1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Oct 2013 to 04 Dec 2013

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:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of the test substrate: The test item is dispersible in water. Therefore, test solutions were made by dispersing weighed amounts of the test item in deionised water, immediately prior to application. The test item was dispersed in sufficient deionised water such that the addition of the test solutions to the test substrate resulted in a final water content of 40-60 % of WHC. The treated substrate was thoroughly mixed using a laboratory mixer immediately after application.
- Order of application and cleaning procedure:
> deionised water (control)
> the test item in ascending order
The mixer was cleaned with a brush following application of each test item concentration and with cleaning agents after the last mixing. Cleaning agents used were a alkaline cleaning agent for laboratory equipment, water, deionised water and spirit for glass equipment.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Reared under ambient laboratory conditions in the test facility (original breeding animals were purchased from “W. Neudorff GmbH KG”, An der Mühle 3, 31860 Emmerthal, Germany)
- Age at test initiation: Adult worms (approximately 4 months old with clitellum); to avoid systematic errors in distributing the worms to the test vessels the homogeneity of the population was determined by weighing individual worms sampled at random from the synchronised population; having ensured homogeneity, batched worms were then selected, weighed and assigned to test vessels using a randomisation procedure
- Weight at test initiation: 398 – 560 mg/worm
- Breeding medium: Breeding medium: mixture of horse manure, straw, peat (1:1:1); origin: horse manure and straw were purchased from farmers
- Breeding conditions: breeding containers (50 cm x 40 cm x 30 cm) constant diffuse light, temperature: approximately 20 °C, moist soil, pH: approximately 7
- Food used during the test: air-dried and finely ground horse manure

ACCLIMATION
- Acclimation period: approximately 24 hours in the artificial substrate (with food)

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20.2 – 22.0 °C

pH:

-Test start: 5.98 – 6.03
-Test end: 5.73 – 5.82

Moisture:

- Test start: 24.9 – 25.0 % (equivalent to 58.6 – 58.8 % of WHC)
- Test end: 24.3 – 24.6 % (equivalent to 57.2 – 57.9 % of WHC)

Details on test conditions:

TEST SYSTEM
- Test container: Plastic vessel of Bellaplast (inside dimensions: about 16.5 cm x 12 cm x 6 cm) with a lid pervious to air and light
- Amount of soil: 750 g wet weight corresponding to 600 g dry weight
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8


SOURCE AND PROPERTIES OF SUBSTRATE
- 5 % sphagnum peat; origin: “Torfwerk Moorkultur Ramsloh”, 26683 Saterland, Germany, classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant parts remained, finely ground, dried to measured moisture content)
- 20 % kaolin clay (kaolinite content > 30 %)
- 0.3 % calcium carbonate
- 74.7 % industrial quartz sand (fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
- deionised water
- Max. water holding capacity WHC (g/100 g dry soil): 42.5
- Water content (%):*
Guideline requirement: 40-60 % of WHC corresponding to approximately 25 % water content of dry soil
test start: 24.9 – 25.0 % (equivalent to 58.6 – 58.8 % of WHC)
test end: 24.3 – 24.6 % (equivalent to 57.2 – 57.9 % of WHC)

OTHER TEST CONDITIONS
- Photoperiod: Light : dark = 16 h : 8 h
- Light intensity: 450 lux

EFFECT PARAMETERS MEASURED
Time and parameters
At test start:
- individual fresh weight (mg/worm)
- behaviour of earthworms
- determination of physico-chemical parameters (water content, pH) of the artificial soil
Weekly:
- observation of behavioural and pathological symptoms (including the feeding activity)
4 weeks after start of exposure:
- number of surviving adult earthworms per replicate
- observation of behavioural and pathological symptoms (including morphological alterations)
- fresh weight of surviving adult earthworms per replicate
8 weeks after start of exposure:
- number of juveniles per replicate
- observation of behavioural and pathological symptoms (including morphological alterations)
- determination of physico-chemical parameters (water content, pH) of the artificial soil

Nominal and measured concentrations:

- Nominal concentrations: 1.28, 2.30, 4.14, 7.45, 13.42, 24.15, 43.47 and 78.24 mg formulation/kg soil dw, corresponding to 0.327, 0.588, 1.06, 1.91, 3.43, 6.17, 11.1, 20 mg test substance in formulation/kg soil d.w.

