N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

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:

12 Jul 2002 to 06 Sep 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Short term deviation of temeprature( -1 ˚C), no impact is expected.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of the test substrate: An exactly weighed amount of the test item was dispersed in water, without addition of solubility mediators, immediately before application. This stock solution was diluted with deionised water in a way that the resulting solutions contained the amount of test item required for 556 g d.w. articial soil (25 % water content). After adding the required amount of test item solution into the mixing vessel to adjust the selected concentration and a water content of about 35 %, the test item dispersed in deionized water was thoroughly mixed with the basic substrate using a mixing machine. The control was treated with deionised water only.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: The organisms were originally purchased. Then they were hold and breed under ambient laboratory conditions in the test facility:
> Breeding medium: Mixture of horse manure, straw, peat (1:1 :1) all ingredients were free from pesticides
> Breeding condition: Breeding cages (50 cm x 40 cm x 30 cm); diffuse light; temperature: about 20 °C; moist soil; pH: about 7
- Age at test initiation: Adult worms (about 4 months old with clitellum)
- Weight of animals used in the test: 329 - 461 mg/worm
- Food used during the test: Air-dried finely ground horse manure

ACCLIMATION
- Acclimation period: About 24 hours in the test substrate

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Exposure included 4 weeks adult mortality and 4 weeks juvenile development

Test temperature:

17 - 22 °C (test room)

pH:

- Start: 6.2
- End: 5.8 - 6.2

Moisture:

- WHC:60.1%
- Water concent: 36.1% (start); 35.3 - 36.6% (end)

Details on test conditions:

TEST SYSTEM
- Test container: Plastic vessel of Bellaplast (18.3 cm x 13.6 cm x 6 cm) with a lid which is pervious to air and light
- Amount of substrate/test vessel: 750 g wet weight corresponding to about 556 g dry weight of articial soil with about 35 % water content
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
The test vessels were set up at random in the test room.

PROPERTIES OF SUBSTRATE (artificial soil)
- Composition of the artificial soil: 10 % Sphagnum peat; 20 % kaolinite clay; 0.5 % calcium carbonate; 69.5 % industrial quartz sand; deionized water
- Maximum water holding capacity of the soil: 60.1%

OTHER TEST CONDITIONS
- Photoperiod: Light : dark = 16 : 8 h
- Light intensity: 600 lx

EFFECT PARAMETERS MEASURED
- At the start of the test, the following parameters were analysed: individual fresh weight (mg/earthworm); behaviour of earthworms and physico-chemical parameters (water content, pH) of the articial soil.
- Behavioural and pathological symptoms were observed weekly.
- The following parameters were analysed 4 weeks after application: the number of surviving adult earthworm for each test vessel; observation of behavioural and pathological symptoms; individual fresh weight of surviving adult earthworms (mg/worm)
- The following parameters were analysed 8 weeks after application: the number of surviving juveniles for each test vessel; observation of behavioural and pathological symptoms; morphological alterations; physico-chemical parameters (water content, pH) of the articial soil.

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.12, 0.24, 0.6 and 1.2 mg test subtance/kg dw

Reference substance (positive control):

yes

Remarks:

Benomyl 50%

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

and growth

Details on results:

An overview of the results is provided in Table 1 – Table 2 in ‘Any other information on results incl. tables’
Effects of the test item on mortality, growth and reproduction of earthworms were determined at concentrations of 0.12, 0.24, 0.6 and 1.2 mg test substance/kg dry weight of soil giving five treatment groups including one untreated control group. Four replicates per treatment group were used. At the beginning of the test, the fresh weight of the earthworms was in the range of 329-461 mg/earthworm. The physico-chemical parameters measured at the start and at the end of the test corresponded to the methodical requirements. No effects of the test item on adult worm survival were observed (0 % mortality) at doses of 0.12, 0.24, 0.6 and 1.2 mg/kg dry weight of soil during 4 weeks. The earthworm growth was not statistically significantly affected at any concentration. (change in fresh weight after 4 weeks relative to initial fresh weight), i.e. a weight increase of 23 %, 15.9%, 13.6 % and 21.6 % was recorded at doses of 0.12, 0.24, 0.6 and 1.2 mg test substance/kg d.w., respectively. No statistically significant effects on number of juveniles compared to the control group were recorded at doses of 0.12, 0.24, 0.6 and 1.2 mg test substance/kg d.w. Therefore, the no-observed-effect-concentration (NOEC) is assumed to be 1.2 mg/kg dry weight of artificial soil. No effects on behaviour and morphology of the adult worms and on feeding activity were observed during the first four weeks of the test.

