2-(1-(3',3'-dimethyl-1'-cyclohexyl)ethoxy)-2-methyl propyl propanoate

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:

From December 14, 2017 to September 17, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 16 December 2015; Date of signature: 31 May 2016

Specific details on test material used for the study:

- Water solubility: 4.7 mg/L at 20°C (experimental study, OECD Guideline 105, flask method modified with slowstirring)
- Vapour pressure: Low volatility: 17.2 Pa at 20°C (experimental study, EU Method A.4, isoteniscope method)
- Log Kow: 4.68 (experimental study, EU Method A.8, flask method)
- Relative density (D 20/4): 0,938 (experimental study, Pycnometer method)
- Chemical stability: Stable in water (SDS)
- Storage conditions: Closed vessel, preferably full, container away from heat sources, and protected from extremes of temperature (SDS). Protected from light, in a refrigerator (temp 2-8 °C), store under Nitrogen in a closed container after first opening (CoA).

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
Sieved and spiked soil was amended with powdered plant material (lupine-green meal) at a plant-soil ratio of 5 g plant per kilogram of soil (dry mass). Three incubation containers per treatment were filled up with an amount of moist spiked soil representing 500 g of dry mass. Four containers were filled up with control soil.

APPLICATION OF TEST SUBSTANCE TO SOIL
The quantity of the liquid test item required to obtain the desired concentrations was mixed with a portion of quartz sand of about 10 g / kg dry mass soil. The mixture and the respective
amount of water needed to adjust the test substrate to 40 – 60 % WHC were added to the test soil, mixed thoroughly and placed into the test container.

VEHICLE:
Not applicable

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

The incubation temperature was measured continuously with a thermograph. With 20.0 – 21.0 °C the permitted range of 20 ± 2 °C was kept.

Moisture:

The moisture content of the soil was maintained during the test at 50% of the WHCmax with a range of 5%.

Details on test conditions:

TEST SYSTEM
- Testing facility: Incubation was done in darkness.
- Test container (type, material, size): no data
- Amount of soil: 10 g / kg dry mass soil
- No. of replicates per concentration: 3
- No. of replicates per control: 4 (the blank control consisted of test soil adjusted to 40 - 60% of the maximum Water Holding Capacity (WHC), 10 g Quartz sand / kg dry mass soil, plant material, but no test item)
- No. of replicates per vehicle control: not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
One single, common, agricultural soil was used for the study. The soil, a loamy sand soil, was taken from the reference soils at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), 57392 Schmallenberg, Germany, and is representative for an agricultural field soil. For at least one year prior to soil sampling at the field, no plant protection products had been applied to the sampling site. No organic and mineral fertilizer had been applied to the soil for six and three months prior to soil sampling, respectively. The soil was sieved to a particle size of 2 mm.
- Soil parameters:
Soil name = RefeSol 01-A
Soil batch = IME-05
Soil texture = Loamy sand
Clay (%) = 7
Silt (%) = 20
Sand (%) = 73
WHC (g H2O/kg soil dry weight) = 293
CECeff (mmol/kg) = 5.8
pH (H2O) = 6.6
Total Org. C (%) = 0.89
Microbial biomass (mg C/kg dry mass soil), calculated from respiration activity = 103
Microbial biomass (% of total Org. C) = 1.2
Total Nitrogen (%) = 0.08
NO3- (mg/kg dry weight) = 31.6

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Samples of each treated and control replicate were analysed for nitrate (powdered plant amended samples) and glucose-induced respiration (samples without powdered plant amendment) at the beginning (day 0) and at the end of the exposure period (28 days).
- Measurements of nitrogen transformation rates: Nitrate was extracted from soil by shaking samples (30 g dry mass) with 0.1 M KCl solution at a ratio of 5 mL of KCl solution per gram dry weight for 60 minutes at 150 rpm. The mixtures were filtered (0.45 μm PET, Graphic Controls®) and the liquid phases were analysed for nitrate photometrically (Spectroquant® PHARO 100). When the analyses could not be applied immediately, the soil samples were stored at minus 20 ± 5 °C for one month at a maximum.

