Imidazolium compounds, 2-C17-unsatd.-alkyl-1-(2-C18-unsatd. amidoethyl)-4,5-dihydro-N-methyl, Me sulfates

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:

2021-04-08 to 2021-06-03 (final report date xx-xx-xxxx)

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:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: trout worm
- Source: Breeding stock culture maintained at the test facility
- Age at test initiation (mean and range, SD): 2-12 months
- Weight at test initiation (mean and range, SD): 0.36 to 0.51 g

ACCLIMATION
- Acclimation period: Earthworms were adapted to the artificial soil to be used for the test two days prior to test start.
- Acclimation conditions (same as test or not): same as test

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

10 % peat, air-dried and finely ground - 20 % kaolin, kaolinite content > 30 % - 69 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm) - 0.40 % calcium carbonate (CaCO3) to achieve a pH of 6.0 ± 0.5

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

20 ± 2 °C

pH:

Between 5.86 and 6.44

Moisture:

between 21.1 % and 26.9 %

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and a height of 14 cm
- Amount of soil or substrate: Test units containing about 600 g soil dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
- 10 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 69 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.40 % calcium carbonate (CaCO3) to achieve a pH of 6.0 ± 0.5

OTHER TEST CONDITIONS
- Photoperiod: 16h/8h
- Light intensity: 400-800 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At experimental starting and experimental completion, the pH-value and moisture content of the test medium were determined in each treatment and the control from pooled samples of all replicates.
The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water.
The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate.
After the first four weeks, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate.
After a further four weeks, the number of offspring (juveniles) hatched from the cocoons was counted for each test container.


VEHICLE CONTROL PERFORMED: The test item was dispersed in demineralised water and added to the artificial soil. The control was treated like the test groups except for the test item.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor 2
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: control, 10, 100, 1000 mg i.a./kg soil dry weight
- Results used to determine the conditions for the definitive study: No mortality occurred in the preliminary test, thus, the highest concentration in the main test was also 1000 mg i.a./kg SDW

Nominal and measured concentrations:

31.25 – 62.5 – 125 – 250 – 500 – 1000 mg a.i./kg SDW

Reference substance (positive control):

yes

Remarks:

CARBENDAZIM: The test is conducted once within twelve months. The most recent test was carried out from 2021-02-19 to 2021-04-16 using artificial soil containing 10 % peat. Application into the artificial soil by mixing. 0.5 - 1 - 2 - 4 mg a.i./kg SDW.

Key result

Duration:

8 wk

Dose descriptor:

EC50

Remarks:

reproduction

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

EC10

Remarks:

reproduction

Effect conc.:

164 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Remarks:

reproduction

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

NOEC

Remarks:

mortality, biomass

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: 0
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Control: 32.4 ± 5.81%; 31.25 mg i.a./kg SDW: 35.2 ± 6.58%; 62.5 mg i.a./kg SDW: 23.9 ± 3.91%; 125 mg i.a./kg SDW: 29.5 ± 4.92%; 250 mg i.a./kg SDW: 26.7 ± 7.74%; 500 mg i.a./kg SDW: 31.25 ± 9.73%; 1000 mg i.a./kg SDW: 29.6 ± 5.89%
- No. of offspring produced: 46-80
- Morphological abnormalities: None reported
- Behavioural abnormalities: None reported

Results with reference substance (positive control):

- Results with reference substance valid? yes; significant effects were observed between 1 and 5 mg i.a./kg SDW.
- Relevant effect levels: releative number of juveniles [%]: 0.5 mg i.a./kg SDW: 74.6% of control; 1 mg i.a./kg SDW: 79.8% of control; 2 mg i.a./kg SDW: 64.9 % of control; 4 mg i.a./kg SDW: 0.0 % of control

Reported statistics and error estimates:

The arithmetic mean and NOEC were determined. Since no mortality occurred, no further computations have been performed.
Biomass / Reproduction
- 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 Sequentially t-test Procedure
The α-value (acceptable probability of incorrectly concluding that there is a difference) was α = 0.05. For the endpoint of biomass and reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10/20/50 - value for reproduction were calculated using 3-param. normal CDF.