Results with reference substance (positive control):

For controlling the sensitivity of the test system the reference item is regularly tested at a concentration of 1.25 kg product/ha. In the most recent test at the time of the experiment, the number of juveniles was reduced by 95.2 % after 8 weeks of test duration when compared to control.

Reported statistics and error estimates:

The endpoints are growth (fresh weight of surviving worms) and reproduction (the number of juveniles produced). The arithmetic mean and the variance per treatment and per control for reproduction and biomass were calculated. The statistical analysis was performed with EASY ASSAY computer program (RATTE 1998). The multiple sequential test procedure after Dunnett was used to compare the control with the independent test item groups and shows a non-significant difference between both.

Table 1. Effects of the test substance on growth (biomass increase during 4 weeks exposure) of adult earthworms

Test substance [mg/kg d.w.]
Replicate		Control			0.12		0.24			0.6 I			1.2
Initial fresh weight / worm [mg] (mean per
1		393.6			372.3		395.6			396.2			421.8
2		408.6			390.5		380.9			393.1			382.4
3		373.1			365.2		381.5			390.0			373.2
4		359.6			359.5		388.5			363.4			359.5
average		383.7			371.9		386.6			385.7			384.2
SD		23.1			20.0		30.2			27.3			27.4
cv%		6.0			5.3		7.8			7.1			7.2
Fresh weight / worm [mg] after 4 weeks (mean per replicate)
1		465.8				425.1	502.6			433.8			453.2
2		489.7				455.8	447.8			451.8			440.3
3		429.8				448.5	436.5			438.4			502.1
4		419.9				496.7	406.6			426.6			465.2
average		451.3				456.5	448.4			437.7			465.2
SD		72.7				78.2	75.0			68.5			72.3
cv%		16.1				17.2	16.6			15.7			15.5
Biomass increase (change in fresh weight after 4 weeks relative to initial fresh weight weight / worm [mg] (mean per replicate)
	1		72.2		52.8				107.0			37.6			31.4
	2		81.1		65.3				66.9			58.7			57.9
	3		56.7		83.3				55.0			48.4			128.9
	4		60.3		137.2				18.1			63.2			105.7
	average		67.6		84.7				61.8			52.0			81.0
	SD		11.2		37.2				36.6			11.4			44.3
	cv%		16.6		43.9				59.3			22.0			54.8
	Biomass increase (change in fresh weight after 4 weeks relative to initial weight) [%] mean per replicate
	1		18.3					27.0			9.5			7.4
	2		19.8	16.7				17.6			14.9			15.1
	3		15.2	22.8				14.4			12.4			34.5
	4		16.8	38.2				4.7			17.4			29.4
	average		17.5	23.0				15.9			13.6			21.6
	SE			-				-			-			-

SD: standard deviation; cv %: coefficient of variation; SE: statistical evaluation

- not statistically significantly different (DUNNETT-Test, p < 0.05)

Table 2. Effects of test substance on mortality of adult earthworms and reproduction

		Test substance [mg/kg d.w.]
Replicate	Control	0.12	0.24	0.6	1.2
Number of living adult worms per replicate (4 weeks after test initiation)
1	10	10	10	10	10
2	10	10	10	10	10
3	10	10	10	10	10
4	10	10	10	10	10
average	10	10	10	10	10
Mortality per treatment (%)
average	0	0	0	0	0
Number of juveniles per replicate
1	97	118	1 13	102	85
2	93	95	74	91	76
3	89	101	89	109	82
4	102	98	71	75	77
average	95.3	103.0	86.8	94.3	80.0
SD	5.6	10.3	19.2	14.8	4.2
cv%	5.84	10.00	22.12	15.72	5.30
SE		-	-	-	-

SD: standard deviation; cv %: coefficient of variation; SE: statistical evaluation

- not statistically significantly different (DUNNETT-Test, p < 0.05)

Validity criteria

The study is considered to be valid, since the validity criteria were accomplished:

For the control group:

- Adult mortality: ≤ 10 % (being 0 % after 4 weeks).

