cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug 1986 to 5 Nov 1986

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:

not specified

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Tween 80

Details on preparation and application of test substrate:

The test article (5.555 g) was suspended in water (1000 mL) containing Tween 80. The final concentration of Tween in the soil was 0.18% (dry weight basis). The preparations were made immediately prior to the application.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Age at test initiation: Adult
- Weight at test initiation: 200 - 312 mg

ACCLIMATION
- Acclimation period: 24 hours

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

21.5 °C

pH:

7.4 to 7.6

Moisture:

23.0% to 37.4%

Details on test conditions:

TEST SYSTEM
- Test container: 1 L glass beakers
- Cover: Coverlid with a perforated plastic film to prevent the test medium from drying
- Amount of soil: 750 g wet artificial soil (corresponding to 555 g 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

SOURCE AND PROPERTIES OF SUBSTRATE
The sail was prepared according ta the OECD TG 207 consisting of the fallowing constituents (dry weight basis):
- Sand (particle size 0.2 mm): 389 g (70%)
- Clay-loam soil: 111 g (20%)
- Sphagnum peat: 55 g (10%)
- Sand (particle size > 0.05 mm): 22.9%
- Silt (particle size 0.05 - 0.002 mm): 36.1%
- Clay (particle size < 0.002 mm): 41.0%
After weighing of the above given constituents, the soil was mixed in a mixer. During mixing first 100 mL of water containing 1.0 g Tween 80 and the suspended test article at the desired concentration was added drapwise followed by addition of 69 mL of distilled water. By this procedure, the sail moisture contents of the artificial soil was adjusted ta 35%.

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Soil Moisture and pH: Soil moisture and pH of the soil were determined for each test sample at the end of the test. At the start, the moisture of the soil samples was adjusted to 35% (based on soil dry weight). The pH measured for the test medium at the beginning of the test was 6.5.

EFFECT PARAMETERS MEASURED
- Mortality: Mortality was determined at day 7 and 14 of the exposure period. Therefore, the contents of the beakers was emptied onto a stainless steel plate and the living worms counted. After the 7-day assessment, worms and medium were replaced in the test beakers. Based on the data, the LC50 was estimated by means of Logit analysis.
- Body weights: Body weights of 10 worms of each batch or of surviving worms were recorded at the beginning and at the end of the test, respectively.
- Symptoms: At days 7 and 14 of the exposure period, the symptoms of the worms were recorded and the reaction of the worms to a mechanical stimulus tested.

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 62.5, 125, 250, 500 and 1000 mg/kg soil dw

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

335 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

343.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

An overview of the results is provided in Table 1 – Table 2 in ‘Any other information on results incl. tables’.

- Mortality: After 14 days exposure, ≤2 dead organisms were observed in control, 62.5, 125 and 250 mg/kg dry soil groups. The mortality was 100% in 500 and 1000 mg/kg dry soil treatment.

- Body weights: The average weights of worms determined at the beginning and the end of the study were:
Day 0: 200 – 312 mg
Day 14: 111 – 249 mg
When compared with the control (Tween 80), no influence on the average body weights of worms up to a concentration of the test article in the soil of 125 mg/kg was observed.
In conclusion, body weights of all live worms were lower at the end of the study than at the beginning. They were reduced by 24.7% for worms of the untreated control. For treated worms up to a concentration of the test article in the soil of 125 mg/kg, the reduction of the body weight accounted for 14.9%. to 19.9%. Only at a concentration of the test article of 250 mg/kg, a slight influence (34.7%) of the test article on the body weight was observed, indicating that the concentration of the test article exceeded the NOEC in the soil. However, these effects are not considered critical since body weight losses even of controls in this order of magnitude are often observed. They have to be attributed mainly to adaptation problems for worms transferred from a nutrient-rich, wet soil to a test medium poor in organic nutrients and with lower humidity.

- Symptoms: No abnormal symptoms were detected in live worms.

