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Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
according to guideline
EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Details on preparation and application of test substrate:
Quartz sand was used as carrier. The required amounts of the test substance were added to about 10 g quartz sand (equal to 10 g/kg soil dry weight) and thoroughly mixed. The control assay was prepared with the same amount of quartz sand without test substance. The test substance was mixed with soil with a carrier.
Test organisms (inoculum):
Total exposure duration:
28 d
Test temperature:
20.2 - 20.9 °C
40 ± 5 % of the WHCmax.
Details on test conditions:
TEST SYSTEM- Soil type: 5M- Batch no.: F5M 0116- Treatment: The soil was not treated with crop protection products for more than 4 years. The sampling field was not cultivated for 4 years and no fertilizer application was performed during the last 4 years before sampling the soil. The information regarding to the soil were provided by the supplier of the soil. The analyses were performed according to GLP.- Sampling depth: Approximately 20 cm- Sampling date: 04 Jan 2016- Weather condition on sampling: No details- Preparation of the soil by the supplier: Drying for 3 days at room temperature, Presieving with 10 mm screen, Last sieving 2 mm screen, water content 9.5 g/100 g dry weight, dry weight 91.4 %.- Supplier: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Obere Langgasse 40, 67346 Speyer, Germany.- Arrival in the testing facility: 19 Jan 2016- Storage: After arrival in the laboratory the soil was stored in a cold storage room in the building Z570 in a closed plastic sack at a temperature from 5.3 to 6.4 °C in the dark for 27 days.- Sand (particle size >0.063 -2.0 mm): 53.5 ± 3.2 % (German DIN) - Soil type according German DIN: Loamy sand (IS)- pH-value (0.01M CaCl2): 7.3 ± 0.1- Cation exchange capacity: 17.4 ± 3.6 meq/100g- Water holding capacity: 40.0 ± 2.3- Grain size: 2 mm- Plant cover: Meadow for the last 5 years (within 2016)- Water content of the soil (measured): 9.3 g/100g Dw (mean value of 2 values)- Initial microbiological biomass (measured): 466.6 ± 1.1 mg carbon/kg Dw (mean value of 3 values, correspond to 4.6 % of carbon content of the soil).- Pre-arrangement: Two days before start of exposure 7651.7 g of the soil were mixed with 470 mL deionized water (the resulting water content corresponds to 40 % of the water holding capacity). The mixture was stored in a laboratory room at room temperature until usage. 35.06 g luzerne meal was added to 8121.7 g of the watered soil at the start of exposure.NITROGEN SOURCELuzerne green gras meal- Supplier: Tock GmbH Futtermühle; 66798 Wallerfangen-Ihn, Weinbachstr. 18-20 - Date of delivery: 18 June 2003- Crude protein: 17 %- Carotene: 80 mg/kg- Content of carbon: 41.9 g/100g- Content of nitrogen: 2.4 g/100g- C/N ratio: 17/1- Size: Dried and pulverizedSAMPLING PROCEDUREThree samples from each test assay were taken at the start of exposure, after 7, 14 and 28 days for determination the content of nitrate. Therefore amounts of about 23.2 g were weighed into glass bottles and 100 mL deionized water were added for extraction. The soil suspensions were shaken for about 1 hour at 150 rpm with a laboratory shaker. The temperature during the extraction were 20.5-20.6 °C at the start of exposure 20.6 °C at day 7 20.5-20.7 °C at day 14 and 20.5-20.6 °C at the end of the exposure. Aliquots of the supernatant of the suspensions were taken and centrifuged for about 30 minutes at 4500 rpm.MEASURING METHODThe content of nitrate was measured as nitrate nitrogen in each supernatant of the test mixtures using the analytical kit no. LCK 339 and LCK 340 from company Hach Lange GmbH, Germany. An appropriate amount of the supernatant was pipetted to the reaction tube and mixed well. After 15 minutes reaction time the test tubes within the reaction solution were placed in the photometer type LPW2 at the 340 nm from company Hach Lange GmbH, Germany. According to the parameters for the test kits which were stored in the photometer the content of nitrate nitrogen was displayed.EXPERIMENTAL PROCEDURE- Test vessels for incubation: As test vessels for the incubation of the prepared soil 2 L glass container with a wide opening were used. They were covered with a perforated aluminum lid.- Preparation of the test assays: The required amounts of the test substance for each test concentration were mixed with quartz sand and then with the soil. The first portion of the prepared soil with luzerne meal was mixed with quartz sand without test substance (BC). Five portions of the prepared soil with luzerne meal were mixed with amounts of quartz sand/test substance mixture (TS1-TS5). The blending of the test mixtures was done with a mixer. At start of exposure the test assays were filled with 928 ± 1 g of the test mixtures (~ 800 g d.w.) 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 eight times. The loss of weight in the test assays was corrected with deionized water.
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/kg soil d.w.- Measured concentrations: no analysis performed.
Reference substance (positive control):
Key result
28 d
Dose descriptor:
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
The test batches remained visually unchanged during exposure period. The coefficient of variation in the replicates of the blank control at the end of the exposure was 1.3 %.
Reported statistics and error estimates:
The statistical evaluation of the data was carried out using ToxRat. If the %inhibition was lower than 0% it was set to 0%. If the %inhibition was greater than 100% it was set to 100%. A curve was fitted between the concentration and the %inhibition via the probit model. This curve was used for the estimation of the EC10, EC25 and EC50. Appropriate weighting for the model fitting via linear transformation were used. The confidence intervals were determined via normal approximation.
Validity criteria fulfilled:

Description of key information

In a 28-day toxicity study, natural soil was exposed to the test substance at nominal concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/kg (dry weight) under conditions in accordance with the guidelines. Luzerne meal was used as nitrogen source. The moistness, pH and temperature were within acceptable guideline specifications. The amount of nitrate formed by microbial processes was determined at start of exposure, after 7, 14 and after 28 days at the end of exposure. The results of the nitrate analyses of the treated replicates were compared to control assays and the nitrate production in the treated replicates was expressed as an inhibition of the process of nitrogen oxidation. On day 28 the EC10 and EC50 values were determined to be >1000 mg/kg dry weight. The results in this study are consistent with all relevant validity criteria and the test is valid according to the guidelines of this study.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
1 000 mg/kg soil dw

Additional information