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate [mg a.i./kg  SDW]	Replicate
	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
31.25	0	0	0	0	-				0
62.5	0	0	0	0					0
125	0	0	0	0					0
250	0	0	0	0					0
500	0	0	0	0					0
1000	0	0	0	0					0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate [mg a.i./kg  SDW]	Replicate
	Observation parameter	1	2	3	4	5	6	7	8
Control	A	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
31.25	A	10/10	10/10	10/10	10/10	-
62.5	A	10/10	10/10	10/10	10/10
125	A	10/10	10/10	10/10	10/10
250	A	10/10	10/10	10/10	10/10
500	A	10/10	10/10	10/10	10/10
1000	A	10/10	10/10	10/10	10/10

A = no obvious pathological symptoms

Body Weight Changes of the Adult Earthworms

Application rate [mg a.i./kg  SDW]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.44	0.54	0.10	22.7	0.14 ± 0.03	32.4 ± 5.81	–
	2	0.44	0.60	0.16	36.4
	3	0.44	0.55	0.11	25.0
	4	0.44	0.58	0.14	31.8
	5	0.44	0.58	0.14	31.8
	6	0.44	0.61	0.17	38.6
	7	0.44	0.60	0.16	36.4
	8	0.44	0.60	0.16	36.4
31.25	1	0.44	0.57	0.13	29.5	0.16 ± 0.03	35.2 ± 6.58	No
	2	0.44	0.57	0.13	29.5
	3	0.44	0.62	0.18	40.9
	4	0.44	0.62	0.18	40.9
62.5	1	0.44	0.54	0.10	22.7	0.11 ± 0.02	23.9 ± 3.91	No
	2	0.44	0.57	0.13	29.5
	3	0.44	0.54	0.10	22.7
	4	0.44	0.53	0.09	20.5
125	1	0.44	0.57	0.13	29.5	0.13 ± 0.02	29.5 ± 4.92	No
	2	0.44	0.59	0.15	34.1
	3	0.44	0.54	0.10	22.7
	4	0.44	0.58	0.14	31.8
250	1	0.44	0.56	0.12	27.3	0.12 ± 0.03	26.7 ± 7.74	No
	2	0.44	0.57	0.13	29.5
	3	0.44	0.51	0.07	15.9
	4	0.44	0.59	0.15	34.1
500	1	0.44	0.55	0.11	25.0	0.14 ± 0.04	31.25 ± 9.73	No
	2	0.44	0.64	0.20	45.5
	3	0.44	0.55	0.11	25.0
	4	0.44	0.57	0.13	29.5
1000	1	0.44	0.54	0.10	22.7	0.13 ± 0.03	29.6 ± 5.89	No
	2	0.44	0.58	0.14	31.8
	3	0.44	0.60	0.16	36.4
	4	0.44	0.56	0.12	27.3

SD  = Standard deviation; Sig. = Statistical Significance (Williams Multiple Sequentially t-test Procedure, α = 0.05) compared to control

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg a.i./kg dry soil weight]	Replicate	Number of juveniles	Mean ± SD	CV               [%]	[%] of control	Sig.
Control	1	58	82 ± 18.9	23.1	-	-
	2	70
	3	104
	4	108
	5	59
	6	88
	7	78
	8	89
31.25	1	104	78 ± 19.7	25.3	95.1	No
	2	62
	3	81
	4	63
62.5	1	84	80 ± 26.3	32.9	97.6	No
	2	114
	3	69
	4	52
125	1	66	74 ± 10.4	14.0	90.2	No
	2	71
	3	89
	4	69
250	1	67	69 ± 9.90	14.4	84.2	No
	2	58
	3	82
	4	69
500	1	80	58 ± 15.2	26.2	70.7	Yes
	2	46
	3	50
	4	57
1000	1	40	46 ± 15.2	33.0	56.1	Yes
	2	66
	3	47
	4	30

SD = Standard deviation; CV = Coefficient of variation; Sig. = Statistical Significance (Williams Multiple Sequential t-test Procedure, α = 0.05) compared to the control

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, neither earthworm mortalities nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and any test item concentration. Furthermore, no statistically significant differences in earthworm body weights were observed at any test item concentration compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 82 in the control and ranged from 46 to 80 in the test item treatment rates. No statistically significant differences in earthworm reproduction were observed in the treatment rates 31.25 to 250 mg a.i./kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg a.i./kg SDW reproduction of earthworms was statistically significantly reduced.

Executive summary:

In a 28 days toxicity and subsequent 8 weeks reproduction study, earthworms (Eisenia fetida) were exposed to the test chemical at 0, 31.25, 62.5, 125, 250, 500, 1000 mg a.i./kg dry weight of artificial substrate. The reference chemical used was Carbendazim at 0.5, 1, 2, 4 mg a.i./kg dw of the substrate. The experiment was carried out in accordance with OECD Test Guideline 222 (2016). The 28-day LC50 was ≥1000 mg a.i./kg dw of substrate. The 28-day EC50 was ≥ 1000 mg a.i./kg dw of substrate. The 28-day NOEC, based on mortality/biomass was ≥ 1000 mg a.i./kg dw of substrate. The LOEC, based on mortality/biomass was > 1000 mg a.i./kg dw of substrate.  The a.i. is considered to be non-toxic to earthworms up to a concentration of 1000 mg a.i./kg dw of substrate within 28 days.