- Weight loss of adult worms: ≤ 20 % (being increased by 17.5 % after 4 weeks)

- Number of juveniles/vessel: ≤ 30 (being 97, 93, 89, 102 / replicate 1, 2, 3 and 4)

- Coefficient of variation of juvenile number: ≤ 30 % (being 5.8 %)

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

In this toxicity study to the earthworm Eisenia fetida, performed in accordance with ISO 11268-2, the 8-week NOEC was determined to be 1.2 mg/kg soil dw. for reproduction.

Executive summary:

The toxicity of the test substance to earthworms (Eisenia fetida) was tested according to ISO 11268-2 and was in compliance with GLP criteria. Four replicates of 10 earthworms per treated group were exposed in artificial soil to nominal concentrations of 0.12, 0.24, 0.6 and 1.2 mg test substance/kg dw soil, for a 8-week study. An untreated group was included as control. The test organisms were adult earthworm, Eisenia fetida, of about 4 months old with clitellum and weighing 329 – 461 mg. They were acclimatised in an artificial soil for 24 hours prior to exposure. One day after application, 3 g dry ground horse manure was scattered on the soil surface of each test vessel, and this was sprinkled with 5 mL deionised water. The feeding interval was weekly during the first four weeks of the study. After four weeks, the adult worms were removed, and the number of surviving worms and the bodyweight of worms per vessel determined. The juvenile worms remained in the test vessels for a further 4 weeks, when the test was terminated and the number of juvenile worms determined. The test conditions were maintained at 17 - 22 ˚C; 16 hour light and 8 hour darkness (light intensity: 600 lux), and pH 5.8 – 6.2. The endpoints are growth (fresh weight of surviving worms) and reproduction (the number of juveniles produced). The arithmetic mean and the variance per treatment and per control for reproduction and biomass were calculated.

No effects of the test item on adult worm survival were observed in any of the test concentrations during the 4 weeks of exposure. The earthworm growth change (change in fresh weight after 4 weeks relative to initial fresh weight) was not statistically significantly affected at any test concentration. Exposure to the test substance at all test concentrations caused no statistically significant effects on earthworm reproduction. Based on this information, the chronic toxicity and reproduction NOEC for the earthworm was determined to be 1.2 mg test substance/kg (the highest concentration tested).

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jun 1989 to 13 Jul 1989

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:

4 April 1984

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

A dry stock mixture was prepared for each test concentration by premixing the corresponding amount of test substance with 200 g fine quartz sand.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Age at test initiation: Adult (> 2 months of age), mature with clitellum
- Weight at test initiation: 249 - 284 mg

ACCLIMATION
- Acclimation period: 24 hours conditioning in artificial soil

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 ± 1 °C

pH:

6.9 - 7

Moisture:

34 - 38 % (at the start and at the end of the test, respectively)

Details on test conditions:

TEST SYSTEM
- Test container: Beakers of 1 liter filled with 750 soil (wet weight), covered with perforated plastic film
- Amount of soil or substrate: 750 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity: 400—800 Lux, cold white fluorescent light

EFFECT PARAMETERS MEASURED:
Mortality and flaccidity - day 7 and day 14; pH and moisture content of soil and average live weight of test organisms - the beginning and the end of the test

Nominal and measured concentrations:

Nominal: 12.3, 37.0, 111, 333, 1000 mg/kg soil dry weight

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

There were no notable mortalities in any test concentrations, giving an LC50 value of >1000 mg/kg dry soil. Additionally, neither sub-lethal effects, such as flaccidity, nor treatment-related differences in body weight developments (0-14 d) occurred in the treatments.

Table 1. Acute toxicity of the test substance to Eisenia fetida

Concentration (mg/kg dw soil)	Cumulative mortality (%)	Mean body weight per worm (mg)		Body weight change (%)
	14 d	0 d	14 d
Control	0	263	249	- 5.3
12.3	0	281	232	- 17.4
37	0	277	258	- 6.9
111	3	284	277	- 2.5
333	3	249	227	- 8.8
1000	0	276	268	- 2.9

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

In a short-term toxicity study in earthworm (Eisenia fetida), the 14-day LC50 value for the test substance was determined to be >1000 mg/kg soil.

