2,6,10-Trimethyldodecane (Farnesane)

Bioaccumulation: terrestrial

Administrative data

Endpoint:

bioaccumulation: terrestrial

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

other: Please refer to 'justification for type of information'

Rationale for reliability incl. deficiencies:

other: Preliminary investigation in to the uptake potential of the test item.

Justification for type of information:

The OECD TG 222 study detailed in 6.3.1 is assinged as reliability score 1; the results and analytical method detailed in this section were not to GLP, however, they were detailed and validated according to relevant guidelines (SANCO 3029/99 rev.4 (2000))

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-07-12 to 2018-09-07

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 17GLY03173080182

Analytical monitoring:

no

Remarks:

Analytical monitoring was not conducted as part of the study. Only the highest test concentration treatment was analysed for the test substance at the end of the study (28 days) (please refer to IUCLID section 5.3.3 for the methods and results).

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Method of mixing into soil (if used):
On day of insertion of the earthworms, the respective test item amount was weighed out for a stock solution (9150 mg/50mL) and dissolved in acetone. The stock solution was further diluted with acetone to receive the respective spiking solutions. The appropriate amount of spiking solution (2.5 mL per replicate) was mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil. Additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax, each.
Subsequently, the test medium was mixed thoroughly for 3 minutes with a handheld mixer and a stainless steel spoon to ensure a homogenous distribution and about 610 g soil dry weight were filled into each test vessel.

- Controls: positive and negative controls included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): quartz sand 10 g per 600 g dw.
- Evaporation of vehicle before use: yes.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Breeding stock culture maintained at the test facility (Noack Laboratorien GmbH)
- Age at test initiation: Synchromised adults (with developed clitellum) of 2-12 months age.
- Weight at test initiation (range): 0.42 - 0.60 g

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions were same as the test conditions.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

28 days for adult earthworms

Post exposure observation period:

28 days for reproductive output (juvenile growth)

Test temperature:

20 +/- 2 °C

pH:

6.0 +/- 0.5

Moisture:

ca. 50 - 55 % of the water holding capacity of the artificial soil

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Glass dishes (volume 1.5 L, wide mouth, mason jar) were used. Transparent and perforated lids enabled sufficient gas exchange as well as light input and prevented the test substrate from drying.
- Amount of soil or substrate: 610 g dw
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Artificial soil prepared at the test facility according to the guideline (OECD TG 222)
- % sand: > 50, < 74
- % silt: > < 24
- % clay: 20
- Composition (if artificial substrate):
- Organic carbon (%): 5 % peat
- Maximum water holding capacity (in % dry weight): 37.6 g/100g DW
- Pretreatment of soil: Adjusted with 0.18 % calcium carbonate (CaCO3) to achieve pH of 6.1 +/- 0.5
- Storage (condition, duration): The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.
OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 400 - 800 lx
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality, reproduction, biomass
VEHICLE CONTROL PERFORMED: yes
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: A preliminary range finding test was performed with 4 test item concentrations each containing 2 replicates with 10 earthworms each.
-Test concentrations: 1, 10, 100, 1000 mg/kg dw soil including solvent and negative control.
- Results used to determine the conditions for the definitive study: Yes, effect on biomass observed at 1000 mg/kg dw.

Nominal and measured concentrations:

Nominal: 5.86 – 11.7 – 23.4 – 46.9 – 93.8 – 188 – 375 - 750 mg/kg soil dry weight.
The results are interpreted using nominal concentrations, despite one concentration being measured at the end of the study (28 days). The highest treatment (750 mg/kg soil dw) was analysed for the test substance concentration for the purposes of assessing the ratio of test substance in soil and in earthworm tissue.

Reference substance (positive control):

no

Remarks:

The reference test with carbendazim is conducted once within twelve months.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

93.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

188 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

biomass

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 750 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

The effect to 50 % of the population is estimated to be more than the highest concentration tested.

