Cyclohexanone, 4-methoxy-

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-07 to 2019-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

Since no data on the water solubility of the test item are available, an exact weighed amount of the test item was mixed with finely ground quartz sand, immediately prior to application. This stock mixture was diluted with quartz sand in a way that 10 g of the mixture contained the amount of test item required for one replicate to adjust the selected concentration. The treated quartz sand (10 g per replicate) was added to the prepared amount of artificial soil (665 g wet weight) yielding 675 g wet treated artificial soil (corresponding to 500 g dry weight). The test item mixture was then mixed thoroughly with the artificial soil by intensive stirring in a laboratory mixer (Appendix 1).
The order of application was as follows: - quartz sand (untreated control)- test item mixtures (increasing concentrations

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

Origin of the test organism: reared under ambient laboratory conditions in the test facility (original breeding of animals was purchased from “W. Neudorff GmbH KG”, An der Mühle 3, 31860 Emmerthal, Germany) Holding and breeding under laboratory conditions Breeding medium: mixture of horse manure, straw, peat (1:1:1) origin: horse manure and straw were purchased from farmers, peat was purchased from the company “Torfwerk Moorkultur Ramsloh”, Germany Breeding conditions: breeding containers (50 cm x 40 cm x 30 cm) diffuse light. Temperature: about 20 °C moist soil; pH: about 7 Age of animals used in the test: adult worms (approximately 4 months old with clitellum)

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

19.5 – 21.4 °C

pH:

guideline requirement: 6.0 ± 0.5; test start: 5.97 – 6.00; test end: 5.66 – 5.77

Moisture:

guideline requirement: 40 – 60 % of WHC (g/100 g soil d.w.):*(difference between start and end of the test: ≤ 10 %) test start: 34.9 – 35.1 (equivalent to 56.1 – 56.4 % of WHC) test end: 34.4 – 34.9 (equivalent to 55.3 – 56.1 % of WHC) (difference between start and end of the test: max. 2.0 %)

Details on test conditions:

Test vessel: plastic vessel of Bellaplast (inside dimensions: about 16.5 cm x 12 cm x 6 cm) with a lid pervious to air and light ;Number of animals/test vessel (= replicate): 10; Number of replicates/ control group: 8; Number of replicates/ treated group: 4; Number of animals/ treatment group: 40 (control group: 80); The test vessels were set up at random in the test room.

Nominal and measured concentrations:

10 – 18 – 32 –56 – 100 mg pure metabolite (corresponding to 10.08, 18.15, 32.26, 56.45, 100.81 mg test item)/kg dry weight mixed into artificial soil.

Reference substance (positive control):

yes

Remarks:

Toxic standard: 5 and 10 mg Maypon Flow/kg soil d.w.; control: untreated

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass change

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

At test start, the fresh weight of the earthworms used was in a range of 300 – 490 mg/worm. The physico-chemical parameters measured at the start and at the end of the tests met the guideline requirements. Mortality rates of 0.0 % to 2.5 % and 0.0 % were recorded in the test item treatment groups and in the control group, respectively (Table 3). No statistically significant mortality compared to the control was observed at any test item concentration (Multiple Sequentially-rejective Fisher Test After Bonferroni-Holm,  = 0.05,one-sided greater). No pathological symptoms and no effects on behaviour (including feeding activity) of the worms were observed during the test. The weight change of adult worms ranged between 26.8 and 31.6 % in the treated groups and 29.1 % in the control group. The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control groups at any concentration tested (Williams-t-test,  = 0.05, two-sided, Table 2). No statistically significant effects (Williams-t-test,  = 0.05, one-sided smaller) on the number of juveniles compared to the control group were found at any concentration tested (Table 3). Therefore, the No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 100 mg pure metabolite/kg soil dry weight, and the Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 100 mg pure metabolite/kg soil dry weight.

Results with reference substance (positive control):

To verify the sensitivity of the test system, the reference item Maypon Flow (Carbendazim, SC 500) is routinely tested at concentrations of 5 and 10 mg product/kg soil dry weight. In the most recent study with Maypon Flow (BioChem project No. 19 48 TEC 0009, reported 24 January 2019), the number of juveniles was reduced by 58 and 99 % at concentrations of 5 and 10 mg product/kg soil dry weight (mean number of juveniles = 71.3 and 1.5) after 8 weeks of test duration when compared to control (mean number of juveniles = 169).

Validity criteria fulfilled:

yes

Conclusions:

4-methoxycyclohexanone showed no statistically significantly adverse effects on survival, growth and reproduction of the earthworm Eisenia andrei in artificial soil up to and including 100 mg pure metabolite/kg soil dry weight, i.e. the highest concentration tested. Therefore, the No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 100 mg pure metabolite/kg soil dry weight, and the Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 100 mg pure metabolite/kg soil dry weight.

Executive summary:

4-methoxycyclohexanone showed no statistically significantly adverse effects on survival, growth and reproduction of the earthworm Eisenia andrei in artificial soil up to and including 100 mg pure metabolite/kg soil dry weight, i.e. the highest concentration tested. Therefore, the No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 100 mg pure metabolite/kg soil dry weight, and the Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 100 mg pure metabolite/kg soil dry weight.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-11-29 to 2008-01-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

With respect to the properties of the test item (Log Pow> 2) 5% peat instead of 10% peat were used considering the influence on bioavailability (EPPO 2003). At the start of the study, the individual body weight of some of the earthworms was lower than 300 mg, as recommended in the guideline.

