cis-2-tert-butylcyclohexyl acetate

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Oct 2015 to 11 Feb 2016

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))

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

aceton

Details on preparation and application of test substrate:

Artificial soil:
- 10 % Sphagnum-peat, air-dried and finely ground (2 mm with no visible plant remains)
- 20 % Kaolin clay (Kaolinite content > 30 %)
- 69.6 % fine quarts-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm.
- 0.4 % Calcium carbonate (CaCO3) was added to adjust pH to 6.0 +/- 0.5

The artificial soil was moistened to approximately half of the final water content 1 day before the application. The additional water required to achieve the final water content was added when applying the test item.

Maximum WHC of the Articifial soil was 54 % of the dry weight of artificial soil.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Adult worms of approximately 7 months, with well-developed clitellum. Age range between test individuals not differing by more than 4 weeks. Body weights ranged between 322 and 596 mg. Bred under standardised conditions in Ibacon laboratories in a breeding medium of cattle manure, peat, sand , calcium carbonate and straw, fed with catlle manure, stored at RT. Worms were acclimatised for 1 day in artificial soil under test conditions.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Adults removed after 4 wks, offspring remained in test containers for another 4 weeks

Test temperature:

18 - 22 degrees °C.

pH:

At start of study pH 6.2 - 6.3
After 8 wks pH 6.2-6.3 - 6.4

Moisture:

At experimental start: 32.0-32.6% (50.0% to 51.0% of the maximum WHC, i.e. within the recommended range 40-60 % of the total WHC)
At experimental end: 32.9 % to 36.4 % (51.4 % to 56.9 % of the maximum WHC)

Details on test conditions:

Controlled environment room, in a ventilated area.
Light intensity was within the range of 400 lux to 800 lux.
Light regime: 16 h light and 8 h dark
Once a week the water content of the soil was checked by weighing each container and evaporated water was replenished. In this way, it was ensured that the difference in water content between experimental start and end wass less than 10%.

Nominal and measured concentrations:

A stock solution was prepared by dissolving 1738 mg of Iso E Super in 25 mL acetone. A sequential dilution series was prepared to achieve following nominal concentrations: 3.16, 10, 31.6, 100 and 316 mg test item/kg soil. Then 10 mL of the corresponding dilutions were added to 20 g fine quartz sand. The treated sand was left for approximately four hours in a fume hood until the solvent has evaporated and was mixed with a spoon. Then artificial soil equivalent to 2180 g dry weight was added to test item blended sand.
Samples were taken in case analyses would be considered to be necessary. No analyses were carried out.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

45.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Conf interval

Remarks:

21.0-65 mg/kg soil dw

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

158 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality

Remarks:

and weight change

Details on results:

See also tables at 'any other informtion on results incl. tables'.
Mortality and body weight: The body weight changes in the test item treated groups were not statistically significantly different compared to the control up to and including the test concentration of 158.1 mg test item/kg soil (Dunnett's t-test, α = 0.05, two-sided). The weight changes at the concentration of 500.0 mg test item/kg soil were not evaluated because all worms were dead after 28 days. The NOEC for body weight changes was determined to be 158.1 mg test item/kg soil.

Reproductive assessment:
The reproduction rates were not statistically significantly different compared to the control up to and including the test concentration of 50.0 mg test item/kg soil. At 158.1 mg test item/kg soil and above reproduction was statistically significantly reduced compared to the control (Bonferroni-Welch t-test, α = 0.05, one-sided smaller). The NOEC for reproduction was determined to be 50.0 mg test item/kg soil. The EC10 was determined to be 45.1 mg test item/kg soil (95% confidence intervals of 21.0 to 65.0 mg test item/kg soil), the EC20 was determined to be 65.4 mg test item/kg soil (95% confidence intervals of 37.8 to 86.9 mg test item/kg soil) and the EC50 was determined to be 133.1 mg test item/kg soil (95% confidence intervals of 103.8 to 169.7 mg test item/kg soil
Feeding activity:
In all treatment groups, food was consumed. The results show that the turnover of biomass of those earthworms up to and including the concentration of 50.0 mg test item/kg soil was comparable to the control whereas the food intake at the concentration of 158.1 mg test item/kg soil appeared to be slightly reduced. The feeding activity was reduced at 500.0 mg test item/kg soil

Behavioural abnormalities:
At the concentration of 500.0 mg test item/kg soil worms did not burrow into the soil after introduction and were observed still at the soil surface one day after the application. No additional behavioural abnormalities were observed and the worms at all other concentrations burrowed into the soil within 15 minutes after introduction.

