Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

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:

25-08-2010 to 23-11-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met or with acceptable deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Remarks:

Microbial carbon content of the bulk soil : lower than recommended (0.7% instead of 2:1% of TOC). However, nitrate formation rates from added lucerne meal revealed a metabolically active microbial biomass in the test soil. Considered acceptable deviation.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2007 ; signature: March 2009

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Not applicable. The test system was the autochthonous microbial community of a freshly sampled field soil (standard soil Lufa 2.3).
- Other: Finely ground lucerne meal was incorporated in to the bulk test soil on day 0 of the test (test start) immediately before test item application at a concentration of 5.0 g/kg dry weight soil. The pellets were ground to a fine powder (particles < 1 mm) with an electric mill. Nitrogen content (%): 2.79 ; Carbon content (%): 41.03 ; C/N ratio: 14.7. Preparation: Air drying (only until sievable) from day of collection + 4 days with; final sieving to 2 mm on day 4. Then soil moisture and microbial biomass carbon determination.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item is not soluble in water; therefore, a volatile organic solvent was used. The stock solution was prepared in acetone. All concentrations to be tested were based on the test item. The test item was dissolved in an amount of acetone sufficient to prepare a stock solution. This stock solution was used to produce the various dosage solutions of the test item. The stock solution (SL) was prepared by dissolving 265.5 mg of the test item in 20 mL acetone (nominal concentration 13.3 mg/mL). The stock solution was also used as test solution for treatment T5. See table 1 for treatment codes relative to test item concentrations relative to soil and sand and stock solution volumes. Volumes of 10 mL of the higher concentrated test solution were diluted with 10.0 mL of acetone to achieve the next lower test concentration. For each treatment a volume of 1.0 mL of the appropriate test solution was used to spike 4.0 g of fine quartz sand. After complete evaporation of the solvent under a fume cupboard (passive evaporation for approximately 2 hours) the sand was mixed into 400 g (dry weight) of soil. Thereafter, the soil was moistened with deionised water to adjust a soil moisture of 45% of the maximum water holding capacity (WHCmax) and distributed equally to four test vessels (=four replicates).

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable. See above.
- Evaporation of vehicle before use: Yes. See above.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Remarks:

In accordance with the relevant guidelines.

Test temperature:

20 ± 2 °C in the dark

Moisture:

Initial soil moisture [% dry mass]: 7.8 equivalent to 22% of WHC
Soil moisture adjusted for test [% dry mass]: 15.8 equivalent to 45% of WHC

Following test item application, the test vessels were incubated for 28 days at 20 ± 2 °C in the dark. All test vessels were weighed once per week and weight loss was compensated with deionised water to keep the soil moisture within a range of ± 5% of 45% WHCmax.

Organic carbon content (% dry weight):

0.99

Nitrogen content (% dry weight):

0.08

Details on test conditions:

TEST SYSTEM
- Testing facility:
- Test container (type, material, size): Glass jars (volume 1.7 L, height 186 mm, diameter 121 mm) with airtight lids
- Amount of soil: loaded with 100 g soil (d.w. equivalent)
- No. of replicates per concentration: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4

SOIL INCUBATION
- Method: series of individual subsamples ; following test item application, the test vessels were incubated for 28 days at 20 ± 2 °C in the dark. All test vessels were weighed once per week and weight loss was compensated with deionised water to keep the soil moisture within a range of ± 5% of 45% WHCmax.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LUFA, Oberc Langgasse 40, 67346 SPEYER (Batch number: F23310) ; Note: F = field fresh.
- History of site: The sampling location of the soil was uncultivated during the last four years and has neither received fertilisers nor pesticides within the last 4 years
- Vegetation cover: None (in past 4 years).
- Treatments with pesticides or fertilizers: No.
- Accidental contamination: None, indicated during the past 4 years (soil characterisation detailed in the full study report).
- Other: Weather conditions during sampling: sunshine (date recorded in the full study report).
- Depth of sampling: Depth: ea. 20 cm
- Soil texture (USDA particle sizes used to define relative distribution %): of test system:
- % sand: 62.5 ± 4.7
- % silt: 28.7 ± 4.5
- % clay: 8.9 ± 1.5
- Soil taxonomic classification: sandy loam (USDA)
- Soil classification system: silty sand (uS) [German DIN]
- pH (in water): 6.7 +/- 0.3
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): Day 0: Nominal: 26.6 and reported Min: 26.2 ; Max: 26.7 and Mean: 26.5 (99.6% nominal)
- Maximum water holding capacity (in % dry weight): WHCmax = 35.6 ± 3.0 g/100g
- Cation exchange capacity (mmol/kg): 10.0 ± 2.0 meq/100 g (reported in the study report) this equates to: 100.0 ± 20.0 mmol/kg (multiply traditional unit by x10)
- Pretreatment of soil: The test system was the autochthonous microbial community of a freshly sampled field soil (standard soil Lufa 2.3). Finely ground lucerne meal was incorporated in to the bulk test soil on day 0 of the test (test start) immediately before test item application at a concentration of 5.0 g/kg dry weight soil. The pellets were ground to a fine powder (particles < 1 mm) with an electric mill. Nitrogen content (%): 2.79 ; Carbon content (%): 41.03 ; C/N ratio: 14.7. Preparation: Air drying (only until sievable) from day of collection + 4 days with; final sieving to 2 mm on day 4. Then soil moisture and microbial biomass carbon determination.
- Storage (condition, duration): See above.
- Initial microbial biomass as % of total organic C: Microbial carbon content of the bulk soil : lower than recommended (0.7% instead of 2:1% of TOC). However, nitrate formation rates from added lucerne meal revealed a metabolically active microbial biomass in the test soil. Considered acceptable deviation.