Results with reference substance (positive control):

Not applicable

Table 1. Mortality of earthworms exposed to the test substance in artificial soil (values cumulatively).

Treatment group	Replicate	Conc. of test article in soil (mg/kg)	No. of animals exposed	No. of dead animals
				Day 7	Day 14
1	1	0	10	1*	2*
1	2	0	10	0*	0*
1	3	0	10	0*	1*
1	4	0	10	0*	0*
2	1	0**	10	1*	1*
2	2	0**	10	0*	1*
2	3	0**	10	0*	0*
2	4	0**	10	0*	1*
3	1	62.5	10	0*	0*
3	2	62.5	10	0*	1*
3	3	62.5	10	0*	0*
3	4	62.5	10	0*	0*
4	1	125	10	0*	0*
4	2	125	10	0*	0*
4	3	125	10	0*	0*
4	4	125	10	1*	1*
5	1	250	10	0	0
5	2	250	10	0	0
5	3	250	10	1	2
5	4	250	10	0	0
6	1	500	10	10	10
6	2	500	10	10	10
6	3	500	10	10	10
6	4	500	10	10	10
7	1	1000	10	10*	10*
7	2	1000	10	10*	10*
7	3	1000	10	10*	10*
7	4	1000	10	10*	10*

 * Not used for Logit estimation.

** Control with Tween 80.

Table 2. Average body weights of earthworms determined at the beginning and the end of the exposure.

Treatment group	Replicate	Conc. of test article in soil (mg/kg)	Average weight per worm		Loss in body weight (%)
			Start	End
1	1	0	312	249	20.2
1	2	0	247	182	26.3
1	3	0	222	160	27.9
1	4	0	236	179	24.2
Mean			254	193	24.7
2	1	0*	270	233	13.7
2	2	0*	279	228	18.3
2	3	0*	252	202	19.8
2	4	0*	237	219	7.6
Mean			260	221	14.9
3	1	62.5	230	188	18.3
3	2	62.5	237	212	10.5
3	3	62.5	250	213	14.8
3	4	62.5	258	218	15.5
Mean			244	208	14.8
4	1	125	210	174	17.1
4	2	125	218	174	20.2
4	3	125	218	182	16.5
4	4	125	241	179	25.7
Mean			222	177	19.9
5	1	250	224	158	29.5
5	2	250	228	135	40.8
5	3	250	223	174	22.0
5	4	250	207	111	46.4
Mean			221	145	34.7
6	1	500	200	---	---
6	2	500	218	---	---
6	3	500	217	---	---
6	4	500	226	---	---
Mean			215	---	---
7	1	1000	229	---	---
7	2	1000	224	---	---
7	3	1000	222	---	---
7	4	1000	273	---	---
Mean			237	---	---

* Control with Tween 80.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 14-day LC50 was determined to be 335 mg/kg soil dw.

Executive summary:

The toxicity of the test substance to earthworms (Eisenia fetida) was tested according to the OECD TG 207. The study was in compliance with GLP criteria. Four replicates of 10 earthworms per treated group were exposed in artificial soil to nominal concentrations of 62.5, 125, 250, 500 and 1000 mg a.i./kg soil, for a 14-d study. Two additional groups were used as control and solvent control (Tween 80). Temperature, pH and soil moisture content were maintained at 21.5 ˚C, 7.4 -7.6 and 23.0% to 37.4%, respectively. Assessments of mortality, effects on bodyweight and symptoms of toxicity were made at days 7 and 14. Results were calculated by Probit analysis.

