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Ecotoxicological information

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:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
adopted 21 January 2000
Deviations:
yes
Remarks:
The ratio of quartz sand to dry soil has been increased from 10 g/kg to 67 g/kg in order to ensure homogeneous distribution of the test item-sand mixture in the soil. This deviation had no impact on the outcome of the study.
Qualifier:
according to
Guideline:
EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008
Deviations:
yes
Remarks:
The ratio of quartz sand to dry soil has been increased from 10 g/kg to 67 g/kg in order to ensure homogeneous distribution of the test item-sand mixture in the soil. This deviation had no impact on the outcome of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: not applicable (UVCB substance)
Analytical monitoring:
not required
Vehicle:
yes
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: lucerne meal with a C:N ratio of approx. 15.8:1

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Application solutions for the different test concentrations were prepared by dispersing the test material in dichloromethane. A volume of 20 mL of the respective application solution was applied dropwise on the surface of 50 g of quartz sand and mixed using a mortar. After evaporating the solvent overnight, the test material-quartz sand mixture was thoroughly mixed with 750 g dry soil. The ratio of quartz sand to dry soil has been increased from 10 g/kg to 67 g/kg in order to ensure homogeneous distribution of the test item-sand mixture in the soil. This deviation had no impact on the outcome of the study.

VEHICLE:
- Chemical name of vehicle: dichloromethane
- Evaporation of vehicle before use: yes
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
19.5 – 19.9 °C (mean 19.6 °C)
Moisture:
42% of the maximum water holding capacity (MWHC)
Details on test conditions:
TEST SYSTEM
- Testing facility: climate cabinet
- Test container (type, material, size):1L glass incubation flasks stoppered with cotton wool plugs
- Amount of soil: 160.75 g dry soil per flask (incl. 0.75 g lucerne meal and 10 g quartz sand)
- No. of replicates per concentration: 1
- No. of replicates per control: 1
- No. of replicates per vehicle control: 1

SOIL INCUBATION
- Method: bulk

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), Speyer/Germany.
- History of site
- Sampling date: 04 October 2016
- Arrival at testing site: 10 October 2016
- Vegetation cover: no cultivation between 2012 and 2016
- Treatments with pesticides or fertilizers: no pesticides between 2012 and 2016; no fertilizers between 2015 and 2016
- Depth of sampling: approx. 20 cm
- Soil texture
- % sand: 59.6 ± 1.4
- % silt: 33.6 ± 0.5
- % clay: 6.8 ± 1.6
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- pH (in water): 6.7
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
control: 41.3 mg NO3-/kg dry soil
solvent control: 43.1 mg NO3-/kg dry soil
treated soil: 48.9 – 51.8 mg NO3-/kg dry soil
- Maximum water holding capacity (in % dry weight): 35.6%
- Cation exchange capacity: 7.6 ± 0.8 meq/100 g
- Pretreatment of soil: sieved to 2 mm;
- Storage (condition, duration):
After arrival of the soil at the testing laboratory, the soil was stored in the dark under aerobic conditions at approx. 4°C for three days.
- Initial microbial biomass as % of total organic C: 4.5 %

DETAILS OF PREINCUBATION OF SOIL (if any): the soil was acclimatised to the test conditions (dark, approx. 20°C) for 27 days until test start on (DAT 0)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : nitrate formation after 28 days of exposure

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY: not performed
Nominal and measured concentrations:
nominal: 62.5, 125, 250, 500, and 1000 mg/kg dry soil
Reference substance (positive control):
yes
Remarks:
6.7 mg nitrapyrin/kg dry soil
Key result
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrate concentration at Day 28
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrate concentration at Day 28
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: nitrate concentration at Day 28
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: nitrate formation rate inhibited by 166% versus control
Reported statistics and error estimates:
Differences in nitrate concentrations were tested
- between the control and the solvent control using Student t-tests (two-sided at DAT 0 and DAT 28, α = 0.05);
- between the test item treatments and the solvent control using Dunnett t-tests (two-sided at DAT 0 and DAT 28, α = 0.05);
- between the solvent control and the reference item treatment using Student t-tests (two-sided at DAT 0, one-sided smaller at DAT 28, α = 0.05).

NOEC and LOEC were determined according to the results of the statistics.

Calculations of ECx of nitrate concentration at day 28 were performed using probit analysis and linear maximum likelihood regression).

No nitrites were detectable in the soil solutions (since nitrites may impact the colorimetric analysis of nitrates, nitrite analysis was performed beforehand).

Validity criteria fulfilled:
yes
Remarks:
The variation between replicate control samples was less than ± 15%.

Description of key information

Toxicity to soil microorganisms: not toxic to soil microorganisms; EC25 for nitrate formation: 1000 mg/kg (OECD 216)

Key value for chemical safety assessment

Additional information

As the test material showed no adverse effects towards soil microorganisms at the highest recommended concentration (EC25 = 1000 mg/kg dry soil weight) in laboratory tests, no NOEC and thus no PNEC could be derived.