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Diss Factsheets

Administrative data

toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-06 - 2017-07-05
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
The test system (soil) was stored in the required range of 4 ± 2 °C with one aberration of 6.5 °C. An influence to the test result due to this deviation can be excluded.
GLP compliance:
yes (incl. QA statement)

Test material

Chemical structure
Reference substance name:
Bis(2-propylheptyl) phthalate
EC Number:
EC Name:
Bis(2-propylheptyl) phthalate
Cas Number:
Molecular formula:
1,2-bis(2-propylheptyl) benzene-1,2-dicarboxylate
Details on test material:
- Name of test material (as cited in study report): Palatinol 10-P
- Analytical purity: 98.3 area-% (GC, DB-1), 98.8 area-% (GC, DB-5 HT), see report No 17L00129
- Homogeneity: given
- Lot/batch No.: F151_2017
- Expiration date of the lot/batch: 07 Dec 2018

Sampling and analysis

Analytical monitoring:
Details on sampling:
Three 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 22.8 g were weighed into 250 mL 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. During the extraction the temperatures were 21.0 °C at the start of exposure, 21.0 °C at day 7, 20.9 °C at day 14 and 20.9 °C at day 28. Samples of the soil/water suspensions were taken and centrifuged for about 30 minutes at 4500 rpm.

Test substrate

Details on preparation and application of test substrate:
Test system: Natural soil, Type 2.3

Test substance preparation:
Preparing a stock solution in acetone.
Quartz sand was used as carrier. The required amounts of the test substance stock solution/solvent were added to about 10 g quartz sand (equal to 10 g/kg soil dry weight) and thoroughly mixed. The solvent was completely evaporated. The blank control assay was prepared with the same amount of quartz sand without test substance and the blank solvent control assay was prepared with the same amount of quartz sand with 8 mL acetone without test substance.

Application of the test substance::
The test substance was dosed as aliquots of a stock solution with acetone.

Measuring times of Nitrate nitrogen content: 4
Nitrate extraction method: Extraction the nitrate with deionized water

The first portion of the prepared soil with luzerne meal was not mixed with test substance stock solution or organic solvent (BC). The second portion of the prepared soil with luzerne meal was not mixed with test substance stock solution but with organic solvent (BC solv.). Five portions of the same soil were mixed with the stock solution aliquots with carrier (TS1-TS5). The blending of the test mixtures was done with a mixer. At start of exposure the test assays were filled with 914 ± 1 g of the test mixtures (~ 800 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 eight times. The loss of weight in the test assays was corrected with deionized water.

Test organisms

Test organisms (inoculum):

Study design

Total exposure duration:
28 d

Test conditions

Test temperature:
20.8 – 22.3 °C
Water content of the soil (measured): 7.5 g/100g dw (mean value of 2 values)
Water content of the prepared soil with luzerne meal: 14.2 g/100 g dw.
40 ± 5 % of the WHCmax
Details on test conditions:
- Testing facility: BASF SE, Ludwigshafen, Germany
- No. of replicates per concentration: 3
- No. of replicates per control: 3
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.

- Method: Series of individual subsamples

- Source of substrate: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Obere Langgasse 40, 67346 Speyer, Germany
- Soil type: 2.3
- Batch no.: F2.3 0717
- Depth of sampling: Approx. 20 cm
- Sampling quantity: Approximately 1000 kg
- Sampling date: 14 Feb 2017
- Storage: After arrival in the laboratory the soil was stored in a cold storage room in a closed plastic sack at a temperature from 5.6 to 6.5 °C
Two days before start of exposure 7525.4 g of the soil were mixed with 469 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. 35.0 g
luzerne meal was added to 7994.4 g of the watered soil at the start of exposure.

- Soil taxonomic classification: Silty sand (uS) according to German DIN
- pH: 5.9 ± 0.6
- Plant cover:: Uncultivated for the last 5 years (within 2016)
- Initial nitrate concentration for nitrogen transformation test: 0.08 ± 0.01%
- Cation exchange capacity: 7.6 ± 0.8 meq/100g
- Pretreatment of soil: Drying for 6 days at room temperature, presieving with 10 mm screen, last sieving 2 mm screen, water content 6.7 g/100 g dry weight, dry weight 93.7 %.
- Initial microbial biomass: 352.6 ± 1,6 mg carbon/kg Dw (mean value of 2 valid values of 3 measured values, correspond to 5.3 % of carbon content of the soil)

EFFECT PARAMETERS MEASURED: Nitrogen transformation after 7, 14 and after 28 days.

Nominal and measured concentrations:
Nominal concentrations: 62.5, 125, 250, 500 and 1000 mg of the test substance per kg dry weight of soil.
Additionally soil without test substance as blank control (BC) and soil without test substance but with solvent as blank solvent control (BC solv.).
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
Key result
28 d
Dose descriptor:
Effect conc.:
265 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CL: 240 - 289 mg/kg dw
28 d
Dose descriptor:
Effect conc.:
536 mg/kg soil dw
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CL: 510/562 mg/kg dw
Details on results:
The test batches remained visually unchanged during exposure period. The coefficient of variation in the replicates of the blank solvent control at the end of the exposure was 0%. The results in this study are consistent with the validity criteria and the test is valid according to the guidelines of this study.
Reported statistics and error estimates:
The statistical evaluation was carried out using Student’s t-test for a comparison of the blank control with the blanc solvent control. The test was performed two-sided using a significance level of 5%.
Because there was a significance at day 7 and day 28 in the Student’s t-test the base value of the blank solvent control was used for the statistical evaluation.
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.

Applicant's summary and conclusion

Validity criteria fulfilled:
Variation of nitrate content in the blank control replicates should be less than ± 15 % at the end of the exposure: The coefficient of variation in the replicates of the blank solvent control at the end of the exposure was 0%.
Executive summary:

In a 28-day toxicity study, natural soil was exposed to Palatinol 10-P at nominal concentrations of 62.5 mg/kg, 125 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg (dry weight) and additionally blank control and solvent blank control, under conditions in accordance with the guidelines were tested. 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 solvent blank control assay and the nitrate production in the treated replicates was expressed as an inhibition of the process of nitrogen oxidation.

On day 28 an EC10 value of 265 mg/kg dry weight, EC25 value of 536 mg/kg dry weight was calculated. The EC50 values were estimated to be >1000 mg/kg dry weight.