After 14 days exposure, the control mortality was below 10% which is regarded as the limit for natural mortality. After 14 days exposure, ≤2 dead organisms were observed in control, 62.5, 125 and 250 mg/kg dry soil groups. The mortality was 100% in 500 and 1000 mg/kg dry soil treatment. No abnormal symptoms were detected in live worms. Body weights of all live worms were lower at the end of the study than at the beginning. When compared with the control (Tween 80), no influence on the average body weights of worms up to a concentration of 125 mg/kg was observed. At the 250 mg ai/kg test concentration, body weight was reduced by 34.7% on average. No statistics were provided to determine the significance of this reduction. However, this decrease is not considered critical since body weight losses in this order of magnitude are often observed in controls. Such effects is likely to be attributable mainly to adaptation problems for worms transferred from a nutrient-rich, wet soil to a test medium poor in organic nutrients and with lower humidity. Based on the mortality findings, the 14-day LC50 was determined to be 335 mg/kg soil dw with 95% confidence limit of 304.5 - 368.5 mg/kg of dry soil.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Nov 2002 to 17 Jan 2003

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)

Version / remarks:

1998

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 solution: The preparation of the test substrate based on a wet weight of artificial soil/vessel of 750 g. Based on a water content of about 35 % the dry weight of soil per vessel was 556 g artificial soil.
- 1129 g a.i./ha in 600 L/ha: Weighing 10.575 g of the test substance; Filling with deionised water to a total volume of 500 mL (stock solution A); Final concentration of stock A: 2.1150 % (w/v) spray solution
- 564 g a.i./ha in 600 L/ha: Weighing 5.283 g test substance; Filling with deionised water to a total volume of 500 mL (stock solution B); Final concentration of stock B: 1.0566 % (w/v) spray solution
- 283 g a.i./ha in 600 L/ha: Weighing 2.651 g test substance; Filling with deionised water to a total volume of 500 mL (stock solution C); Final concentration of stock C: 0.5302 % (w/v) spray solution
- 171 g a.i./ha in 600 L/ha: Weighing 1.602 g test substance; Filling with deionised water to a total volume of 500 mL (stock solution D); Final concentration of stock D: 0.3203 % (w/v) spray solution
- 87 g a.i./ha in 600 L/ha: Weighing 0.815 g test substance; Filling with deionised water to a total volume of 500 mL (stock solution E); Final concentration of stock E: 0.1630 % (w/v) spray solution
Also see Table 1 in ‘Any other information on materials and methods incl. tables’.

- Method of application: The application cabin was calibrated three times with deionised water immediately before application in order to achieve the application rate of 600 L/ha. Therefore, 4 glass plates of known surface area were sprayed with deionised water. The weight of each glass plate was determined before and after application and the amount of spray deposit per cm2 was calculated as the difference between the weight before and after spraying. The deviation in the spray deposit did not exceed ± 10% for 3 consecutive applications without adjusting. During calibration the mean output of the application cabin was 614 L/ha (which is corresponding to 102%). The actual application rate is provided in Table 2 in in ‘Any other information on materials and methods incl. tables’.

- Order of application and cleaning procedure
The order of application was as follows:
> deionised water (control)
> from the lowest to the highest concentration of the test item
Spray equipment and application cabin were cleaned following application of the test item. Cleaning agents used were alkaline cleaning agent for laboratory equipment, and deionised water. Deionised water was used finally to flush through the sprayer. The sprayer was flushed with the spray solution to be applied next to guarantee undiluted spraying even for the first treatment of the test item.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Holding and breeding under ambient laboratory conditions in the test facility
- Age at test initiation: Adult worms (about 6 months old with clitellum)
- Weight at test initiation: 313 - 433 mg/worm
- Food used during the test: Air-dried finely ground horse manure

BREEDING AND ACCLIMATION
- Breeding medium: mixture of horse manure, straw, peat (1 : 1 : 1), all ingredients were free from pesticides
- Breeding conditions: breeding cages (50 cm x 40 cm x 30 cm); diffuse light; about 20 °C; moist soil; about pH 7
- Time for acclimatisation to test conditions: About 24 hours in the artificial soil

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

4 weeks adult mortality; 4 weeks juvenile development

Test temperature:

19.0 - 23.5 °C

pH:

6.2 - 6.3

Moisture:

- Test initiation: 35.0 % (equivalent to 53.8 % WHC)
- Test termination: 35.3 % - 35.8 % (equivalent to 54.2 - 55.0 % WHC)