Reference substance (positive control):

yes

Remarks:

Nutdazim 50 FLOW (carbendazim, SC 500), 5 and 10 mg product/kg soil dry weight.

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

2.99 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: test substance concentration in formulation

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

6.17 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: test substance concentration in formulation

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

11.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: formulation

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

20 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: test substance concentration in formulation

Basis for effect:

mortality

Remarks:

and biomass

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

78.24 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: formulation

Basis for effect:

mortality

Remarks:

and biomass

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

24.15 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: formulation

Basis for effect:

reproduction

Details on results:

An overview of the results is provided in Table - Table in 'Any other information on results incl. tables'.
- Mortality: The mortality of adult worms was 0 – 5.0% in the treated variants and 1.3 % in the control group. No statistically significant mortality compared to the control was observed at any test item concentration (Fisher’s Exact Binomial Test with Bonferroni Correction, p > 0.05, one-sided greater). No pathological symptoms and no effects on the behaviour (including feeding activity) of the worms were observed during the test.
- Biomass: The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control group, i.e. a mean weight increase of 28.3 % was recorded in the control group and 28.8, 25.8, 29.5, 27.7, 28.5, 27.9, 27.1 and 26.3 % at the tested concentrations of 1.28, 2.30, 4.14, 7.45, 13.42, 24.15, 43.47 and 78.24 mg formulation/kg soil d.w., respectively (Williams-t-test, p > 0.05, one-sided smaller).
- Effect on juveniles: Statistically significant effects (Williams-t-test, p ≤ 0.05, one-sided smaller) on the number of juveniles compared to the control group were recorded at concentrations of 43.47 and 78.24 mg formulation/kg soil d.w..

The EC10, EC20 and EC50 values for reproduction were calculated to be 11.70, 23.43 and > 78.24 mg formulation/kg soil dry weight, respectively.

Results with reference substance (positive control):

To verify the sensitivity of the test system, the reference item Nutdazim 50 FLOW (Carbendazim, SC 500) is routinely tested at concentrations of 5 and 10 mg product/kg soil dry weight. In the most recent study with Nutdazim 50 FLOW, the number of juveniles was reduced by 39 and 100 % at concentrations of 5 and 10 mg product/kg soil dry weight (mean number of juveniles = 77 and 0) after 8 weeks of test duration when compared to control (mean number of juveniles = 127).

Reported statistics and error estimates:

Fisher’s Exact Binomial Test with Bonferroni Correction
Williams-t-test (p ≤ 0.05, one-sided), Probit analysis (Finney 1971)
Statistical program: ToxRat Professional 2.10.06 (2010)

Table 1. Effect of the test substance on the growth of adult earthworms

Treatment group	mg formulation/kg soil d.w.
	control	1.28	2.30	4.14	7.45	13.42	24.15	43.47	78.24
Replicate	Initial fresh weight / worm [mg] (mean per replicate)
1	443.1	468.3	470.4	467.1	470.1	472.0	462.7	468.3	462.1
2	480.6	474.0	505.5	478.9	475.4	475.9	473.9	480.0	481.3
3	490.1	494.5	483.4	490.5	491.2	489.9	494.6	493.8	482.7
4	506.9	499.6	496.0	504.0	496.6	496.1	482.5	496.1	513.8
5	471.7
6	474.9
7	494.5
8	495.2
mean	482.1	484.1	488.8	485.1	483.3	483.5	478.4	484.6	485.0
SD	19.6	15.3	15.3	15.8	12.6	11.4	13.5	13.0	21.4
cv %	4.1	3.2	3.1	3.3	2.6	2.4	2.8	2.7	4.4
	Fresh weight/worm [mg] after 4 weeks (mean per replicate)
1	628.6	604.2	595.7	615.4	624.6	604.5	633.8	560.8	569.8
2	574.8	655.3	598.8	605.9	615.5	621.5	576.3	617.3	622.8
3	609.1	605.6	618.9	664.8	619.1	626.9	607.3	618.7	637.0
4	621.3	626.0	644.3	626.6	608.4	632.6	627.5	668.1	619.5
5	594.9
6	623.7
7	629.2
8	662.3
mean	618.0	622.8	614.4	628.2	616.9	621.4	611.2	616.2	612.3
SD	26.0	23.9	22.4	25.8	6.8	12.1	25.9	43.9	29.3
cv %	4.2	3.8	3.6	4.1	1.1	2.0	4.2	7.1	4.8
	Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight) weight/worm [mg] (mean per replicate)
1	185.5	135.9	125.3	148.3	154.5	132.5	171.1	92.5	107.7
2	94.2	181.3	93.3	127.0	140.1	145.6	102.4	137.3	141.5
3	119.0	111.1	135.5	174.3	127.9	137.0	112.7	124.9	154.3
4	114.4	126.4	148.3	122.6	111.8	136.5	145.0	172.0	105.7
5	123.2
6	148.8
7	134.7
8	167.1
mean	135.9	138.7	125.6	143.0	133.6	137.9	132.8	131.7	127.3
SD	29.9	30.2	23.5	23.7	18.1	5.5	31.3	32.9	24.4
	Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight) [%] (mean per replicate)
1	41.9	29.0	26.6	31.7	32.9	28.1	37.0	19.8	23.3
2	19.6	38.2	18.5	26.5	29.5	30.6	21.6	28.6	29.4
3	24.3	22.5	28.0	35.5	26.0	28.0	22.8	25.3	32.0
4	22.6	25.3	29.9	24.3	22.5	27.5	30.1	34.7	20.6
5	26.1
6	31.3
7	27.2
8	33.7
mean	28.3	28.8	25.8	29.5	27.7	28.5	27.9	27.1	26.3

