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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:
17 March 2015 to 07 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study According to GLP
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
See "details on results". The deviations did not affect the validity or integrity of the study.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Vehicle:
yes
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: For each treatment a volume of 2.0 mL of the appropriate test solution was used to spike 8.0 g of fine quartz sand. After complete evaporation of the solvent under mild conditions (passive evaporation under a slow-running fume cupboard) the sand was mixed into 800 g (dry weight) of soil. The soil was amended with finely ground lucerne meal at 5.0 g/kg dw soil.
Thereafter, the soil was moistened with deionised water to adjust the soil moisture to 45% of the maximum water holding capacity (WHCmax) and distributed equally to the four test vessels per treatment (= four replicates).

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): ethanol
- Concentration of vehicle in test medium (stock solution and final test solution): Stock solution = 203 mg test item/mL in ethanol. The stock solution was also used as test solution for treatment T5 (500 mg test item/kg soil dry weight). To prepare test solutions for the lower treatments defined volumes of the stock solution or higher test solutions were diluted with ethanol.
- Evaporation of vehicle before use: Yes
Test organisms (inoculum):
other: the autochthonous microbial community of a freshly sampled field soil (Lufa standard soil type 2.3).
Total exposure duration:
28 d
Remarks:
following test item application
Test temperature:
20 ± 2 °C
Moisture:
Max. water holding capacity (WHCmax) [g/100 g]: 35.4 ± 1.5
Initial soil moisture [% dry mass]: 6.6, equivalent to 18.6% of WHCmax
Soil moisture adjusted for test [% dry mass]: 16, equivalent to 45% of WHCmax
Details on test conditions:
TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH
- Test container (type, material, size): Glass jars with lids, volume 370 mL, diameter 100 mm
- Amount of soil: Each test vessel was loaded with 233.9 g soil fresh weight equivalent.
- No. of replicates per concentration: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4

SOIL INCUBATION
- Method: Following test item application the soils were incubated for a test period of 28 days at 20 ± 2 °C in the dark. All test vessels were weighed once per week and weight loss was compensated with deionised water to keep the soil moisture within a range of ± 5% of 45% WHCmax.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Supplier: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Obere Langgasse 40, D-67346 Speyer, Germany.
- Geographical reference of sampling site (latitude, longitude): Germany, Rhineland-Palatinate, Offenbach, field name „rechts der Landauer Str.“ field number 826/7.
- Treatments with pesticides or fertilizers: The sampling location of the soil was uncultivated during the last four years and has not received pesticides within the last 4 years; in 2014, the sampling location was fertilised three times (June, September and December) with 3500 kg/ha CaO, each, and once (December) with 1463 kg/ha MgO.
- Depth of sampling: 20 cm
- Soil Texture:
- % sand: 59.7 ± 1.6
- % silt: 33.8 ± 0.1
- % clay: 6.5 ± 1.6
- Organic Carbon % 0.67 +/- 0.03%
- Soil taxonomic classification: Sandy Loam
- pH (0.01 M CaCl2): 5.7 ± 0.6
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in g/100 g dry matter): 35.4 ± 1.5
- Cation exchange capacity [meq/100 g]: 7.5 ± 0.9
- Pretreatment of soil: Finely ground lucerne meal was incorporated in to the bulk test soil on day 0 of the test (test start) at a dosing of 5.0 g/kg dry weight soil immediately before test item application.
- Storage (condition, duration): The soil arrived at the Test Facility on March 05, 2015 and was acclimated at a temperature of 20±2°C until March 12, 2015.
- Initial microbial biomass as % of total organic C: 1.07

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Soil nitrate concentration was determined in soil extracts on day 0 and day 28 after test item application.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10.0, 100 and 1000 mg test item/kg soil dry weight
- Results used to determine the conditions for the definitive study: At a test item concentration up to and including 10.0 mg/kg soil dw results were comparable to the solvent control. A slight reduction in nitrate was observed in soil treated at 100 mg/kg soil dw. Nitrate concentration was clearly reduced compared to the solvent control by 53.9% on day 28 at a test item concentration of 1000 mg/kg soil dw soil.
Nominal and measured concentrations:
Nominal: 6.17, 18.5, 55.6, 167 and 500 mg/kg soil dw
Control and solvent control.
Reference substance (positive control):
no
Remarks:
not required
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
167 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
VALIDITY CRITERIA
The coefficient of variation (CV) of control samples regarding the nitrate concentration was 2.7% on day 0 and 17.1% on day 28 of the test. The CV of solvent control samples was 0.4% on day 0 and 11.2% on day 28. Although the maximum deviation in the control on day 28 was slightly above the 15% validity criterion, the validity criterion was fulfilled for the solvent control. Since all data were related to the solvent control, the validity criterion was fulfilled.

