Cyclohexanone, 4-methoxy-

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-08-21 to 2019-10-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 226 (Predatory Mite (Hypoaspis (Geolaelaps) Aculeifer) Reproduction Test in Soil)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

soil

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

All test item solutions were prepared freshly on the day of the application. The test item was dissolved in water Solution 1: 0.2526 g test item filled up to a volume of 250 mL with deionised water (100 mg pure metabolite/kg dry weight artificial soil). Solution 2: 140 mL solution 1 was filled up to 250 mL with deionised water (56 mg pure metabolite /kg dry weight artificial soil) Solution 3: 143 mL solution 2 was filled up to 250 mL with deionised water (32 mg pure metabolite /kg dry weight artificial soil) Solution 4: 141 mL solution 3 was filled up to 250 mL with deionised water (18 mg pure metabolite /kg dry weight artificial soil) Solution 5: 139 mL solution 4 was filled up to 250 mL with deionised water (10 mg pure metabolite /kg dry weight artificial soil) A uniform volume of 50 mL was used for all application solutions (starting with the lowest application rate and ending with the highest application rate). The test item was thoroughly mixed into 500 g dry weight artificial soil of each application rate using a laboratory mixer. The control group was treated first in the same way as described above but with 50 mL deionised water only. Afterwards the treated artificial soil of each application rate and the control was portioned out. Each test vessel of the 8 control replicates and the 4 treatment replicates of each concentration plus the one for measurement purpose was filled up with 20±1 g dry weight artificial soil avoiding compression of the artificial soil. The remaining artificial soil was disposed.

Test organisms (species):

Hypoaspis aculeifer

Animal group:

Acari (soil-dwelling predatory mite)

Details on test organisms:

The soil mite Hypoaspis aculeifer (Acari: Laelapidae) used in this test has been bred at Bayer AG (formerly Bayer CropScience AG) since 2002. The strain was originally obtained from ECT Oekotoxikologie GmbH, 65439 Flörsheim am Main. Mites from a synchronised culture at an age of 28 days after start of egg laying were used in this study.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 +/- 2 °C

pH (if soil or dung study):

5.4 to 6.2

Humidity:

16.65 to 18.89 %

Photoperiod and lighting:

16-hour light to 8-hour darkness photo period. The light intensity at light period was between 400 - 800 Lux

Details on test conditions:

Directly after application of the test item, the adult, fertilized, females (28 days after start of egg laying for three days) were exposed to the control and treatment vessels. This was achieved by putting 10 females individually onto the surface of the artificial soil using a fine brush. The transfer of the test animals was finished within two hours after the application of the test item. After a period of 14 days, the surviving adults and the living juveniles per test vessel were extracted, applying a temperature gradient. The content of each test vessel was carefully transferred to sieve vessels (mesh size approximately 0.8 mm). The vessels were positioned in MacFadyen-Extractor. The temperature was increased from approximately 25 to 40 °C within two days. Extracted mites were collected in a fixing solution (20% ethylene glycol, 80% deionised water, 2 g detergent/L). All Hypoaspis aculeifer (adult females and juveniles) were counted under a binocular.

Nominal and measured concentrations:

Solution 1: 0.2526 g test item filled up to a volume of 250 mL with deionised water (100 mg pure metabolite/kg dry weight artificial soil).

Solution 2: 140 mL solution 1 was filled up to 250 mL with deionised water (56 mg pure metabolite /kg dry weight artificial soil).

Solution 3: 143 mL solution 2 was filled up to 250 mL with deionised water (32 mg pure metabolite /kg dry weight artificial soil).

Solution 4: 141 mL solution 3 was filled up to 250 mL with deionised water (18 mg pure metabolite /kg dry weight artificial soil).

Solution 5: 139 mL solution 4 was filled up to 250 mL with deionised water (10 mg pure metabolite /kg dry weight artificial soil)

Reference substance (positive control):

no

Key result

Duration:

14 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:

14 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:

Mortality: In the control group 3.8% of the adult Hypoaspis aculeifer died which is below the allowed maximum of ≤ 20% mortality. Concerning the mortality of the adult test organisms statistical analysis (Fisher ́s Exact Binomial Test with Bonferroni Correction, one-sided greater, α = 0.05) revealed no significant difference between control group and any treatment group up to and including 100 mg pure metabolite/kg dry weight artificial soil. Therefore, the No-Observed-Effect-Concentration (NOEC) for mortality is ≥100 mg pure metabolite/kg dry weight artificial soil. The Lowest-Observed-Effect-Concentration (LOEC) for mortality is >100 mg pure metabolite/kg dry weight artificial soil. Reproduction: Concerning the number of juveniles statistical analysis (Williams t-test, one-sided smaller, α = 0.05) revealed no significant difference between control group and any treatment group up to and including 100 mg pure metabolite/kg dry weight artificial soil. Therefore, the No-Observed-Effect-Concentration (NOEC) for reproduction is ≥100 mg pure metabolite/kg dry weight artificial soil. The Lowest-Observed-Effect-Concentration (LOEC) for reproduction is >100 mg pure metabolite/kg dry weight artificial soil. Due to the lack of a concentration-response relationship no reliable ECx-calculation is possible. Therefore no EC10/EC20-value can be reported.

Validity criteria fulfilled:

yes

Conclusions:

NOEC adult mortality: ≥ 100 mg pure metabolite/kg dry weight artificial soil

LOEC adult mortality: > 100 mg pure metabolite/kg dry weight artificial soil

Executive summary:

NOEC _{adult mortality}: ≥ 100 mg pure metabolite/kg dry weight artificial soil

LOEC _{adult mortality}: > 100 mg pure metabolite/kg dry weight artificial soil

Reference 2

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-10 to 2019-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

soil

Analytical monitoring:

yes

Vehicle:

no

Details on preparation and application of test substrate:

Since data on the water solubility of the test item are not available, the test item was thoroughly mixed with quartz sand (= stock mixture). Subsequently the obtained stock mixture was diluted with quartz sand to prepare further test item mixtures (serial dilution) containing the amount of test item which was required to adjust the test item concentration of the respective treatment group (Appendix 1). Afterwards, the test item mixtures were thoroughly mixed with the artificial soil. The preparation of the test substrate was performed in the following order: first the untreated control and thereafter the test item treated groups with increasing concentration. Immediately after application, the test soil was filled into the test vessels and incubated under test conditions.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

Origin of the test organisms: originally purchased from “Biologische Bundesanstalt (BBA)”, Berlin-Dahlem, Germany in May 2000
Origin of the test organisms reared in the laboratory of the test facility used in the test: under ambient laboratory conditions

Limit test:

no

Total exposure duration:

28 d

Test temperature:

19.3 – 21.6 °C

pH (if soil or dung study):

guideline requirement: 6.0 ± 0.5; test start: 5.89 – 5.97; test end: 5.76 – 5.77

Photoperiod and lighting:

source: artificial light (Lumilux L58W) intensity: 580 lux duration: light : dark = 16 h : 8 h

Details on test conditions:

Two days before the start of the test, the dry artificial soil was moistened by adding deionised water to adjust the water content to 40-60 % of WHC. On the day of the test start, the test item was mixed with a small quantity of finely ground quartz sand, such that the required test concentration was achieved once mixed with the artificial soil (10 g treated sand per treatment group). The control substrate contained the corresponding amount of untreated quartz sand only. After thorough mixing, 30 g (dry weight) of the test substrate was placed into each vessel, avoiding compression. The test was started using juvenile collembolans, Folsomia candida, well-fed and 9 - 12 days old.Test organisms of a uniform age were obtained by transferring egg clusters from the breeding containers to new containers with fresh breeding substrate 12 days before test start. After 72 hours, these egg clusters were removed from the containers and the juveniles that had hatched during the preceding 72 hours were fed with granulated dry yeast. After a further 9 days, the test organisms were collected and used for the test. Ten test organisms were randomly introduced to each vessel, using an aspirator. After addition of the test organisms, the test vessels were positioned randomly in a controlled-environment test room, and these positions were re-randomised weekly. The test containers were tightly covered with a lid and briefly opened twice a week for aeration. The test organisms were fed twice during the test (at the start of the test and after 14 days) with approximately 2 mg of granulated dry yeast per test vessel. The pH and water content of the test substrate were determined at the start and at the end of the test. The water content was checked weekly by reweighing the additional test vessels. Water loss was compensated if exceeding 2 % of the initial water content. Four weeks after introducing the test organisms, the parental and juvenile collembolans in the test and control vessels were counted. The test substrate of each replicate was poured into an individual container (with a volume of about 200 mL) and the test organisms were floated off the substrate by the addition of water. To improve the contrast between the white collembolans and surrounding water surface, the water was stained dark with ink. After gentle stirring, the number of parental and juvenile collembolans floating on the surface was determined. Missing parental collembolans were assumed to have died during the test period. Surviving adults and juveniles were counted using a digital image processing system (LemnaTec Scanalyzer), an automated counting technique based on a video camera connected to a digital image storage and analysis system. The extraction efficiency of the extraction method was determined to be 99 % in a separate extraction run using vessels containing a known number of juvenile collembolans kept in untreated test substrate.

