test substance on the nitrogen formation rates of soil microorganisms

Nominal concentration (mg/kg)

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 (%)

Control

60.6

260

7.12

NA

1.2

59.5

250

6.81

-4.4

3.7

59.9

257

7.05

-1.02

11

59.1

257

7.06

-0.89

33

57.1

245

6.71

-5.85

100

58.2

243

6.59

-7.50

Validity criteria fulfilled:
yes
Remarks:
Standard deviation in the control group was 1.50-2.50%, which is less than 15% outlined in the OECD TG 216 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

EC25 and EC50 (28 d) >100 mg/kg dwt (nominal), nitrate formation rate, soil microorganisms.

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

Key value for chemical safety assessment

Additional information

An OECD TG 216 soil microorganisms (nitrogen transformation) is available for the registration substances.

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.

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