Rape oil, oxidized

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2016 - 17 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The requirement of the study is based on:
 Corrigendum to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. Official Journal of the European Union L 136/3.
 Commission Regulation (EU) No 640/2012 of 6 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Official Journal of the European Union L193/1.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

2000

Deviations:

yes

Remarks:

The lower range (18°C) by maximal 0.7°C during the first 20 hours of the test is considered as not affecting the integrity of the study.

Principles of method if other than guideline:

- Principle of test:
The study was conducted in order to determine possible effects of the test item on soil microorganisms through measuring microbial nitrate formation in test item treated versus untreated soils after 28 days of incubation at 20 ± 2°C.
- Short description of test conditions:
The test soil was amended with lucerne meal at 5 g/kg soil dry mass to serve as N-source for the soil microorganisms.
The test item was mixed via spiked quartz sand into sieved field soil (Lufa standard soil type 5M) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg soil dry weight, and incubated for a test period of 28 days at 17.3°C to 21.1°C in the dark.
Nominal test item concentrations in test item treated soil were 95, 171, 309, 556 and 1000 mg/kg soil dry mass. Soil for the control and solvent control was not treated with test item.
- Parameters analysed / observed:
The nitrate concentration in soil was determined on day 0 and at the end of the test on day 28.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is included in report (page 5)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Rape Oil, oxidized (Product name RÜMANOX 804 (Type 18poise/20°C)), Product number ORaps1820, batch number SP5M4616
- Expiration date of the lot/batch: April 2017
- Purity test date: 100% (UVCB substance)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No information
- Solubility and stability of the test substance in the solvent/vehicle: No information
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was insoluble in water. Therefore, a stock solution was prepared by dissolving the test item in acetone
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: The nominal test item concentration of the stock solution was 402 mg test item/mL
- Final preparation of a solid: 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. Sand for the solvent control was spiked with 2.0 mL of acetone without test item. After complete evaporation of the solvent under mild conditions (passive
evaporation under a slow-running fume cupboard) the sand was mixed into 804 g (dry weight) of lucerne amended soil (5.0 g finely ground lucerne meal per kg dry weight of soil).

FORM AS APPLIED IN THE TEST (if different from that of starting material): No

OTHER SPECIFICS: None

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone was used to dilute item, but evaporated before inclusion of the coated quartz into the soil.

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Germany, Rhineland-Palatinate, Mechtersheim, field name „In der Speyerer Hohl“, field number 977. The sampling location of the soil was used as a meadow during the last four years at least and has not received any fertiliser or pesticides within this time.
- Other information: The finely ground lucerne meal (purchased from Semhof, 86753 Möttingen, Germany) 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.

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. Sand for the solvent control was spiked with 2.0 mL of acetone without test item. After complete evaporation of the solvent under mild conditions (passive evaporation under a slow-running fume cupboard) the sand was mixed into 804 g (dry weight) of lucerne amended soil (5.0 g finely ground lucerne meal per kg dry weight of soil).

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2 ml /804g dw soil
- Evaporation of vehicle before use: YES

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20±2°C. Temperature fell below the lower range (18°C) by maximal 0.7°C during the first 20 hours of the test.

Moisture:

Soil moisture adjusted for test [% dry mass]: 18.0 (*), equivalent to 45% of WHCmax

Details on test conditions:

TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH, Flörsheim am Main, Germany
- Test container (type, material, size): /
- Amount of soil: /
- No. of replicates per concentration: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 8