Not statistically significant compared to control regarding biomass (Williams-t-test, p > 0.05, one-sided smaller) SD: standard deviation, cv %: coefficient of variation, d.w.: dry weight (of artificial soil)

Table 2. Effects of the test substance on Mortality and Reproduction of Earthworms

Treatment	mg formulation/kg soil d.w.
group	control	1.28	2.30	4.14	7.45	13.42	24.15	43.47	78.24
Replicate	Number of surviving adult worms per replicate (4 weeks after test initiation)
1	10	10	10	10	10	10	10	10	10
2	10	10	10	9	10	10	8	10	10
3	10	10	10	10	10	9	10	10	9
4	9	10	10	10	10	10	10	10	10
5	10
6	10
7	10
8	10
mean	9.9	10.0	10.0	9.8	10.0	9.8	9.5	10.0	9.8
SD	0.4	0.0	0.0	0.5	0.0	0.5	1.0	0.0	0.5
cv %	3.6	0.0	0.0	5.1	0.0	5.1	10.5	0.0	5.1
	Mortality (%)
mean	1.3	0.0	0.0	2.5	0.0	2.5	5.0	0.0	2.5
	Number of juvenile worms per replicate
1	98	103	87	109	98	74	92	57	48
2	121	127	109	79	83	102	69	95	65
3	105	119	97	104	127	97	88	59	31
4	85	94	115	123	114	111	98	66	74
5	113
6	75
7	137
8	103
mean	104.6	110.8	102.0	103.8	105.5	96.0	86.8	69.3*	54.5*
SD	19.6	15.0	12.5	18.4	19.1	15.8	12.5	17.6	19.0
cv %	18.8	13.5	12.2	17.7	18.1	16.4	14.4	25.4	34.9
	Change of reproduction (%)
% to control	-	-5.9	2.5	0.8	-0.8	8.2	17.1	33.8	47.9

Not statistically significant compared to control regarding mortality (Fisher’s Exact Binomial Test with Bonferroni Correction, p > 0.05, one-sided greater)

* statistically significant compared to control (Williams-t-test, p ≤ 0.05, one-sided smaller)

SD: standard deviation, cv %: coefficient of variation, d.w.: dry weight (of artificial soil)

Negative values = increase, relative to control

Table 3. Effects of the test substance on Eisenia fetida: Summary of Statistical Analysis

NOEC (mortality)	78.24 mg formulation/kg soil d.w.
NOEC (biomass)	78.24 mg formulation/kg soil d.w.
NOEC (reproduction)	24.15 mg formulation/kg soil d.w.
LC50	> 78.24 mg formulation/kg soil d.w.
EC10	11.70 mg formulation/kg soil d.w. * (95 % confidence limits 6.51 to 21.02 mg test item/kg soil d.w.)
EC20	23.43 mg formulation/kg soil d.w. * (95 % confidence limits 16.30 to 33.69 mg test item/kg soil d.w.)
EC50	> 78.24 mg formulation/kg soil d.w. *

*Based on reproduction

Validity Criteria
The validity criteria for the control group were accomplished:
adult mortality: ≤ 10 % (being 1.3 % after 4 weeks)
number of juveniles per replicate (1, 2, 3, 4, 5, 6, 7 and 8): ≥ 30 (being 98, 121, 105, 85, 113, 75, 137 and 103)
coefficient of variation for reproduction: ≤ 30 % (being 18.8 %)

Therefore, the study is considered valid.