Executive summary:

This short-term toxicity study in the earthworm Eisenia fetida was performed in accordance with OECD TG 207 and in compliance with GLP. The study consisted of six groups: five concentrations of the test substance (12.3, 37.0, 111, 333 and 1000 mg/kg) and a 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. Forty adult earthworms (>2 months with clitellum) (group mean wet mass of 249 - 284 mg) were exposed per concentration in 1 L beakers containing 750 g of soil (wet weight), with 10 worms per container. The test duration was 14 days. Mortality was determined on days 7 and 14, and weight of worms was determined on day 14.

The temperature was maintained at 20±1°C, with continuous illumination at 400-800 lux. The pH at test start ranged from 6.9 to 7.0, and the soil moisture at test start and end ranged from 34 to 38 %. There were no notable mortalities in any test concentration, giving an LC50 value of >1000 mg/kg dry soil. Additionally, neither sub-lethal effects, such as flaccidity, nor treatment-related differences in body weight developments (0-14 d) occurred in the treatments. The 14-day LC50 value for the earthworm Eisenia fetida for the test substance was determined to be >1000 mg/kg soil.

Description of key information

8-wk, NOEC = 1.2 mg/kg soil dw, Eisenia fetida, growth and reproduction, ISO 11268 -2, Papaja 2002

14-d, LC50 > 1000 mg/kg soil dw, Eisenia fetida, mortality, OECD TG 207, Rufli 1989

Key value for chemical safety assessment

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

1.2 mg/kg soil dw

Additional information

One acute and one chronic toxicity study of the test substance on soil macroorganisms, Eisenia fetida, are available. Both of them are Reliability 1 studies, which followed OECD TG 207 and ISO 11268-2, respectively, and met the GLP criteria.

In the 14-d acute toxicity test, forty earthworms (> 2 months old with clitellum; group mean wet mass of 249 - 284 mg) were exposed per concentration (10 worms/replicate). The study consisted of six groups: five concentrations of the test substance (12.3, 37.0, 111, 333 and 1000 mg/kg) and a blank control. The temperature was maintained at 20 ± 1°C, with continuous illumination at 400 - 800 lux. The pH at test start ranged from 6.9 to 7.0, and the soil moisture at test start and end ranged from 34 to 38 %. No effects were observed in the highest concentration tested (1000 mg/kg soil dw). Thus, the effect value, LC50, was determined to be > 1000 mg/kg soil dw.

In the 8-wk long-term toxicity test using a formulation, four replicates of 10 earthworms (approximately 4 months old with clitellum and weighing 329 – 461 mg) per treated group were exposed in artificial soil to nominal concentrations corresponding to 0.12, 0.24, 0.6 and 1.2 mg a.s./kg dw soil. An untreated group was included as control. The test conditions were maintained at 17 - 22 ˚C; 16 hour light and 8 hour darkness (light intensity: 600 lux), and pH 5.8 – 6.2. The endpoints are growth (fresh weight of surviving worms) and reproduction (the number of juveniles produced). No mortality or effects on growth of adult worms, no effects on behaviour or morphology and no statistically significant effects on reproduction were observed up to the highest treatment. The arithmetic mean and the variance per treatment and per control for reproduction and biomass were calculated. The results indicate a NOEC value of 1.2 mg a.s./kg dw for growth and reproduction, this value was selected for CSA as well.

Metabolites – available information

In addition to the studies with the test substance, four toxicity studies on earthworm (Eisenia fetida, 3 acute and 1 long-term) are available with the major degradation products (i.e. M4, M6 and M7). As the dossier has been prepared to address the test substance itself, these studies are not summarized as endpoint study records but are briefly discussed here. The metabolite M6 had the highest acute toxicity to earthworms of the three major soil metabolites, showing an LC50 value of 530 mg/kg soil dw (Rufli 1997). The LC50 values for M4 and M7 were determined to be 610 and > 1000 mg/kg soil dw, respectively (Pfeifle 2001a and 2001b). A long-term earthworm reproduction study was additionally carried out with M6. The 56-d NOEC of M6 for growth and reproduction was determined to be 3.0 mg/kg soil dw (the highest concentration tested) (Friedrich 2003).