NITRATE CONCENTRATION IN SOIL
Throughout the 28 day study, soil moisture of all treatments was within the range of ±5% of the soil moisture adjusted at the start of the test.
At test start (day 0) nitrate was measured in sub-samples of the bulk soil before adding lucerne meal, and after adding lucerne meal in sub-samples of all test-item treated soil and control soils, and in a NO3 standard solution.
At the end of the test (day 28) nitrate was measured in sub-samples of all treated and control soils, and in a NO3 standard solution.
Measured NO3 concentrations of the NO3 standard solution were 96.2 % of nominal on day 0 and 94.2% on day 28.
Nitrate concentration of the bulk soil used to set up the different treatments and controls was 68.7 ± 1.3 mg kg-1 soil dw on day 0 before adding lucerne meal (mean and standard deviation, n = 4).
After adding lucerne meal on day 0 of the test, mean nitrate concentration of the control soil and the solvent control soil was 83.7 ± 2.3 mg kg-1 soil dw and 82.9 ± 0.3 mg kg-1 soil dw, respectively (mean and standard deviation, n = 4).
Differences between the untreated control and the solvent control were statistically not significant; i.e. the solvent had no measurable effect on nitrate formation. Hence, results of the test-item treated soils were compared to the results of the solvent control in order to determine test-item induced effects on nitrate formation.
Nitrate concentration in the solvent control increased to 101.4 ± 11.3 mg kg-1 soil dw on day 28. Coefficient of variation (CV) of the solvent control was 0.4% and 11.2% on day 0 and day 28, respectively.
There were no statistically significant differences in nitrate content between the solvent control and any of the test item treated soils at the start of the test on day 0.
On day 28, nitrate concentration in soil treated with the test item at 500 mg/kg soil dry weight was statistically significantly reduced compared to the solvent control.
On day 28 there were no significant differences in nitrate concentration between the solvent control and any of the test item treated soils at test item concentrations up to and including 167 mg test item/kg soil dry weight.
Hence, the NOEC was 167 mg/kg soil dry weight. Due to the lacking of a dose-effect relationship, effect concentrations (ECx) could not be calculated.
Validity criteria fulfilled:
yes
Conclusions:
At 500 mg/kg soil dry weight, which was the highest tested concentration in this study, the test item statistically significantly reduced the nitrate concentration in soil by 14.1% after 28 days.

There were no significant differences in nitrate concentration between the solvent control and any of the test item treated soils at test item concentrations up to and including 167 mg test item/kg soil dry weight. Hence the NOEC was considered to be ≥167 mg/kg soil dw.

Due to the lacking of a dose-response relationship effective concentrations (ECx) could not be calculated.

Based on the above results, the test item, Benzyl Salicylate is considered to have no long-term adverse effects on nitrate formation in soil at concentrations up to and including 167 mg test item/kg soil dry weight.

Description of key information

The effects of Benzyl Salicylate on Soil Microorganisms has been determined. The NOEC was 167 mg/kg soil dry weight.

Key value for chemical safety assessment

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

Additional information

A study was conducted in order to determine possible effects of the test item on soil microorganisms through measuring microbial nitrate formation in treated versus untreated soils after 28 days of incubation.


 


The testing was performed in a Speyer 2.3 soil with an Organic Carbon content of 0.67%.


 


At 500 mg/kg soil dry weight, which was the highest tested concentration in this study, the test item significantly reduced the nitrate concentration in soil by 14.1% after 28 days. There were no significant differences in nitrate concentration between the solvent control and any of the test item treated soils at test item concentrations up to and including 167 mg test item/kg soil dry weight. Hence the NOEC can be considered to be ≥167 mg/kg soil dw. Due to the lacking of a dose-response relationship effective concentrations (ECx) could not be calculated.


 


Based on the above results, the test item, Benzyl Salicylate is considered to have no long-term adverse effects on nitrate formation in soil at concentrations up to and including 167 mg test item/kg soil dry weight.