SOIL INCUBATION
- Method: bulk / series of individual subsamples

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): autochthonous microbial community of a freshly sampled field soil (Lufa standard soil type 5M) / Germany, Rhineland-Palatinate, Mechtersheim, field name „In der Speyerer Hohl“, field number 977
- History of site: The sampling location of the soil was used as a meadow during the last four years at least and has not received any fertiliser or pesticides within this time
- Vegetation cover: /
- Treatments with pesticides or fertilizers: No
- Accidental contamination: /
- Other: /
- Depth of sampling: ca. 20 cm
- Soil texture: /
- % sand: 59.3 ± 1.5
- % silt: 30.1 ± 1.4
- % clay: 10.6 ± 0.8
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA
- pH (in water): 7.3 ± 0.1
- Initial Nitrogen content (%): 0.13 ± 0.01
- The finely ground lucerne meal (Nitrogen content: 2.81%) 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.
- Maximum water holding capacity (in % dry weigth): 40.1 ± 2.4
- Cation exchange capacity (meq/100g): 17.7 ± 3.7
- Pretreatment of soil: Air drying (only until sievable) from 16 November 2016 to 17 November 2016; final sieving to 2 mm on 18 November 2016
- Storage (condition, duration): The soil arrived at the test facility on 18 November 2016 and was preincubated at a mean temperature of 20.1°C (range 17.3 to 21.1°C) until the start of the test on 06 December 2016.
- Initial microbial biomass as % of total organic C: 1.6%

DETAILS OF PREINCUBATION OF SOIL (if any): The soil arrived at the test facility on 18 November 2016 and was preincubated at a mean temperature of 20.1°C (range 17.3 to 21.1°C) until the start of the test on 06 December 2016.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : /

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: /
- Results used to determine the conditions for the definitive study: /

Nominal and measured concentrations:

Control, Solvent control, 95, 171, 309, 556, 1000 mg/kg soil dw.

Reference substance (positive control):

not specified

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

< 95 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

other: LOEC

Effect conc.:

<= 95 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.:

582 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

271 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

182 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

Treatment code       Test item concentration       Day 0                     Day 28              Inhibition %
BS                            0.0                                   30.8 ± 0.3               n.d.                           n.a.
C                              0.0                                   42.0 ± 1.2           122.8 ± 1.8               n.d.
SC                            0.0                                   45.8 ± 0.4           122.0 ± 1.2               n.a.
T1                            95                                      42.8 ± 2.5              116.9 ± 1.9*             4.1
T2                            171                                   41.6 ± 0.8              108.3 ± 1.4*            11.2
T3                            309                                   40.5 ± 0.7              92.7 ± 2.7*              24.0
T4                            556                                   43.6 ± 0.6              67.5 ± 2.3*              44.6
T5                           1000                                  45.6 ± 1.4              30.0 ± 1.9*              75.4
BS = bulk soil without lucerne; C = control; SC = solvent control; n.d. = not determined; n.a. not applicable; mean ± standard deviation (sd) n = 4; * significantly different from the solvent control Williams multiple sequential t-test, p<0.05, two-sided).

Results with reference substance (positive control):

None

Reported statistics and error estimates:

STUDENT-t test for Homogeneous Variances: Pair-wise comparison of the two controls by the t test procedure. Significance was Alpha = 0,05, two-sided; Conclusion was that there is no statistically significant difference between Control and SC.
Williams Multiple Sequential t-test Procedure: Comparison of treatments with "SC" by the t test procedure after Williams. Significance was Alpha = 0,05, twosided; Conclusion was that The NOEC is lower than 95 mg/kg.
Probit analysis to select the effective concentrations (ECx) of the test item and their 95%- and 99%- confidence limits (according to Fieller`s theorem); Conclusion was that EC10 value is 181.82 mg/kg and its lower and upper 95% confidence values 120.64 and 234.24, respectively.

Validity criteria fulfilled:

yes

Conclusions:

The study was valid.
The dose dependent reduction in nitrate was observed at all test item concentrations ranging from only 4% at 95 mg/kg soil dry weight to 75% at 1000 mg/kg soil dry weight.
LOEC and NOEC: ≤95 and <95 mg/kg soil dry weight, respectively
EC50 / EC20 / EC10: 582 / 271 / 182 mg/kg soil dry weight, respectively.

Executive summary:

Rape Oil, oxidized effects on Soil Microorganisms were tested according to OECD Guideline for the Testing of Chemicals No. 216 “Soil Microorganisms: Nitrogen Transformation Test” (1998) and under the OECD Principles of Good Laboratory Practice (1997).