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 (The test vessels were set up at random in the test room).
- Amount of soil or substrate: 750 g wet weight corresponding to about 556 g dry weight of artificial 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

SOURCE AND PROPERTIES OF SUBSTRATE
Composition of the artificial soil:
- 10 % sphagnum peat
- 20 % kaolinite clay
- 0.5 % calcium carbonate
- 69.5 % industrial quartz sand
- deionised water

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 600 lux

TEST METHOD DESCRIPTION
The day before starting the test, the artificial soil was prepared. Deionised water was added to the dry components to give a moisture content of about 35 % of the dry weight. Earthworms were preconditioned to the artificial soil (mixed with horse manure, which was also used as food source in the test) for about 24 hours before the test begun. Each test vessel was filled with the moist artificial soil (500-600 g d.w./vessel) and the weighed worms were placed onto the soil surface. After 30 minutes any worms remaining on the surface were replaced since it could not be excluded that they were damaged during transfer, the application of the test item mixed with deionised water on the soil surface was performed in an automatic track sprayer. About 1 hour after application, the test vessels were closed with a lid, to prevent worms from escaping. This lid gives access to light and allows gaseous exchange between substrate and the atmosphere. The test vessels were then set up at random in a climatic test room.

One day after application, 3g dry, ground horse manure was scattered on the soil surface of each test vessel. The manure on the soil surface was sprinkled with 3 mL deionised water. The feeding interval was weekly during the first four weeks of the test (using horse manure as food). After four weeks, the adult worms were removed from test vessels. The number of surviving worms and the bodyweight of the worms per vessel were determined. After all adult worms had been removed from the test vessels, the soil in each vessel was mixed with 3 g horse manure.
Four additional weeks later the test was terminated. The final assessment included counting of the juveniles per test vessel, determination of the water content and pH measurements of the artificial soil. Juveniles were counted by manual inspection of the substrate

EFFECT PARAMETERS MEASURED
- At the start of the test: individual fresh weight (mg/worm); behaviour of earthworms
- Weekly: Observation of behavioural and pathological symptoms
- 4 weeks after application: Number of adult surviving earthworms for each test vessel;
observation of behavioural and pathological symptoms; individual fresh weight of adult surviving earthworms (mg/worm)
- 8 weeks after application: Number of surviving juveniles for each test vessel; observation of behavioural and pathological symptoms; morphological alterations

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 87, 171, 283, 564 and 1129 g a.i./ha (equivalent to 2, 3, 5, 11 and 21 g test substance/L) See Table 1 and Table 2 in 'Any other information on materials and methods incl. tables'.

Reference substance (positive control):

yes

Remarks:

Benomyl

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 129 g/ha

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality, growth and reproduction

Remarks on result:

other: Equivalent to 1.5 mg a.i./kg soil dw

Details on results:

An overview of the results is provided in Table 3 – Table 4 in ‘Any other information on results incl. tables’.
 
- Effects on adult worm: At the beginning of the test, the fresh weight of the earthworms used was in the range of 413 - 433 mg/earthworm. No effects of the test item on adult worm survival (i.e. 0 % mortality) were observed at doses of 87, 171, 283, 564 and 1129 g a.i./ha in 600 L/ha during 4 weeks. The test item caused no statistically significant decrease in worm growth (change in fresh weight after 4 weeks relative to initial fresh weight) relative to the control treatment at any concentration tested; i.e. a weight increase of 53.2 %, 51.9 %, 54.7 %, 53.1 % and 50.8 % was recorded at doses of 87, 171, 283, 564 and 1129 g a.i./ha, respectively. No effects on behaviour and morphology of the adult worms and on feeding activity were observed during the first four weeks of the test.

- Effects on juveniles: No statistically significant effects on number of juveniles compared to the control group were recorded at all rates tested (more detailed results are presented in table 3). Therefore, the NOEC was 1129 g a.i. in 600 L water/ha.