Details on results:

- Mortality at end of exposure period: No evident earthworm mortalities (< 10 %) were observed in the controls and all test item concentrations.
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
At beginning of the test, the earthworms had individual weights in the recommended range of 0.30 - 0.60 g. Since no statistically significant difference was observed, both controls were pooled for further evaluation. There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the pooled control.
No. of offspring produced
The reproduction rate (average number of juveniles produced per replicate) was 119 in the pooled control and ranged from 57 to 124 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 5.86 to 93.8 mg/kg soil dry weight compared to the pooled control. However, at the test item concentrations 188 to 750 mg/kg soil dry weight the earthworm reproduction was statistically significantly reduced. The number of unhatched cocoons ranged from 0 to 5 per replicate in the controls and from 0 to 10 in the test item concentrations, respectively. The EC50-value for reproduction was determined to be > 750 mg/kg soil dry weight. The coefficient of variation calculated for the reproduction of the pooled control was 18.4 % and thus lower than the 30 %, required for a valid test.
- No. of unhatched cocoons:
All control replicates (incl. solvent control): 34
All test substance treatment replicates (n = 4): 10, 5, 10, 10, 8, 18, 4, 4 in the test item treatments 5.86, 11.7, 23.4, 46.9, 93.8, 188, 375, 750 mg/kg soil dry weight, respectively
- Other biological observations: No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Relevant effect levels: Effect seen between 1-5 mg/kg dry weight soil.

Reported statistics and error estimates:

Data were checked for normality, homogeneity of variance and trend analysis. Dunnett’s Multiple t-test Procedure and Williams Multiple Sequential t-test Procedure was applied to assess differences in reproduction and biomass.
The p-value was set at 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10/20/50-values for reproduction were calculated by sigmoidal concentration response using SigmaPlot software.

Table 1: NOEC, LOEC and EC-values derived for the test substance (nominal concentrations) from the OECD TG 222

Endpoint	[mg/kg SDW]
LOECreproduction	188
LOECmortality,biomass	> 750
NOECreproduction	93.8
NOECmortality,biomass	≥ 750
EC-valuesreproduction (95 % Confidence Interval)	EC10: 46.1 (14.8 – 115) EC20: 134 (59.9 – 239) EC50: > 750

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test substance did not induce earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms in the controls and all test item concentrations. There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the pooled control. After a further four weeks, the mean reproduction rate (average number of juveniles produced per replicate) was 119 in the pooled control and ranged from 57 to 124 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 5.86 to 93.8 mg/kg soil dry weight compared to the pooled control. However, at the test item concentrations 188 to 750 mg/kg soil dry weight the earthworm reproduction was statistically significantly reduced.

Executive summary:

The effects of the test substance on mortality, biomass and the reproductive potential of the earthworm speciesEisenia fetida(Annelida, Lumbricidae) were determined according to OECD 222 (2016).

The study was conducted under static conditions over 8 weeks with the test item concentrations 5.86 – 11.7 – 23.4 – 46.9 – 93.8 – 188 – 375 - 750mg/kg soil dry weight (factor 2).Each application rate was mixed into the artificial soil containing 5 % peat. A control and a solvent control including untreated artificial soil and untreated artificial soil spiked with acetone, respectively, were tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control and 8 solvent control replicates each and 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.30 and 0.60 g at experimental starting.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10 %) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations. There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the pooled control. After a further four weeks, the mean reproduction rate (average number of juveniles produced per replicate) was 119 in the pooled control and ranged from 57 to 124 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 5.86 to 93.8 mg/kg soil dry weight compared to the pooled control. However, at the test item concentrations 188 to 750 mg/kgs oil dry weight, the earthworm reproduction was statistically significantly (Williams Multiple Sequential t-test Procedure) reduced. The 28-day NOEC for reproduction was derived at 93.8 mg/kg dw soil (nominal).