Validity criteria fulfilled:

yes

Remarks:

See Validity criteria in 'Any other information on results incl. tables'

Conclusions:

In a long-term toxicity study in earthworm (OECD TG 222, GLP), the 56-day NOEC and EC10 value for reproduction were determined to be 6.17 and 2.99 mg test substance in formulation/kg soil dw, respectively, based on nominal concentrations.

Executive summary:

This long-term toxicity study on the earthworm Eisenia fetida was performed following OECD TG 222 and in compliance with GLP. The study consisted of nine treatment groups: eight concentrations of the test substance (1.28, 2.30, 4.14, 7.45, 13.42, 24.15, 43.47, 78.24 mg formulation/kg soil) and blank control. The test substance was mixed with artificial soil to produce the required test concentrations, and the required amount of water was added to obtain the specified moisture content. Ten adult earthworms (weight range 398 - 560 mg/worm) were exposed per test vessel containing 750 g of soil (wet weight). There were 4 replicates for the test substance-treated group and 8 for the control group. The test duration was 56 days. Adult worm mortality, behavioural effects and biomass development were determined after 28 days of exposure. The reproduction rate was determined after 56 days of exposure. The test conditions were: temperature 20 - 22 °C, light intensity 450 lux (16h: 8h light-dark cycle), pH 5.73 - 6.03 and the soil moisture at test start and end ranged from 24.3 to 25.0 %.

The mortality of adult worms was 0 – 5.0 % in the treated variants and 1.3 % in the control group. No statistically significant mortality compared to the control was observed at any test item concentration. No pathological symptoms and no effects on the behaviour (including feeding activity) of the worms were observed during the test. The test item caused no statistically significant change in biomass compared to the control group. Statistically significant effects on the number of juveniles compared to the control group were recorded at 43.47 and 78.24 mg formulation/kg soil dw treatment groups. Based on the findings, the 56-day NOEC and EC10 for reproduction were determined to be 6.17 and 2.99 mg test substance in formulation/kg soil dw, respectively, based on nominal concentrations. The NOEC value for mortality and biomass was determined to be 20 mg test substance in formulation/kg soil dw.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Apr 1999 to 07 May 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

PREPARATION OF SOIL MEDIUM
Artificial soil was used as the test substrate. The artificial soil medium consisted (based on mass) of 70% industrial sand (at least 50% of the particles were 50 - 200 pm), 20% kaolin clay (kaolinite > 30%) and 10% sphagnum peat moss. The soil was mixed with a cement mixer. CaCO3 was added in order to adjust the soil pH to approximately 6.0 ± 0.5. The moisture of the completed soil prior to the addition of test solutions was approximately 20% (percent weight of dry soil). The moisture content and pH of the soil were measured at initiation and termination of the definitive test. A sample of the artificial soil made up with the same ingredients as the soil used during the definitive test were analysed for the presence of pesticides, metals and PCB’s. None of these compounds were detected at concentrations considered toxic in the soil sample analysed. No pesticide residue analysis was performed on the deionized water since the presence of pesticide residues was not expected.

STOCK SOLUTION PREPARATION
A stock solution was prepared by diluting 5.6457 g of the test material, with acetone to a total volume of 10 mL (equivalent to 535.7 g a.i./L acetone or 1000 mg a.i./kg dry soil after application). Thereafter, the stock was serially diluted by taking aliquots of 5 mL and diluting it with acetone to a total volume of 10 mL. Following solution concentrations were thus obtained: 267.85, 133.93, 66.96 and 33.48 g a.i./L solvent, corresponding to 500, 250, 125, and 62.5 mg a.i./kg dry soil, respectively.