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 containing 74.7 % quartz sand, 20 % kaolin clay, 5 % sphagnum peat and 0.3 % CaCO3.

Reference substance (positive control):

yes

Remarks:

Boric Acid

Toxic reference:

standard: 44 – 67 – 100 – 150 – 225 mg boric acid/kg soil d.w.; control: untreated, solvent control: none.

Key result

Duration:

28 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:

28 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:

The test item showed no statistically significantly adverse effects on adult mortality and reproduction of the collembolan Folsomia candida in artificial soil at concentrations up to and including 100 mg pure metabolite/kg d.w. Therefore, the No-Observed-Effect-Concentration (NOEC) for mortality was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for mortality was determined to be > 100 mg pure metabolite/kg d.w. The No-Observed-Effect-Concentration (NOEC) for reproduction was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for reproduction was determined to be > 100 mg pure metabolite/kg d.w.

Results with reference substance (positive control):

To verify the sensitivity of the test system the reference item boric acid is routinely tested at concentrations of 44, 67, 100, 150 and 225 mg/kg soil dry weight. The collembolans of the reference test were from the same source culture as those used in the definitive test. In the most recent study (BioChem project No. 19 48 TCC 0057, dated 2019-08-19) the EC50 was determined to be 103 mg/kg soil dry weight. The LC50 was determined to be 161 mg/kg soil dry weight. The NOEC for mortality and for reproduction was determined to be 87 and 122 mg/kg soil dry weight, respectively. The EC50 value for the reproduction was close to the value of 100 mg/kg soil dry weight as stated in OECD 232 (2016). The EC50 therefore showed that the test system is sensitive.

Validity criteria for the measurement of the toxicity to soil arthtopods

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
The duration of the definitive test is 3 weeks for F. fimetaria or 4 weeks for F. candida.	Test species: Folsomia candida Test duration: 28 d	Yes
Mean adult mortality should not exceed 20% at the end of the test in the untreated control group.	The mean adult mortality was 1.3%.	Yes
The mean number of juveniles per vessel should be at least 100 at the end of the test in the untreated control group.	The mean number of juveniles per replicate was > 100 (1174).	Yes
The coefficient of variation calculated for the number of juveniles should be less than 30% at the end of the definitive test in the control group.	The coefficient of variation (mean number of juveniles per replicate) was < 30% (14.9 %).	Yes
A suitable reference substance is boric acid, which should reduce reproduction by 50% at about 100 mg/kg dry weight soil for both species.	Boric acid was used as a reference control. The EC50 of the boric acid in this test was determined to be 103 mg/kg soil dry weight.	Yes

 

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The test item showed no statistically significantly adverse effects on adult mortality and reproduction of the collembolan Folsomia candida in artificial soil at concentrations up to and including 100 mg pure metabolite/kg d.w. Therefore, the No-Observed-Effect-Concentration (NOEC) for mortality was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for mortality was determined to be > 100 mg pure metabolite/kg d.w. The No-Observed-Effect-Concentration (NOEC) for reproduction was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for reproduction was determined to be > 100 mg pure metabolite/kg d.w.

Executive summary:

The test item showed no statistically significantly adverse effects on adult mortality and reproduction of the collembolan Folsomia candida in artificial soil at concentrations up to and including 100 mg pure metabolite/kg d.w.

Therefore, the No-Observed-Effect-Concentration (NOEC) for mortality was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for mortality was determined to be > 100 mg pure metabolite/kg d.w.

The No-Observed-Effect-Concentration (NOEC) for reproduction was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for reproduction was determined to be > 100 mg pure metabolite/kg d.w.

Description of key information

The test item 4-methoxycyclohexanone showed no statistically significantly adverse effects on adult mortality and reproduction of the collembolan Folsomia candida in artificial soil at concentrations up to and including 100 mg pure metabolite/kg d.w. Therefore, the No-Observed-Effect-Concentration (NOEC) for mortality was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for mortality was determined to be > 100 mg pure metabolite/kg d.w. The No-Observed-Effect-Concentration (NOEC) for reproduction was determined to be ≥ 100 mg pure metabolite/kg d.w., and the Lowest-Observed-Effect-Concentration (LOEC) for reproduction was determined to be > 100 mg pure metabolite/kg d.w.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil dwelling arthropods:

100 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

100 mg/kg soil dw

Additional information

Should read "> 100 mg/kg soild dw"