DETAILS OF PREINCUBATION OF SOIL (if any): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : (1) Soil Organic Carbon and Microbial Biomass Carbon ; (2) Measured NO3 concentrations of the NO3 standard solution on day 0 and on day 28 AND Mean, minimum and maximum nitrate concentration of the untreated soil (control), and maximum deviation from the mean at the start (day 0) and on 28 of the test (n = 4) ; (3) Absolute [mg/kg soil dw] and relative [% of solvent control] nitrate concentration and relative inhibition [% of solvent control] in soils treated with test item and incubated for 28 days ; (4) Daily nitrate formation rate and relative inhibition [%] in soils treated with test item and incubated for 28 days.

VEHICLE CONTROL PERFORMED: Yes.

RANGE-FINDING STUDY
- Test concentrations: Not performed.
- Results used to determine the conditions for the definitive study: Definitive test concentrations ranged from : 2.0 mg/kg soil dry weight (equal to the predicted environmental concentration, PEC) to 32 mg/kg soil dry weight (16 times the PEC).

Nominal and measured concentrations:

Nominal concentrations: Blank control, Solvent control, 2.0, 4.0, 8.0, 16.0, 32.0 mg test item/kg soil (dw).

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 32 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: Not applicable.
- Other: Nominal concentrations: Blank control, Solvent control, 2.0, 4.0, 8.0, 16.0, 32.0 mg test item/kg soil (dw) ; Definitive test concentrations ranged from : 2.0 mg/kg soil dry weight (equal to the predicted environmental concentration, PEC) to 32 mg/kg soil dry weight (16 times the PEC). Nitrate formation rates in soils treated with test item and incubated for 28 days did not show any dose-related response on day 28.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Effective concentrations for the endpoint nitrate concentration in soil on day 28 were determined by applying the probit analysis to the day 28 data of nitrate concentrations in soil extracts.
Normal distribution and homogeneity of variances was tested by the Kolmogorof-Smirnov test and Cochran's test, respectively. The NOEC was determined by the Student-t test and, in case of inhomogeneous variances, by the Welch-t test (two sided, significance a = 0.05).
All statistical tests were performed with the ToxRat statistical software Version 2.10. Full details of the statistical analysis are available in the full study report.

Results:

1. Soil Organic Carbon and Microbial Biomass Carbon

The microbial biomass carbon (Cmic) of the bulk soil (without lucerne meal added) was determined at the beginning of the test by applying the Fumigation-Extraction (FE) method according to ISO (1997).The Cmic was calculated to be 69 µg/g soil dry weight. The organic carbon content of the soil type Lufa 2.3 used in this study was 0.99% of the soil dry weight. Therefore, microbial biomass carbon represented 0.70% of the total organic carbon of the soil at the start of the test. Although the measured microbial biomass carbon of the soil was slightly below the recommended value of 1% of C org. nitrate formation rate was high, thus indicating an active microbial biomass. Nitrate formation was in the range of activity that is usually found in freshly sampled Lufa 2.3 soil with higher measured biomass values. This was considered acceptable.

 

Results of the nitrogen transformation test

Soil moisture of all treatments was within the range of ±5% of the soil moisture adjusted at the start of the test. Nitrate was measured at test start and test end in sub-samples of all treated soils and the control soils, and in a NO3 standard solution.