Immediately after stock preparation, 1 mL of each solution and 105.1 mL of deionized water were added to the glass beakers containing each 644.9 g of artificial soil with 20.39% soil humidity (based on dry weight) . After the addition of the test item solution and deionized water, the soil was mixed thoroughly with a mixing spindle connected to a drill. The moisture of the soil resulted in 39.1 to 39.8% humidity. Applications were performed proceeding from the control to the test item treatments in increasing concentrations. Since the mixer was progressively used for replicates with increasing concentrations, it was cleaned after all applications were performed. To the solvent control, 1 mL of acetone was added instead of the test item solutions. The control was prepared as described above without adding acetone.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Source: Bred at the test facility
- Age at test initiation: All the earthworms used in this study were mature, at least 2 months old, and possessed clitellae.
- Weight at test initiation: The weight of each earthworm was between 323 and 600 mg, i.e. within the acceptable 300 to 600 mg range. Before weighing, the earthworms were washed with deionised water and gently dried on blotting paper.

ACCLIMATION
The earthworms were cultured in a medium consisting of peat and commercial garden soil and fed with horse manure (from horses untreated with antihelmintics or other medication for at least four weeks), dregs and lucern meal.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

18.5 - 24 °C

pH:

5.94 - 6.15

Moisture:

39.1 - 39.8%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): The test was conducted in clean 1.5 L glass beakers. Test vessels were covered with a loose-fitting glass lid to prevent excessive water evaporation, but also allowing gas exchange.
- Amount of soil or substrate: Each replicate test vessel contained 750 ± 2 g of moist artificial soil (moisture content 35-40% of the dry soil weight), which was approximately equivalent to 535.7 g of dry soil.
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4
- Introduction of individuals: One day before test initiation, the earthworms were transferred to artificial OECD test soil and held under test conditions to acclimatise. At test initiation, the weight of each earthworm was determined and only those having weights between 300 and 600 mg were used for the test.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
See 'Details on preparation and application of test substrate'.

OTHER PARAMETERS
- Lighting: Light was provided continuously, with an intensity of 608 to 790 lux. by fluorescent lights to ensure that the earthworms remained in the test medium throughout the duration of the study.

SOIL QUALITY MEASUREMENTS
Moisture and pH of the artificial OECD soil were monitored and recorded on day 0 (after the addition of the treatment solutions) and on day 14 (completion of the test) . The moisture content and the pH-value were measured from two soil samples. The first sample was taken from replicates A and B and the second sample from replicates C and D. The pH was measured using a pH meter. The water content was determined by weighing the samples as they were taken and after drying at approximately 105 °C and taking the difference between the two weights. After that, the water content as percent moisture of dry soil weight was calculated.
Air temperature in the environmental chamber was monitored continuously using a Thermohygrometer. Light intensity at the soil surface was measured at beginning of the study using a light meter.

EFFECT PARAMETERS MEASURED: burrowing time, mortality, signs of toxicity
- Burrowing time: The burrowing time of the earthworms was observed on days 0 and 7. It was defined as the time needed for all ten earthworms within each treatment replicate to burrow completely underneath the soil surface. This was achieved by observing the worms 10, 30, and 60 minutes after being placed on the soil surface.
- Mortality and signs of toxicity: After 7 and 14 days, earthworm mortality and health assessments were performed. This was achieved by emptying the test medium onto a tray, sorting the earthworms from the medium, and testing their reaction to a mechanical stimulus. Mortality was defined as the lack of visible movement after gentle mechanical stimulation was applied. The general health of the earthworms was assessed and recorded by observing colour changes, lethargy, lesions and the absence of burrowing. On day 7, earthworms and medium were returned to the test vessels. Earthworm weights were measured at the beginning (day 0) and end (day 14) of the test after removing adhering soil with deionized water and blotting them dry with paper towels.

RANGE-FINDING STUDY
A range-finding test (at test item concentrations of approximately 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry soil) was conducted in order to determine definitive test concentrations. At termination of this preliminary test (day 14), 100% mortality was observed in the 1000 mg a.i./kg dry soil treatment. In all other treatments no mortality was observed. Based on the results of mortality, the definitive test was performed at the test material, concentrations of 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil weight, including a solvent control as well as a deionized water control.

Nominal and measured concentrations:

- Nominal test concentrations: 0 (negative control), 0 (solvent control), 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil

Reference substance (positive control):

yes

Remarks:

2-Chloracetamide

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

686.47 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% C.I.: 500 - 1000 mg/kg soil dw

Details on results:

The results are tabulated in 'Any other information on results incl. tables'.

