A mixture of cis- and trans-cyclohexadec-8-en-1-one

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:

2021-09-23 to 2021-11-17

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:

yes

Remarks:

Food was provided on day 0 instead of day 1 due to good experience with this procedure. At test end, the soil moisture deviated by more than 10% from the initial value in all treatments. These deviations are considered to have no impact on the quality.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC-MS method

Details on sampling:

Each concentration level and the control were analysed via GC-MS at start of the exposure using the respective spiking solutions and an additional likewise spiked portion of quartz sand, each.

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

- Controls: solvent control (acetone) and Artificial soil
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 2.5 mL per replicate
- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Age at test initiation: adult earthworms (with clitellum) aged 2 - 12 months
- Weight at test initiation: 0.33 to 0.55 g

ACCLIMATION
- Acclimation period: two days prior to test start
- Acclimation conditions: same as test
- Health during acclimation, any mortality observed: no

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

20+/-2 °C

pH:

day 0: 6.58-6.94;
day 55: 6.24-6.60

Moisture:

day 0: 22.2-26.16 % of dry weight;
day 55: 27.6-31.4 % of dry weight.

Details on test conditions:

TEST SYSTEM
- Test container: Test units containing about 600 g soil dry weight were round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom
surface area of 177 cm2 and a height of the containers of 14 cm. Transparent and perforated lids enabled sufficient gas exchange, light input and prevented the test substrate from drying.
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 400 - 800 lx in the area of the test container

EFFECT PARAMETERS MEASURED:
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms (mortality)
I) number of juveniles produced
J) body weight

Nominal and measured concentrations:

Concentrations:
9.38 – 18.75 – 37.5 – 75 – 150 – 300 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

CARBENDAZIM

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 300 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 300 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 300 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

See below

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 1-5 mg/kg soil dw.

Reported statistics and error estimates:

Adult Mortality:
-Qualitative Trend Analysis by Contrasts (Monotonicity of Concentration/Response)
-Tarone’s Test Procedure
-Step-down Rao-Scott-Cochran-Armitage Test Procedure

Biomass:
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend analysis by Contrasts (Monotonicity of Concentration/Response)
- Williams Multiple Sequential t- Test Procedure

Reproduction:
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis by Contrasts (Monotonicity of Concentration/Response)
- Dunnett’s Multiple t-test Procedure

Reproduction rate (number of Juveniles after 8 weeks):

Application rate [mg test item/kg soil dry weight]	Replicate	Number of juveniles	Mean ± SD	CV               [%]	[%] of pooled control	Inhibition to pooled control*	Sig.
Control	1	176	156 ± 19.9	12.8	-	-	-
	2	157
	3	158
	4	125
	5	129
	6	181
	7	163
	8	155
Solvent Control	1	131	142 ± 21.5	15.2	-	-	-
	2	140
	3	131
	4	190
	5	128
	6	154
	7	134
	8	125
Pooled Control			149 ± 21.3	14.3	-	-	-
9.38	1	109	93 ± 12.5	13.4	62.4	37.6	Yes
	2	97
	3	86
	4	81
18.75	1	144	161 ± 27.7	17.2	108	-8.05	No
	2	161
	3	139
	4	200
37.5	1	185	136 ± 38.5	28.3	91.3	8.72	No
	2	91
	3	134
	4	132
75	1	159	137 ± 14.8	10.8	92.0	8.05	No
	2	132
	3	129
	4	128
150	1	158	148 ± 17.1	11.5	99.3	0.67	No
	2	160
	3	150
	4	123
300	1	143	148 ± 17.2	11.6	99.3	0.67	No
	2	168
	3	153
	4	127

SD = Standard deviation       CV = Coefficient of variation 

Sig. = Statistical Significance (Dunnett’s Multiple  t-test Procedure,  α = 0.05) compared to the pooled control According to the definition of the LOEC all test concentrations above the LOEC should normally show an effect that is statistically different from the control (OECD GD 222, 2016). Therefore, the LOEC was assessed to be ≥ 300 mg/kg SDW.

Validity criteria fulfilled:

yes

Conclusions:

The NOEC (reproduction) of the test item to earthworm (Eisenia fetida) was determined to be >= 300 mg/kg soil dry weight.

Executive summary:

The long term effect of the test item to soil macroorganisms (earthworm: Eisenia fetida) was determined according to OECD guideline 222 (2016) under GLP. The study was conducted under static conditions over 8 weeks with the test item concentrations 9.38 – 18.75 – 37.5 – 75 – 150 – 300 mg test item/kg soil dry weight (SDW). A control and a solvent control were tested under the same conditions as the test item treatments. 160 test organisms were inserted into 8 control and 8 solvent control replicates. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). All inserted worms had an individual body weight between 0.33 and 0.55 g at experimental starting. After 28 days of exposure in soil, neither evident earthworm mortalities (< 10 %) nor evident pathological  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 at all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 149 in the pooled control and ranged from 93 to 161 in the test item treatment rates. A statistically significant difference in reproduction compared to the pooled control was observed in the lowest test item concentration 9.38 mg/kg SDW. However, no statistically significant differences in earthworm reproduction were determined in the test item concentrations 18.75 to 300 mg/kg SDW compared to the pooled control. According to the definition of the LOEC all test concentrations above the LOEC should normally show an effect that is statistically different from the control (OECD GD 222, 2016). Therefore, the LOEC was determined to be > 300 mg/kg SDW. Overall, the NOEC for mortality, biomass and reproduction was determined to be >= 300 mg/kg SDW.

Description of key information

The long term effect of the test item to soil macroorganisms (earthworm: Eisenia fetida) was determined according to OECD guideline 222 (2016) under GLP. The study was conducted under static conditions over 8 weeks with the test item concentrations 9.38 – 18.75 – 37.5 – 75 – 150 – 300 mg test item/kg soil dry weight (SDW). A control and a solvent control were tested under the same conditions as the test item treatments. 160 test organisms were inserted into 8 control and 8 solvent control replicates. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). All inserted worms had an individual body weight between 0.33 and 0.55 g at experimental starting. After 28 days of exposure in soil, neither evident earthworm mortalities (< 10 %) nor evident pathological  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 at all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 149 in the pooled control and ranged from 93 to 161 in the test item treatment rates. A statistically significant difference in reproduction compared to the pooled control was observed in the lowest test item concentration 9.38 mg/kg SDW. However, no statistically significant differences in earthworm reproduction were determined in the test item concentrations 18.75 to 300 mg/kg SDW compared to the pooled control. According to the definition of the LOEC all test concentrations above the LOEC should normally show an effect that is statistically different from the control (OECD GD 222, 2016). Therefore, the LOEC was determined to be > 300 mg/kg SDW. Overall, the NOEC for mortality, biomass and reproduction was determined to be >= 300 mg/kg SDW.

Key value for chemical safety assessment

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

300 mg/kg soil dw