In conclusion, the study showed the test substance is not acutely toxic to the test item and induces reproductive effects but not effects on biomass at nominal concentrations > 188 mg/kg dw soil.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

- Principle of test:
Please refer to to detail methods of experimental design in IUCLID Section 6.3.1, at the end of the 28 day exposure period, where all adult earthworms were removed from the treatments and cocoons were left to hatch, worms from the highest test substance treatment, 750 mg/kg dw soil, were collected for analysis.
All worms of each 750 g/kg dw replicate were washed with demineralised water, dried gently on a paper towel and weighed per replicate including gut content. The worms were left to purge for ca. 24 h at 20 + 2°C (dark) into a Bellaplast box containing moist filter paper, that was changed after approximately 7 hours of the purging period.
At the end of the purging period the wet weight of the worms of each replicate was determined after worms were washed in demineralised water and patted dry. Afterwards the worms were killed by freezing in centrifuge tubes and the samples were stored in the freezer until further chemical analyses of earthworm tissue.

- Short description of test conditions:
Refer to IUCLID Section 6.3.1
- Parameters analysed / observed:
Total test substance concentration in earthworm tissue (mg/kg fresh weight)
Total test substance concentration in soil (mg/kg dry weight soil)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,6,10-trimethyldodecane

EC Number:

622-542-2

Cas Number:

3891-98-3

Molecular formula:

C15H32

IUPAC Name:

2,6,10-trimethyldodecane

Test material form:

liquid

Details on test material:

Clear colorless liquid
Density: 0.77 g/cm3 (20 degC)

Specific details on test material used for the study:

Batch number: 17GLY03173080182

Radiolabelling:

no

Sampling and analysis

Details on sampling:

- Sampling intervals/frequency for test organisms: Once, following 28 days exposure
- Sampling intervals/frequency for test medium samples: Once, following 28 days exposure
- Sample storage conditions before analysis: soil samples were stored at 6°C in the dark; earthworms were stored in the freezer at -20°C.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Liquid standards: A stock solution of 1000 mg/L ISTD in acetone was diluted to 1000 µg/L with cyclohexane. A stock solution of 20 g/L in acetone (for method validation and day 28 extraction) and 1 g/L in acetone (for the QC sample), respectively, was diluted in cyclohexane to obtain the calibration standards, which where fortified with 100 µL ISTD solution. For the calibration range using the above standards, see Table 34.
Preparation of soil samples: The QC soil samples was prepared as described in the section “ASE conditions” below, extracting 5.0 g wet soil, corresponding to approx. 4.1 g dry weight - the correct moisture was taken into account for. The sample was subsequently diluted (Dilution factor 100). The method was validated accoridng to SANCO 3029/99 rev.4 (2000).
ASE conditions: The soil in the ASE vials were mixed with approximately 10 g sodium sulphate and extracted as described below. After extraction, the samples were filled up to the mark with the solvent.
Preparation of standards for SPME samples: A stock solution of 1000 mg/L ISTD in acetone was diluted to 500 µg/L with acetone. A stock solution of 20 g/L test item in acetone was diluted to the standard spiking solutions with the acetone extract of the control. 1 ml of the spiking solution was fortified with 0.1 ml ISTD solution. 100 µL of each standard spiking solution was pipetted subsequently into a 20 mL headspace vial containing 9.9 mL A. demin (saturated with NaCl). For the calibration range using the standards above, see Table 35.
Preparation of the worm samples: A whole worm (Control: 2.26 g, Solvent Control: 2.16 g, 750 mg/Kg sample: 2.07 g) was weighted into a centrifuge vial. The samples were extracted as described in the section “Extraction of the worm”. Afterwards each solution was first diluted including the internal standard and finally diluted for SPME analysis prior to analysis.
Extraction of the worm sample: The samples were extracted with 10 mL acetone first with ultrasonic for 10 min and afterwards for 20 min on a rotary shaker. After centrifugation (4000 rpm, 10 min) the supernatant was collected in a 25 mL volumetric flask. This procedure was repeated once, afterwards the samples were filled up to the mark with acetone.

Test substrate

Vehicle:

yes

Remarks:

acetone (allowed to fully evaporate)

Details on preparation and application of test substrate:

Refer to the cross referenced study in IUCLID Section 6.3.1.