MORTALITY
Table 1 presents the mortality and health of earthworms on test days 7 and 14. On test day 7, all earthworms were healthy in the solvent control, the control, the 62.5, 125 and 250 mg a.i./kg dry soil test item treatments. In the 500 mg a.i./kg dry soil treatment 97.5% of the earthworms were healthy and 2.5% (i.e. one worm) had lesions. This was not significantly different from the control (Yates corrected Chi-square test: p = 1.000). In the 1000 mg/kg dry soil treatment, 95.0% of the earthworms were dead. Of the surviving earthworms 50% were healthy and 50% had lesions. Mortality and the percent number of worms with lesions in the 1000 mg a.i./kg dry soil treatment were statistically significantly different to the solvent control (Yates corrected Chi-square tests: p < 0.05). On test day 14, all earthworms in the controls, the 62.5, 125 and 250 mg a.i./kg dry soil were alive and healthy. Levels of mortality of 2.5% and 100% were observed in the 500 and 1000 mg a.i./kg dry soil treatments, respectively. In the 500 mg a.i./kg dry soil treatment 90% of the surviving earthworms were healthy and 10% were lethargic, with no statistically significant difference form the solvent control (Yates corrected: Chi-square test: p = 0.118). There was a statistically significant difference in mortality between the solvent control and the 1000 mg a.i./kg dry soil test item treatment (Yates corrected Chi-square test: p < 0.05), but not between the solvent control and the 500 mg a.i./kg dry soil test item treatment (Yates corrected Chi-square test, p = 1.000).

WEIGHTS
The mean weights of the earthworms on test day 0 and the weight differences between test days 0 and 14 are presented in Table 2. On day 0, the mean earthworm body weight were 467, 464, 441, 420, 427, 420 and 457 mg for the solvent control, the deionized water control, the 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil treatments, respectively. There were statistically significant differences between the solvent control and the 125 and 500 mg a.i./kg dry soil test item treatments (Kruskal-Wallis ANOVA: H = 25.441, p = 0.0003 / Tukey HSD by ranks: a < 0.05). Therefore, only weight differences between day 14 and day 0 were analysed to compare the treatments. During the 14-day test, the solvent control earthworms lost an average of 33 mg weight per individual. The mean body weight losses of the earthworms treated with deionized water, 62.5, 125, 250 and 500 mg a.i./kg dry soil were 28, 30, 28, 27 and 78 mg per individual, respectively. No statistically significant difference in weight loss were observed between the treatments (Kruskal-Wallis ANOVA: H = 10.470, p = 0.063). This corresponds to decreases in weight of 7.1, 6.0, 6.8, 6.7, 6.3 and 18.6% in the respective treatments.

BURROWING TIME
Table 3 represents the data of earthworm burrowing time recorded during the study. On test day 0, the maximum burrowing time of the earthworms exposed to the solvent control, the deionized water control, the 62.5 and the 125 mg a.i./kg dry soil test substance, treatments was lower than 10 minutes. For the 250 mg a.i./kg dry soil test item treatment, the maximum burrowing time was between 30 and 60 minutes and for the 500 and 1000 mg a.i./kg dry soil test item treatments the burrowing time was more than 60 minutes. On day 7, the earthworms of the solvent control, the control, the 62.5, 125 and 250 mg a.i./kg dry soil test item treatments needed less than 10 minutes to burrow into the soil. The earthworms exposed to the 500 mg a.i./kg dry soil and the 2 surviving worms exposed to the 1000 mg a.i./kg dry soil test substance, treatment needed more than 60 minutes to burrow into the soil.

Results with reference substance (positive control):

The sensitivity of earthworms used for the present study was evaluated using the reference item 2-Chloracetamide (>98% purity). The study was conducted separately, at the same facility, and under GLP. The LC50 of 2-Chloracetamide was determined to be 42.24 mg test item/kg dry soil (95%-confidence interval of 25 to 50 mg test item/kg), which is within the range of 20-80 mg/kg stated by Ma and Bodt (1993). The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) for 2-Chloracetamide were determined to be 25 mg test item/kg dry soil and 50 mg test item/kg dry soil, respectively.