Results with reference substance (positive control):

To verify the sensitivity of the test system, the reference item Benomyl is routinely tested at a rate of 1.25 kg product/ha. In the most recent test, dated 19.11.2002, the number of juveniles was reduced by 91.2 % 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 the computer program EASY ASSAY Multiple Testing. The multiple sequential test procedure after DUNNETI-test was used to compare the control with the independent test item groups and shows a non-significant or a significant difference between both.

Table 3. Effects of the test substance on growth (biomass increase during 4 weeks exposure) of adult earthworms

Test substance (g a.i./ha in 600 L water/ha)
Replicate	Control	87	171	283	564	1129
1	337.7	353.5	348.5	355.2	337.9	334.2
2	359	357.4	362.9	358.3	363.7	362.6
3	366.2	366.2	371.6	370.6	368.2	364.9
4	358.3	379.7	375	371.8	385.3	390.5
average	355.3	364.2	364.5	364	363.8	363.1
SD	29.4	33	31.3	36.5	28.5	31.3
cv%	8.3	9.1	8.6	10	7.8	8.6
Fresh weight/worm [mg] after 4 weeks (mean per replicate)
1	512.9	516.1	564.7	639.4	568.8	557.2
2	539.8	524.6	557.4	543.6	603.9	549.1
3	593	609.9	496.9	536.8	530	574.8
4	642.7	582.2	594.1	528.2	516.8	498.5
average	572.1	558.2	553.3	562	554.9	544.9
SD	75.2	66.4	106	120	102.5	106.3
cv%	13.3	12.1	19.1	21.5	18.5	19.8
Biomass increase (change in fresh weight after 4 weeks relative to initial fresh weight) weight/worm [mg] (mean per replicate)
1	175.2	162.6	216.2	284.2	230.9	223
2	180.8	167.2	194.5	185.3	240.2	186.5
3	226.8	243.7	125.3	166.2	161.8	209.9
4	284.4	202.5	219.1	156.4	131.5	108
average	216.8	194	188.8	198	191.1	181.9
SD	50.7	37.6	43.7	58.7	52.9	51.5
cv%	23.4	19.4	23.2	29.6	27.7	28.3
Biomass increase (% change in fresh weight after 4 weeks relative to initial fresh weight) [%] mean per replicate
1	51.9	46	62	80	68.3	66.7
2	50.4	46.8	53.6	51.7	66	51.4
3	61.9	66.5	33.7	44.8	43.9	57.5
4	79.4	53.3	58.4	42.1	34.1	27.7
average	60.9	53.2	51.9	54.7	53.1	50.8
SE	-	-	-	-	-	-

SD: standard deviation

cv %: coefficient of variation

SE: statistical evaluation

- statistically non-significant (DUNNETT-Test, p < 0.05)

Table 4. Effects of the test substance on mortality of adult earthworms and reproduction

Test substance (g a.i./ha in 600 L water/ha)
Replicate	Control	87	171	283	564	1129
	Number of surviving adult worms per replicate (4 weeks after test initiation)
1	10	10	10	10	10	10
2	10	10	10	10	10	10
3	10	10	10	10	10	10
4	10	10	10	10	10	10
average	10	10	10	10	10	10
	Mortality per treatment (%)
average	0	0	0	0	0	0
	Number of juveniles per replicate
1	117	138	133	90	121	85
2	181	176	157	97	86	59
3	129	115	92	129	147	104
4	192	198	121	106	87	139
average	154.8	156.8	125.8	105.5	110.3	96.8
SD	37.3	37.3	27	17	29.4	33.7
cv%	24.1	23.8	21.5	16.1	26.7	34.8
SE	-	-	-	-	-	-

SD: standard deviation

cv %: coefficient of variation

SE: statistical evaluation

- statistically non-significant (DUNNETT-Test, p < 0.05)

 

Validity criteria fulfilled:

yes

Remarks:

See Validity criteria in 'Any other information on materials and methods incl. tables'.