The 8-week NOEC, based on reproduction was 250 mg i.a./kg SDW. The 8-week LOEC, based on reproduction was 500 mg i.a./kg SDW. The EC10 and EC50 value based on reproduction after 8 weeks were: 164 (97.0-278) and > 1000 (742->1000), respectively.

Under the conditions of this study, neither earthworm mortalities nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and any test item concentration. Furthermore, no statistically significant differences in earthworm body weights were observed at any test item concentration compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 82 in the control and ranged from 46 to 80 in the test item treatment rates. No statistically significant differences in earthworm reproduction were observed in the treatment rates 31.25 to 250 mg a.i./kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg a.i./kg SDW reproduction of earthworms was statistically significantly reduced.

 

This study is classified as acceptable and satisfies the guideline requirements for an 28-day/8-week reproduction toxicity study with earthworms according to OECD TG 222.

 

Results Synopsis

 

Test organism age (mean weight): between 0.36 and 0.51 g

 

Test Type:

28-day NOEC ≥ 1000 mg a.i./kg dw soil

8-week-NOEC =250 mg a.i./kg dw soil

8-week- EC50: > 1000 mg a.i./kg dw soil     95% CI: 742 to > 1000 mg a.i./kg dw soil

 

Endpoint(s) Effected: Mortality and reproduction

Description of key information

Draft study report, OECD 222, (Eisenia fetida) were exposed to the test chemical at 0, 31.25, 62.5, 125, 250, 500, 1000 mg a.i./kg dry weight of artificial substrate for 8 weeks, not classified, Long-term NOEC = 250 mg/kg soil dw

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:

250 mg/kg soil dw

Additional information

No final results for toxicity to soil macroorganisms except anthropods are available, however, draft results were used instead. Please note that due to the equivocal results no new risk assessment could be performed. A new rsik assessment including eventually changed exposure scenarios will be added as soon as possible in form of a spontaneous IUCLID update.

 

In a 28 days toxicity and subsequent 8 weeks reproduction study, earthworms (Eisenia fetida) were exposed to the test chemical at 0, 31.25, 62.5, 125, 250, 500, 1000 mg a.i./kg dry weight of artificial substrate. The reference chemical used was Carbendazim at 0.5, 1, 2, 4 mg a.i./kg dw of the substrate. The experiment was carried out in accordance with OECD Test Guideline 222 (2016). The 28-day LC50 was ≥1000 mg a.i./kg dw of substrate. The 28-day EC50 was ≥ 1000 mg a.i./kg dw of substrate. The 28-day NOEC, based on mortality/biomass was ≥ 1000 mg a.i./kg dw of substrate. The LOEC, based on mortality/biomass was > 1000 mg a.i./kg dw of substrate.  The a.i. is considered to be non-toxic to earthworms up to a concentration of 1000 mg a.i./kg dw of substrate within 28 days.

The 8-week NOEC, based on reproduction was 250 mg i.a./kg SDW. The 8-week LOEC, based on reproduction was 500 mg i.a./kg SDW. The EC10 and EC50 value based on reproduction after 8 weeks were: 164 (97.0-278) and > 1000 (742->1000), respectively.

Under the conditions of this study, neither earthworm mortalities nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and any test item concentration. Furthermore, no statistically significant differences in earthworm body weights were observed at any test item concentration compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 82 in the control and ranged from 46 to 80 in the test item treatment rates. No statistically significant differences in earthworm reproduction were observed in the treatment rates 31.25 to 250 mg a.i./kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg a.i./kg SDW reproduction of earthworms was statistically significantly reduced.

 

This study is classified as acceptable and satisfies the guideline requirements for an 28-day/8-week reproduction toxicity study with earthworms according to OECD TG 222.

 

Results Synopsis

 

Test organism age (mean weight): between 0.36 and 0.51 g

 

Test Type:

28-day NOEC ≥ 1000 mg a.i./kg dw soil

8-week-NOEC =250 mg a.i./kg dw soil

8-week- EC50: > 1000 mg a.i./kg dw soil     95% CI: 742 to > 1000 mg a.i./kg dw soil

 

Endpoint(s) Effected: Mortality and reproduction