Reported statistics and error estimates:

Mortality data on test day 14 were used to estimate the median lethal concentration (LC50), which is the estimated concentration of the test item which would produce 50% mortality in the earthworm population under the experimental conditions. A computer program, modified from the program of C. Stephan (Peltier and Weber, 1985) was used to calculate the LC50 value using three statistical methods (probit analysis, moving average method and binominal probability) including the 95% confidential interval. The method selected here was the binominal test method.

The NOEC (No Observed Effect Concentration) is defined as the highest concentration tested at which no significant test item effects are observed. The LOEC is defined as the lowest concentration tested at which a significant test item effect is observed. The NOEC and LOEC were determined by comparing earthworm mortality and weight loss between control and test item treatments.

Dichotomous data (i .e. mortality, health data) were analysed for significant differences between the solvent control and the test item treatment using the Yates corrected Chisquare test (Zar, 1984) . Earthworm weight was analysed for normality using the Shapiro-Wilk's Test (Zar, 1984). Since assumption of normality was not met, Kruskal-Wallis ANOVA, followed by non-parametric comparison tests (Tukey's Multiple Comparison test by ranks) was used to compare treatments (Zar, 1984). The computer program used to run the statistical analysis was "Statistica for Windows", by StatSoft Inc., Tulsa. OK, USA.

Table 1: Mortality and health of Eisenia fetida during the 14-day acute toxicity test with the test material, (n = sample size) 

Treatment (mg a.i./kg dry soil)	% Mortality (mean ± SD)	Surviving worms
		Healthy (mean ± SD)	Lethergic (mean ± SD)	With lesions (mean ± SD)	n
Day 7
0 (Solvent control)	0 ± 0	100 ± 0	0 ± 0	0 ± 0	40
0 (Control)	0 ± 0	100 ± 0	0 ± 0	0 ± 0	40
62.5	0 ± 0	100 ± 0	0 ± 0	0 ± 0	40
125	0 ± 0	100 ± 0	0 ± 0	0 ± 0	40
250	0 ± 0	100 ± 0	0 ± 0	0 ± 0	40
500	0 ± 0	97.5 ± 5.0	0 ± 0	2.5 ± 5.0	40
1000	95 ± 10*	50.0 ± 0	0 ± 0	50.0 ± 0*	2
Day 14
0 (Solvent control)	0 ± 0	100 ± 0.0	0 ± 0	0 ± 0	40
0 (Control)	0 ± 0	100 ± 0.0	0 ± 0	0 ± 0	40
62.5	0 ± 0	100 ± 0.0	0 ± 0	0 ± 0	40
125	0 ± 0	100 ± 0.0	0 ± 0	0 ± 0	40
250	0 ± 0	100 ± 0.0	0 ± 0	0 ± 0	40
500	2.5 ± 5.0	90.0 ± 14.1	10.0 ± 14.1	0 ± 0	39
1000	100 ± 0.0*	NA	NA	NA	NA

 * Statistically significant different to the solvent control (Yates corrected Chi-square: P < 0.05).

 

 

Table 2: Mean weights of Eisenia fetida determined at test initiation and test end, and mean weight difference of surviving earthworms at test termination of the 14-day acute toxicity test with the test material, (n = sample size).

Treatment (mg a.i./kg dry soil)	Weight (mean ± SD)
	Day 0 (mg) ( n= 40)	Difference Day 14 – Day 0 (mg) (n)	% reductions in body weight
0 (Solvent control)	467 ± 80	-33 ± 13(4)	-7.1%
0 (Control)	464 ± 54	-28 ± 6(4)	- 6.0%
62.5	441 ± 65	-30 ± 5(4)	- 6.8%
125	420 ± 51*	-28 ± 6 (4)	- 6.7%
250	427 ± 69	-27 ± 12 (4)	- 6.3%
500	420 ± 72 *	-78 ± 9 (4)	- 18.6%
1000	457 ± 65	NA	NA

 * Statistically significant difference when compared to the solvent control (Kruskal-Wallis ANOVA: H = 25.441, p = 0.0003 / nonparametric comparison test (Tukey HSD by ranks: a < 0.05))

 

Table 3: Burrowing time of Eisenia fetida on test days 0 and 7 of the 14-day acute toxicity test with the test material

Treatment (mg a.i./kg dry soil)	Burrowing time (minutes)
	Day 0					Day 7
	Replicate				Maximum	Replicate				Maximum
	A	B	C	D		A	B	C	D
0 (Solvent control)	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
0 (Control)	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
62.5	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
125	<10	<10	<10	<10	<10	<10	<10	<10	<10	<10
250	<60	<30	<30	<30	>60	<10	<10	<10	<10	<10
500	>60	>60	>60	>60	>60	<10	<10	>60	<30	>60
1000	>60	>60	>60	>60	>60	>60	NA	NA	NA	>60

 

 

Validity criteria fulfilled:

yes

Conclusions:

In a short-term toxicity study on earthworms, performed according to OECD TG 207, the 14-day LC50 was determined to be 686.47 mg/kg dry soil.