Deviations:

yes

Remarks:

see "Remarks"

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

All application mixtures were prepared freshly on the day of application. The test item was mixed into quartz sand. A sufficient amount of the application mixtures was prepared separately for all test concentrations. Uniform masses of the application mixtures were used for all test concentrations. They were thoroughly mixed into the artificial soil of each replicate using a laboratory mixer in the order lowest application rate to highest application rate. Finally, 50 mL of deionized water were added and mixed into each replicate to achieve soil moisture of between 40 % and 60 % of the maximum water holding capacity (WHCmax). The control group was treated first in the same way as described above but with untreated quartz sand only.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Eisenia fetida was used as the test species (Eisenia fetida andrei; strain of Prof Graff, Forschungsanstalt für Landwirtschaft, 38104 Braunschweig, Germany). This strain has been held in our laboratory for several years under the conditions described in the BBA Proposal for an OECD Guideline (ECO 85, = UPEC 15, February 1981): 20 ±2°C, 12:12 hours light-dark cycle. The breeding substrate consists of about 70 % by weight of natural soil, 25 % peat and 5 % straw (dry weight in each case). The animals are fed on ground, dried cattle manure at 14 day intervals. At the same time, the substrate is also replenished with water. The animals are transferred into fresh substrate at half-yearly intervals.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 +/- 2 °C

pH:

5.74 to 5.78

Moisture:

20.7 % water content

Details on test conditions:

For all moisture determinations a defined weight of 10 g of either shredded peat or of artificial soil was dried up to a constant weight in a microwave (“Bosch”, Inventory-No.: 695293) at appropriate capacity. Moisture content was calculated from the weight difference before and after drying.
Peat moisture was determined prior to preparation of the artificial soil. Artificial soil moisture was determined directly after test start (day 0) and at the end of the test. The maximum water holding capacity (WHC max) of the artificial soil was determined in 2 samples of the artificial soil: the soil was filled into a cylindrical vessel made of 2A-steel (diameter 6 cm, height 7.8 cm). The bottom of the steel vessel is perforated by 19 holes with diameters of 4 mm. The perforated bottom was covered by gauze or filter paper to avoid loss of soil. The vessel was put into a water-filled bucket for at least 3 hours with the water level lying above the soil surface. After this time, the cylinder was removed and was suspended or was hung up to allow the water to drip off for 2 hours. The maximum water holding capacity of the artificial soil was determined by drying the soil and weighing it afterwards. For determination of pH, approx. 50 g of the test soil (two samples of the soil at the start and at the end of the test respectively) were resuspended in 100 ml of 0.1 mol/L KCl solution. The pH was determined with an electronic pH meter.

Nominal and measured concentrations:

nominal concentrations of 62.5, 125, 250, 500 and 1000 mg test item / kg dry weight soil

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

125 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

In this study the LC50 (14 d) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be greater than 1000 mg test item/kg dry weight artificial soil. The Lowest Observed Effect Concentration (LOEC) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be 250 mg test item /kg dry weight soil. The No Observed Effect Concentration (NOEC) was determined to be 125 mg test item /kg dry weight soil, based on effects on bodyweight. No morphological and behavioural effects were observed.

Results with reference substance (positive control):

None reported

Validity criteria for the measurement of the toxicity to soil macroorganisms:

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
Where possible Eisenia foetida should be used. Other species may be used if the necessary methodology is available.	Test speicies was Eisenia foetida.	Yes
The mortality in the controls should not exceed 10 % at the end of either test.	The mortality rate in the control was below 10 % which can be regarded as the limit for natural mortality.	Yes
The test should be performed at a temperature within the range 20 ± 2°C.	The test was performed between 20 ± 2°C.	Yes
One concentration resulting in no mortality and one resulting in total mortality should be used.	In this study the LC50 (14 d) was determined to be greater than 1000 mg test item/kg dry weight soil.	Not applicable

 

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables'.

Conclusions:

In this study the LC50 (14 d) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be greater than 1000 mg test item/kg dry weight artificial soil. The Lowest Observed Effect Concentration (LOEC) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be 250 mg test item /kg dry weight soil. The No Observed Effect Concentration (NOEC) was determined to be 125 mg test item /kg dry weight soil, based on effects on bodyweight. No morphological and behavioural effects were observed.

Executive summary:

In this study the LC50 (14 d) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be greater than 1000 mg test item/kg dry weight artificial soil. The Lowest Observed Effect Concentration (LOEC) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be 250 mg test item /kg dry weight soil. The No Observed Effect Concentration (NOEC) was determined to be 125 mg test item /kg dry weight soil, based on effects on bodyweight.

 

No morphological and behavioural effects were observed.

Description of key information

In this study the LC50 (14 d) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be greater than 1000 mg test item/kg dry weight artificial soil. The Lowest Observed Effect Concentration (LOEC) of Spirotetramat - 4-methoxycyclohexanon to earthworms (Eisenia fetida) was determined to be 250 mg test item /kg dry weight soil. The No Observed Effect Concentration (NOEC) was determined to be 125 mg test item /kg dry weight soil, based on effects on bodyweight. No morphological and behavioural effects were observed.

 

4-methoxycyclohexanone showed no statistically significantly adverse effects on survival, growth and reproduction of the earthworm Eisenia andrei in artificial soil up to and including 100 mg pure metabolite/kg soil dry weight, i.e. the highest concentration tested. Therefore, the No-Observed-Effect-Concentration (NOEC) was determined to be ≥ 100 mg pure metabolite/kg soil dry weight, and the Lowest-Observed-Effect-Concentration (LOEC) was determined to be > 100 mg pure metabolite/kg soil dry weight.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

100 mg/kg soil dw