2. Measured NO3 concentrations of the NO3 standard solution on day 0 and on day 28:

Measured NO3 concentrations of the NO3 standard solution were 99.6% of nominal on day 0 and 99.1% on day 28

Mean, minimum and maximum nitrate concentration of the untreated soil (control), and maximum deviation from the mean at the start (day 0) and on 28 of the test (n = 4):

Mean nitrate concentration of the untreated control soil on day 0 of the test was 41.75 mg/kg soil dw.Which increased to 83.69 mg/kg soil dw on day 28. Maximum deviation from the mean within the four replicates of the untreated control was 9.65%.

Differences between the untreated control and the solvent control were statistically significant; consequently, treatments were compared with the solvent control. Nitrate concentration of treated soils was always higher than nitrate concentration in the solvent control. On day 28, the increase ranged from 3.8% (4.0 mg a.s./kg soil dw) to 37.8% (32.0 mg a.s./kg soil dw) compared to the solvent control.Consequently, there is little or no difference between the controls and the test concentrations when the figures are normalised to the production rate of nitrate. These results rather represent biological variation than an effect caused by the test item. No concentration response relationship was discernible. As no inhibition was observed, effect concentrations (ECx) could not be calculated.

 

3. Table 2.0: Absolute [mg/kg soil dw] and relative [% of solvent control] nitrate concentration and relative inhibition [% of solvent control] in soils treated with test item and incubated for 28 days

Test item concentration / mg/kg soil dw.		NO3 concentration / mg/kg soil dw	±	% of solvent control	±	Inhibition / %
0.0	Control	83.69	2.79	109.0	3.63	-9.0
0.0	Solvent control	76.81	1.63	--	--	--
2.0	PEC	81.94	1.43	106.7	1.87	-6.7
4.0	2x PEC	79.75	8.84	103.8	11.51	-3.8
8.0	4x PEC	86.69	1.09	112.9	1.42	-12.9
16.0	8x PEC	95.06	4.96	123.8	6.46	-23.8
32.0	16x PEC	105.81	3.69	137.8	4.80	-37.8

 

4. Table 3.0: Daily nitrate formation rate and relative inhibition [%] in soils treated with test item and incubated for 28 days.

Test item concentration / mg/kg soil dw.		NO3 formation rate / mg/d kg	±	Relative Inhibition / % of solvent control *
0.0	Control	1.50	0.15	-11.9
0.0	Solvent control	1.34	0.18	--
2.0	PEC	1.52	0.43	-13.4
4.0	2x PEC	1.13	0.40	15.7
8.0	4x PEC	1.36	0.32	-1.5
16.0	8x PEC	1.50	0.26	-11.9
32.0	16x PEC	1.50	0.24	-11.9

* : Negative values indicate advancement

Validity criteria fulfilled:

yes

Conclusions:

The test item had no adverse long-term effects on nitrate formation in soil at concentrations up to and including 32.0 mg test item/kg soil dry weight. The test item 28-day NOEC (nitrate formation rate) ≥ 32.0 mg/kg soil dry weight.

Executive summary:

The toxicity of the test item to soil microorganisms was determined in a 28-day nitrogen transformation test according to OECD TG 216 guideline under GLP. The test item was mixed into sieved field soil (Lufa standard soil 2.3) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg dry weight soil, and incubated for a test period of 28 d at 20±2 °C in the dark. A stock solution was prepared by dissolving the test item in acetone. Test solutions were prepared by a dilution series of the stock solution. The test solutions were used to soak defined amounts of quartz sand which were then mixed into portions of soil. Five test item concentrations, a solvent control (SC) and an untreated control (C) were tested with four replicates, each, and ranged from 2.0 mg test item/mg soil dry weight (predicted environmental concentration, PEC) to 32.0 mg test item/kg soil dry weight (16 x PEC). Soil moisture of test soils was kept within a range of ± 5% of 45% WHCmax. Soil nitrate concentration was determined in soil extracts on day 0 and day 28 day. Daily mean nitrate formation rates were calculated based on the difference in measured soil nitrate concentrations between day 0 and day 28 divided by the number of days. The organic carbon content of the soil type Lufa 2.3 used in this study was 0.99% of the soil dry weight. Therefore, microbial biomass carbon represented 0.70% of the total organic carbon of the soil at the start of the test. Although the measured microbial biomass carbon of the soil was slightly below the recommended value of 1% of organic carbon, nitrate formation rate was high, thus indicating an active microbial biomass. Nitrate formation was in the range of activity that is usually found in freshly sampled Lufa 2.3 soil with higher measured biomass values. This was considered acceptable. The maximum deviation from the mean nitrate concentration in the control was 9.7%. Therefore, the validity criterion of a variability of less than 15% within the control given by the guideline was fulfilled. At the end of the test (day 28) nitrate concentration in soils treated with test item exceeded the nitrate concentration in soil from the solvent control by 3.8% (4.0 mg/kg soil dw) to 37.8% (32.0 mg/kg soil dw). Increased nitrate concentrations in treated soil were already observed at the start of the test (day 0), and, consequently, there is little or no difference between the controls and the test concentrations when the figures are normalised to the production rate of nitrate. Therefore differences observed on day 28 do not seem to be an effect of the test item. It was considered these results rather represent biological variation than an effect caused by the test item. The comparable nitrate production rates in all treatments and the controls during the 28 days of incubation also indicated that the test item had no effect on nitrate production in soil. Hence, effect concentrations (ECx) could not be calculated. Nitrate formation rates in soils treated with test item and incubated for 28 days did not show any dose-related response on day 28. The test item had no adverse long-term effects on nitrate formation in soil at concentrations up to and including 32.0 mg test item/kg soil dry weight. The test item 28-day NOEC (nitrate formation rate) ≥ 32.0 mg/kg soil dry weight.

Description of key information

28d- NOEC (nitrate formation rate) : ≥ 32.0 mg/kg soil (dw) nominal, OECD TG 216, 2010

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

32 mg/kg soil dw

Additional information

Key study : OECD TG 216, 2010 : The toxicity of the test item to soil microorganisms was determined in a 28-day nitrogen transformation test according to OECD TG 216 guideline under GLP. The test item was mixed into sieved field soil (Lufa standard soil 2.3) that was amended with powdered lucerne meal at a concentration of 5.0 g/kg dry weight soil, and incubated for a test period of 28 d at 20±2 °C in the dark. A stock solution was prepared by dissolving the test item in acetone. Test solutions were prepared by a dilution series of the stock solution. The test solutions were used to soak defined amounts of quartz sand which were then mixed into portions of soil. Five test item concentrations, a solvent control (SC) and an untreated control (C) were tested with four replicates, each, and ranged from 2.0 mg test item/mg soil dry weight (predicted environmental concentration, PEC) to 32.0 mg test item/kg soil dry weight (16 x PEC). Soil moisture of test soils was kept within a range of ± 5% of 45% WHCmax. Soil nitrate concentration was determined in soil extracts on day 0 and day 28 day. Daily mean nitrate formation rates were calculated based on the difference in measured soil nitrate concentrations between day 0 and day 28 divided by the number of days. The organic carbon content of the soil type Lufa 2.3 used in this study was 0.99% of the soil dry weight. Therefore, microbial biomass carbon represented 0.70% of the total organic carbon of the soil at the start of the test. Although the measured microbial biomass carbon of the soil was slightly below the recommended value of 1% of organic carbon, nitrate formation rate was high, thus indicating an active microbial biomass. Nitrate formation was in the range of activity that is usually found in freshly sampled Lufa 2.3 soil with higher measured biomass values. This was considered acceptable. The maximum deviation from the mean nitrate concentration in the control was 9.7%. Therefore, the validity criterion of a variability of less than 15% within the control given by the guideline was fulfilled. At the end of the test (day 28) nitrate concentration in soils treated with test item exceeded the nitrate concentration in soil from the solvent control by 3.8% (4.0 mg/kg soil dw) to 37.8% (32.0 mg/kg soil dw). Increased nitrate concentrations in treated soil were already observed at the start of the test (day 0), and, consequently, there is little or no difference between the controls and the test concentrations when the figures are normalised to the production rate of nitrate. Therefore differences observed on day 28 do not seem to be an effect of the test item. It was considered these results rather represent biological variation than an effect caused by the test item. The comparable nitrate production rates in all treatments and the controls during the 28 days of incubation also indicated that the test item had no effect on nitrate production in soil. Hence, effect concentrations (ECx) could not be calculated. Nitrate formation rates in soils treated with test item and incubated for 28 days did not show any dose-related response on day 28. The test item had no adverse long-term effects on nitrate formation in soil at concentrations up to and including 32.0 mg test item/kg soil dry weight. The test item 28-day NOEC (nitrate formation rate) ≥ 32.0 mg/kg soil dry weight.