The test item was mixed via spiked quartz sand into sieved field soil (Lufa standard soil type 5M) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg soil dry weight, and incubated for a test period of 28 days at 17.3°C to 21.1°C in the dark. A stock solution was prepared by dissolving the test item in acetone. Test solutions were prepared from dilutions of the stock solution with acetone. The test solutions were used to soak defined amounts of quartz sand which were mixed into portions of soil after evaporation of the solvent. Five test item concentrations ranging from 95 mg test item/kg soil dry weight to 1000 mg test item/kg soil dry weight, a solvent control (SC) and a water control (C) were tested with four replicates, each. Soil moisture of test soils was kept within a range of ± 5% of 45% of the maximum water-holding capacity (WHCmax). Soil nitrate concentration was determined in soil extracts on day 0 and day 28.

Initial (day 0) measured nitrate concentration in lucerne amended soil ranged from 40.5 to 45.8 mg/kg soil dry weight.

On day 28, nitrate concentration in the control and solvent control was increased to 123 and 122 mg/kg soil dw, respectively, hence confirming the existence of a metabolically active microbial biomass. Nitrate concentration in soils treated with the test item was significantly lower than in the solvent control. The reduction in nitrate was observed at all test item concentrations ranging from 95 mg/kg soil dry weight to 1000 mg/kg soil dry weight.

The LOEC and NOEC were ≤95 and <95 mg/kg soil dry weight, respectively. The concentration-effect relationship observed on day 28 revealed a 50% effect concentration (EC50) of 582 mg/kg soil dw. The EC20 and EC10 were 271 and 182 mg/kg soil dw, respectively.

The study was valid. The dose dependent reduction in nitrate was observed at all test item concentrations ranging from only 4% at 95 mg/kg soil dry weight to 75% at 1000 mg/kg soil dry weight.

LOEC and NOEC: ≤95 and <95 mg/kg soil dry weight, respectively

EC50 / EC20 / EC10: 582 / 271 / 182 mg/kg soil dry weight, respectively.

Description of key information

The N-transformation into nitrate by microorganisms of a naturalsandy loam soil was after 28 days significantly lower at all test item concentrations ranging from 95 mg/kg soil dry weight to 1000 mg/kg soil dry weight.

In consequence, the LOEC and NOEC were ≤95 and <95 mg/kg soil dry weight, respectively. The concentration-effect relationship observed on day 28 revealed EC50 of 582 mg/kg soil dw and EC10 182 mg/kg soil dw.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:

582 mg/kg soil dw

Long-term EC10 or NOEC for soil microorganisms:

182 mg/kg soil dw

Additional information

A toxicity study on Soil Microorganisms N-transformation was performed according to OECD TG 222 and in compliance with GLP.

The microbial nitrate formation by the microorganisms of a natural soil was followed for 28 days while exposed to 200, 300, 450, 670 and 1000 mg test item/kg soil dry weight.

After adding lucerne meal to the bulk soil on day 0, mean nitrate concentration was 42.0 ± 1.2 mg/kg soil dw in the control soil and 45.8 ± 0.4 mg kg-1 soil dw in the solvent control. Nitrate concentration in the test item treated soils on day 0 ranged from 40.5 to 45.6 mg/kg soil dw. Measured nitrate concentrations in soil of the control and of the solvent control were 122.8 ± 2.2 mg/kg soil dw and 122.0 ± 1.5 mg/kg soil dw, respectively, (mean ± standard deviation, n = 4). Compared to the solvent control nitrate concentration in soils treated with the test item was significantly reduced at all tested test item concentrations ranging from 95 to 1000 mg/kg soil dw. Hence, the LOEC and NOEC were ≤95 and <95 mg/kg soil dry weight, respectively, which was the lowest tested test item concentration in soil. There was a clear dose-effect relationship with reductions in nitrate ranging from 4.1% at 95 mg/kg soil dw to 75.4% at 1000 mg/kg soil dw. The 50% effect concentration (EC50) was calculated to be 582 mg/kg soil dw. EC20 and EC10 were 271 and 182 mg/kg soil dw, respectively.