VEHICLE CONTROL PERFORMED: not applicable

RANGE-FINDING STUDY: no

Nominal and measured concentrations:

The nominal concentration in the test containers with test item was 62.5, 125, 250, 500 and 1000 mg/kg dry mass soil.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

161 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% CL: 122 - 190 mg/kg dw

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

304 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% CL: 274 - 338 mg/kg dw

Details on results:

The results of the nitrate measurement are presented as mean values in the Table 6.3.4/1 in "Any other information on results incl. tables".
See Response curve for Nitrogen transformation in the attached graph ("Illustration").

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

A concentration-effect response was obtained and computed. The EC10, EC20 and EC50 for nitrogen transformation were 161 (95% CL = 122 - 190), 200 (95% CL = 164 - 227) and 304 (95% CL = 274 - 338) mg test substance per kg dry soil, respectively.

Table 6.3.4/1: Mean nitrate content (mg/kg) and deviation from control (%)

	Control	62.5 mg TI per kg dw	125 mg TI per kg dw	250 mg TI per kg dw	500 mg TI per kg dw	1000 mg TI per kg dw
Test start
Nitrate [mg/kg]	31.6	31.2	36.8	31.3	35.8	43.8
Day 28
Nitrate [mg/kg]	322.4	291.2	320.8	209.3	46.8	34.3
Deviation [%]		9.7	0.5	35.1	85.5	89.4

TI = Test Item

dw = dry weight

Validity criteria fulfilled:

yes

Remarks:

The variation between replicate control samples was less than +/-15% at test start and test end.

Conclusions:

The EC10 and EC50 for nitrogen transformation were 161 (95% CL = 122 - 190) and 304 (95% CL = 274 - 338) mg test substance per kg dry soil, respectively.

Executive summary:

A study was performed at the Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) to determine the long-term effect of the test substance, after a single exposure, on nitrogen transformation activity of soil microorganisms according to OECD Guideline 216, EU Test Method C.21 and GLP statement.

The inhibitory effect of the test substance on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples and compared to data obtained from control soil samples. The test substance was incubated in loamy sand soil over a period of 28 days in concentrations of 62.5, 125, 250, 500 and 1000 mg/kg dry mass soil. The concentration of 1000 mg/kg dw met the criteria of the most OECD-guidelines dealing with terrestrial test organisms, concerning highest test concentration. A single application of test item was made and the effects on nitrate building rate were determined 28 days after start of incubation.

Four replicates were prepared for a control and three for each concentration. There was a concentration-effect relationship due to the test substance for the relative content of nitrate compared to the control after 28 days of incubation. The EC10 and EC50 for nitrogen transformation were 161 (95% CL = 122 - 190) and 304 (95% CL = 274 - 338) mg test substance per kg dry soil, respectively.

Description of key information

OECD Guideline 216, EU Test Method C.21, GLP, key study, validity 1:

28d-EC10 (nitrogen transformation activity of soil microorganisms) = 161 (95% CL = 122 - 190) mg/kg dry soil, based on nominal concentrations.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

161 mg/kg soil dw

Additional information

Following the European authorities request, a study was performed to determine the long-term effect of the registered substance, after a single exposure, on nitrogen transformation activity of soil microorganisms according to OECD Guideline 216, EU Test Method C.21 and GLP statement. The inhibitory effect of the registered substance on nitrogen transformation was assessed by the determination of nitrate concentration in the soil samples and compared to data obtained from control soil samples. The registered substance was incubated in loamy sand soil over a period of 28 days in concentrations of 62.5, 125, 250, 500 and 1000 mg/kg dry mass soil (4 replicates for a control and 3 replicates for each concentration). A single application of test item was made and the effects on nitrate building rate were determined 28 days after start of incubation. Based on the results of this study, there was a concentration-effect relationship due to the test substance for the relative content of nitrate compared to the control after 28 days of incubation. The EC10 value for nitrogen transformation was 161 (95% CL = 122 - 190) mg/kg dry soil (nominal concentration).