Results with reference substance (positive control):

In a test with reference item Carbendazim 600 g/L SC (June to August 2016) there were statistically significant effects on reproduction at a concentration of 4.16 mg a.i./kg soil and higher, which is in line with the guideline OECD 222 (effects should be observed between 1 and 5 mg a.i./kg soil). The EC50 for reproduction was calculated as 1.91 mg a.i./kg soil.

Reported statistics and error estimates:

Mortality data were analysed for significance by using the Fisher's Exact Binomial Test (multiple comparison, with Bonferroni correction, alpha = 0.05, one-sided greater).
The body weight change and reproduction data were tested for normal distribution and homogeneity of variance (alpha = 0.05) using the Shapiro-Wilk's test and the Levene's test, respectively.
As the data for body weight changes were normally distributed but not homogeneous the Bonferroni-Welch t-test was used to compare treatment and control values (multiple comparison, two-sided alpha = 0.05).
As the data for reproduction were normally distributed and homogeneous the Williams t-test was used to compare treatment and control values (multiple comparison, one-sided alpha = 0.05).
The LC50 and the EC10 and EC20 values for reproduction were calculated by applying Probit-Analysis.

Table 1. Effect of Verdox on earthworms (Eisenia fetida) in a 56-day reproduction study
Test substance [mg/kg soil dry weight]	Control		5.0	15.8		50.0	158.1		500.0
Mortality (day 28) [%]	0		0	0		0	0		100
Statistical Significance1)	-		n.s.	n.s.		n.s.	n.s.		*
Body weight change (day 28) [%]	26.1		26.8	31.5		26.5	29.8		--
Statistical Significance2)	-		n.s.	n.s.		n.s.	n.s.		-
Mean No. of juveniles (day 56)	275		249	272		235	125		0
Statistical Significance3)	-		n.s.	n.s.		n.s.	*		*
Reproduction in [%] of control (day 56)	-		90.6	98.9		85.5	45.3		0
Food consumption [g]	25.0		25.0	25.0		25.0	22.5		5.0
Endpoints [mg test item/kg soil dry weight]
NOEC (day 28 mortality and weight)					158.1
NOEC (day 56 reproduction)					50.0
EC Values (reproduction)4)		EC10= 45.1			EC20= 65.4			EC50= 133.1
95% confidence limits		21.0 to 65.0			37.8 to 86.9			103.8 to 169.7

Validity criteria fulfilled:

yes

Remarks:

Control mortality was 0% and so this validity criterion was met. The number of juvenile worms per replicate was 126 to 227 and so this validity criterion was met. The coefficient of variation of reproduction in control was 16.4% and thus met.

Conclusions:

EC10 for earthworm reproduction is 45.1 mg/kg dw soil. NOEC for earthworm mortality and growth is 158.1 mg/kg soil.

Executive summary:

The long-term toxicity of the substance for earthworm Eisenia foetida was tested in OECD TG 222. The test material was dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.05 -0.2 mm) and 0.4% calcium carbonate to adjust to pH 6.0±0.5. Adult worms were exposed to nominal concentrations in soil of 5, 15.8, 50, 158.1 and 500 mg/kg dw soil. Weights of the adult worms ranged between 322 and 596 mg. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Nominal concentrations are used to present the effects. No worms survived at 500 mg/kg soil dw. The NOEC for mortality and growth was 158.1 mg/kg dw. The EC10 for reproduction was 45.1 mg/kg dw soil (95% confidence interval is 21-65 mg/kg soil dw).

Description of key information

The long-term toxicity of the substance for earthworm Eisenia foetida was tested in OECD TG 222. The test material was dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.05 -0.2 mm) and 0.4% calcium carbonate to adjust to pH 6.0±0.5. Adult worms were exposed to nominal concentrations in soil of 5, 15.8, 50, 158.1 and 500 mg/kg dw soil. Weights of the adult worms ranged between 322 and 596 mg. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Nominal concentrations are used to present the effects. No worms survived at 500 mg/kg soil dw. The NOEC for mortality and growth was 158.1 mg/kg dw. The EC10 for reproduction was 45.1 mg/kg dw soil (95 % confidence interval is 21-65 mg/kg soil dw).

Key value for chemical safety assessment

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

45.1 mg/kg soil dw