Registration Dossier

Administrative data

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:
other: GLP study according to OECD 216

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
yes
Remarks:
The test system (soil) was stored outside the required range of 4 ± 2 °C at 6.1 to 11.0°C. An influence to the test result due to this deviation can be excluded.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
The analyses of the nitrate concentration were carried out within this study. The content of nitrate was measured as nitrate nitrogen in each supernatant of the test mixtures using an analytical kit no. LCK 339 and LCK 340 from company Hach Lange GmbH, Germany.

Three samples from each test assay were taken at the start of exposure, after 7 and 28 days for determination the content of nitrate. Therefore amounts of about 23.4 g were weighed into glass bottles and 100 mL deionized water were added. The soil suspensions were shaken for about 1 hour at 150 rpm with a laboratory shaker at room temperature.
Aliquots of the supernatant of the suspensions were taken and centrifuged for about 30 minutes at 4500 rpm.

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
14996.2 mg test substance were weighed in a 50 mL volumetric flask, dissolved in chloroform and filled up to the mark after solution. The solution was yellowish and clear dissolved. Due to low water solubility application via aqueous stock solution is not possible.

The required amounts of the test substance stock solution for each test concentration were blended with 6 g quartz sand as carrier (equal to 10 g/kg dry weight) and mixed well.
The organic solvent was removed completely by evaporation with compressed-air (checked by smelling test). The test substance / quartz sand mixture was mixed up and added to the soil afterwards.

One portion of soil without luzerne meal was prepared additionally. This portion was mixed with the carrier (quartz sand) containing the test substance in the highest test concentration (prepared like above-mentioned).
The first portion of the prepared soil with luzerne meal was mixed with the carrier without test substance (BC). Five portions of the same soil were mixed with the carrier (quartz sand) containing the test substance in the scheduled test concentrations (TS1-TS5). One portion of the prepared soil without luzerne meal was mixed with the carrier (quartz sand) containing the test substance in the highest test concentrations (TS5-N). The blending of
the test mixtures was done with a mixer.

At start of exposure test assays were filled with ~ 586 g of the test mixtures (~ 500 g Dw) and closed with a perforated aluminum cap. The test assays were incubated in the dark for 28 days respectively at a temperature of 20 ± 2° C. During exposure, the test vessels were weighed to control the water content for seven times (without day 28 of exposure). The loss of weight in the test assays was corrected with deionized water.

Test assays:
703g soil + aliquot of test substance + solvent corresponding to the required dilution of the test substance stock solution + 6g Quartz sand

Test organisms

Test organisms (inoculum):
other: narural soil

Study design

Total exposure duration:
28 d

Test conditions

Test temperature:
20.6-21.2°C
Moisture:
45 ± 5 % of the WHCmax (38.2 +-1.7g/100g)
Details on test conditions:

TEST SYSTEM
pH 5.8-6.0 at start of exposure

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Germany
Batch F2.3 2913 (field fresh)
no fertilizer application for > 4 years
not cultivated > 4 years
Pretreatment of soil: not treated with crop protection products for > 4 years
- soil type: sandy loam (according to USDA), soil type 2.3
- Grain size: sieved to 2mm
- Particle size > 0.063-2mm (sand): 60.5+-5.8%
- Depth of sampling: ca 20 cm
- Sampling quantity: 1300kg
- Weather condition on sampling: Sunshine, air temperature 25°C
- pH (0.01M CaCl2): 6.8
- Initial nitrate concentration for nitrogen transformation test: 0.09 +-0.03%
- Organic carbon content: 1%
- Maximum water holding capacity (in % dry weigth):38.2+-1.7g/100g
- Cation exchange capacity (mmol/kg): 10.7+-0.8meq/100g
- Water content 5.9g/100g dw (measured)
- Preparation of soil by the supplier: Drying 1 day at room temperature, presieving 10mm screen, Last sieving 2 mm screen, water content 6.6g/100g dw
- Storage (condition, duration): in a closed plastic sack at a temperature of 11.0 – 6.1 °C (mv 6.9°C) in the dark
for two month
- Initial microbial biomass as % of total organic C: 538.4 carbon/kg Dw/h (corresponds to 5% of carbon content of the soil)

NITROGEN SOURCE: LUZERNE MEAL
- supplier: Tock GmbH Futtermühle, Wallerfangen-Ihn, Germany
- Crude protein: 17%
- Carotene: 80mg/kg
- Carboncontent: 41.9g/100g
- nitrogen content: 2.4g/100g
- C/N ratio: 17/1

PREPARATION OF THE SOIL
Two days before start of exposure 4766.4 g of the soil were mixed with 509 mL deionized water. The mixture was stored in a laboratory room at room temperature until usage. 18 g luzerne meal was added to 4219.1 g of the watered soil at the start of exposure.
Nominal and measured concentrations:
0 (blank control), 62.5 mg/kg, 125 mg/kg, 250 mg/kg, 500 mg/kg, 1000 mg/kg (dry weight)
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
0 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 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.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
0 d
Dose descriptor:
EC25
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
7 d
Dose descriptor:
EC25
Effect conc.:
207.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
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:
nitrate formation rate
Duration:
0 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 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.:
635.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
EC10 (0 days) was set by expert judgement. EC10 (7 days) was not stated
On day 28 an EC10 value of 636 mg/kg dry weight was calculated. The EC25- and EC50 values were estimated to be >1000 mg/kg dry weight.
The nitrate amounts determined in the test assay without Luzerne meal on day 28 show a significant increase (compared with the same test assay of day 0).
Because the degree of inhibition was less than 25% at the end of exposure the test substance can be evaluated as having no long-term influence on nitrogen transformation in soils.

Applicant's summary and conclusion