Test organisms

Test organisms (species):

Eisenia fetida

Details on test organisms:

Refer to the cross referenced study in IUCLID Section 6.3.1.

Study design

Total exposure / uptake duration:

28 d

Test conditions

Test temperature:

Refer to the cross referenced study in IUCLID Section 6.3.1.

pH:

Refer to the cross referenced study in IUCLID Section 6.3.1.

TOC:

Refer to the cross referenced study in IUCLID Section 6.3.1.

Moisture:

Refer to the cross referenced study in IUCLID Section 6.3.1.

Details on test conditions:

Refer to the cross referenced study in IUCLID Section 6.3.1.

Nominal and measured concentrations:

Refer to the cross referenced study in IUCLID Section 6.3.1.

Results and discussion

Bioconcentration factor

Type:

BCF

Value:

0.5 dimensionless

Basis:

other:

Remarks:

ratio of concentration in test media and earthworm

Calculation basis:

other: ratio of concentration in test media and earthworm

Remarks on result:

other: ratio of concentration in test media and earthworm

Kinetic parameters:

NA

Metabolites:

NA

Details on results:

NA

Reported statistics:

NA

Any other information on results incl. tables

Table 3: Measured Concentrationsand Percent of the Nominal of2,6,10-Trimethyldodecane

Sample	Soil		Worm
Nominal concentration of the soil [mg/kg soil dry weight]	2,6,10-Trimethyldodecane
	Meas. amount [mg/kg soil dry weight]	[%]	Meas. amount [mg/kg Worm fresh weight]
750	411*	55*	33.7
Solvent Control	< LOQ		< LOQ
Control	< LOQ		< LOQ

Meas.amount = Measured amount, enrichment and dilution factor taken into account

% percent of the nominal concentration

LOQ= Limit of quantification of the analytical method (93.6 mg test item/kg SDW for both soil methods and  0.354 mg/kg worm)

*)  = Measured amount of the QC sample (ASE extraction on day 28) was 299 mg/kg soil dry weight and a recovery of 41 % (Nominal concentration of the soil: 738 mg/kg soil dry weight)

The results in earthworm tissue are presented as fresh weight. To compare the results and calculate a hypothetical bioconcentration factor (unitless, ratio between concentration in earthworm and soil), the concentration in fresh weight was calculated in to dry weight according to an assumption 15 % of the earthworm fresh mass is dry weight (van Gestel et al., 2010). This results in a tissue concentration of 224.6 mg/kg dw earthworm.

van Gestel et al. (2010) Soil Biology and Biochemistry, 40: 10, pp 1892 -1893.

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

Earthworms were exposed to the test substance for 28 days under the OECD TG 222 (refer to IUCLID Section 6.3.1) test conditions. Soil and earthworm samples were analysed for test substance concentrations to assess bioaccumulation potential of the substance.
The concentration of the test substance in earthworm tissue following 28 days exposure was 33.7 mg/kg fresh weight (equates to approximately 224 mg/kg dw under the assumption that dry weight is 15 % of fresh weight). The measured test substance concentration following 28 days in soil was 441 mg/g dw (55 % of the nominal 750 mg/kg dw soil). The ratio between the dry weight concentration in earthworm tissue and soil (BCF) is 0.5.

Executive summary:

A preliminary study into the bioaccumulation potential of the test substance was conducted as an additional measurement in an OECD TG 222 study. Earthworms were exposed to the test substance for 28 days under the OECD TG 222 (refer to IUCLID Section 6.3.1) test conditions.

The concentration of the test substance in earthworm tissue following 28 days exposure was 33.7 mg/kg fresh weight (equates to approximately 224 mg/kg dw under the assumption that dry weight is 15 % of fresh weight). The measured test substance concentration following 28 days in soil was 441 mg/g dw (55 % of the nominal 750 mg/kg dw soil). The ratio between the dry weight concentration in earthworm tissue and soil (BCF) is 0.5.