Executive summary:

The short-term toxicity to terrestrial invertebrates was investigated in a study in accordance with OECD TG 207 and in compliance with GLP using earthworms (Eisenia fetida). Adult earthworms were exposed to the test material through artificial soil as substrate for two weeks to a series of treatment rates; 0 (negative control), 0 (solvent control), 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil (spacing factor: 1.8). The toxic standard ( 2-Chloracetamide) was also tested as a separate study to confirm the normal reaction of the earthworms against pesticides. Throughout the study, the environmental conditions were maintained as recommended in the standard guideline.

On days 7 and 14, mortality was statistically significantly different to the solvent control in the 1000 mg a.i./kg dry soil treatment (Yates corrected Chi-square tests: p < 0.05). In this treatment,on day 7, the number of worms with lesions was statistically significantly different to the solvent control (Yates corrected Chi-square test: p<0.05). On day 14, mortality of 2.5% was observed in the 500 mg a.i./kg dry soil treatment whichwasnot statistically significantly different to the solvent control (Yates corrected Chi-square test:p > 0.05). In the test item treatments with concentrations up to and including 250 mg a.i./kgdry soil, no mortality was observed. In the treatments where earthworms survived, i.e.test item treatments with concentrations up to and including 500 mg a.i./kg dry soil and controls, there were no statistically significant differences in the mean weight loss (day 14 - day 0) (Kruskal-Wallis-ANOVA: H = 10.470,p=0.063). On days 0 and 7, earthworm burrowing time was more than 60 minutes in the 500 and 1000 mg a.i./kg dry soil treatments. Based on the fings, the 14-d LC50 was determined to be 686.47 mg/kg soil dw. 

Description of key information

One long-term study and one short-term study on earthworms are included here, and their effect value was used as the key value. 

56-d EC10 = 2.99  mg test substance in formulation/kg soil d.w, reproduction, Eisenia fetida, OECD TG 222, Friedrich 2013

The 14-d LC50 was determined to be 686.47 mg/kg soil d.w, Eisenia fetida, OECD TG 207, Nienstedt 1999

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

686.47 mg/kg soil dw

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

2.99 mg/kg soil dw

Additional information

Two GLP studies following OECD guidelines (one long-term study and one short-term study) on earthworms (Eisenia fetida) are available for this endpoint and included as key studies.

In the long-term study, earthworms were exposed to the test substance formulation (A6097AF) at concentrations of 1.28, 2.30, 4.14, 7.45, 13.42, 24.15, 43.47, 78.24 mg formulation/kg soil dw for 56 days (corresponding to 0.327, 0.588, 1.06, 1.91, 3.43, 6.17, 11.1, 20 mg test substance in formulation/kg soil dw). The test substance was mixed with artificial soil to produce the required test concentrations. Ten adult earthworms (weight range 398 - 560 mg/worm) were exposed per test vessel containing 750 g of soil (wet weight). There were 4 replicates for the test substance-treated group and 8 for the control group. The test conditions were: temperature 20 - 22 °C, light intensity 450 lux (16h: 8h light-dark cycle), pH 5.73 - 6.03 and the soil moisture at test start and end ranged from 24.3 to 25.0 %. The 56-day NOEC and EC10 value for reproduction was determined to be 6.17 and 2.99 mg test substance in formulation/kg soil dw, respectively, based on nominal concentrations (Friedrich 2013, Reliability 1).

In the short-term toxicity study, the adult earthworms were exposed to the test material at concentrations of 0 (negative control), 0 (solvent control), 62.5, 125, 250, 500 and 1000 mg a.i./kg dry soil for 14 days. The 14-d LC50 was determined to be 686.47 mg/kg soil dry weight (Nienstedt 1999, Reliability 1).