Conclusions:

Based on the findings, the 56-day NOEC of the test substance for mortality, growth and reproduction is 1129 g a.i./ha, the highest rate tested. This rate corresponds to a concentration of 1.5 mg a.i./kg soil dw, assuming homogeneous distribution in the top 5 cm of the soil of density of 1.5 g/cm3.

Executive summary:

Adult earthworms of the species Eisenia fetida andrei (about 6 months old, with clitellum) were exposed to different rates of the test substance in formulation applied onto the soil surface of an artificial soil in a single application (automatic track sprayer; applied water volume: 600 L/ha deionised water). This study was in accordance with ISO 11268-2 test guideline and was in compliance with GLP criteria. Four replicates, each with 10 earthworms per treated group (413 - 433 mg body weight), were exposed to nominal levels of 87, 171, 283, 564 and 1129 g a.i./ha (equivalent to 978, 1922, 3181, 6339 and 12690 g test substance/ha or equivalent to 0.12, 0.23, 0.38, 0.75, 1.5 mg a.i./kg soil dw, considering soil depth and density of 5 cm and 1.5 g/cm3, respectively). One additional group was used as control (water only). The number of surviving adults (adult mortality) and the weight loss / increase of the adult earthworms was assessed after exposure under the test conditions for 4 weeks and the number of living juveniles per test vessel recorded after a total of 8 weeks. Behaviour (including feeding activity) and pathological symptoms in the worms were recorded throughout the 8-week test period. The temperature (in the test room), pH and moisture were remained in a range of 19.0 - 23.5 °C, 6.2 - 6.3 and 35.0% – 35.8%, respectively. To demonstrate the sensitivity of the test system, the reference item benomyl was tested under the study conditions at an application rate of 1.25 kg product/ha two days prior to the start of this test substance study.

In the systems treated with the reference item, the number of juveniles was reduced to 8.8% of the control number after 8 weeks. Therefore, the test system was shown to be capable of determining sublethal effects. No effects of the test item on adult worm survival were observed at rates of 87, 171, 283, 564 and 1129 g ai/ha in 600 L water/ha during 4 weeks. The test item caused no statistically significant decrease in worm growth relative to the control treatment at any rate tested. No statistically significant effects on number of juveniles compared to the control group were recorded at all rates tested. No effects on behaviour and morphology of the adult worms and on feeding activity were observed during the first four weeks of the test. Based on the findings, the 56-day NOEC of the test substance for mortality, growth and reproduction is 1129 g a.i./ha, the highest rate tested. This rate corresponds to a concentration of 1.5 mg a.i./kg soil dw, assuming homogeneous distribution in the top 5 cm of the soil of density of 1.5 g/cm3.

Description of key information

14-d LC50 = 335 mg a.i./kg soil dw, Eisenia fetida, mortality, OECD TG 207, Ellgehausen 1986

56-d NOEC = 1.5 mg a.i./kg soil dw, Eisenia fetida, mortality, growth and reproduction, ISO 11268-2, Friedrich 2003

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

335 mg/kg soil dw

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

1.5 mg/kg soil dw

Additional information

Acute toxicity test (Ellgehausen 1986)

The toxicity of the test substance to earthworms (Eisenia fetida) was tested according to the OECD TG 207. The study was in compliance with GLP criteria. Four replicates of 10 earthworms per treated group were exposed in artificial soil to nominal concentrations of 62.5, 125, 250, 500 and 1000 mg a.i./kg soil, for a 14-d study. Two additional groups were used as control and solvent control (Tween 80). Temperature, pH and soil moisture content were maintained at 21.5 ˚C, 7.4 -7.6 and 23.0% to 37.4%, respectively. Assessments of mortality, effects on bodyweight and symptoms of toxicity were made at days 7 and 14. Results were calculated by Probit analysis.

After 14 days exposure, the control mortality was below 10% which is regarded as the limit for natural mortality. After 14 days exposure, ≤2 dead organisms were observed in control, 62.5, 125 and 250 mg/kg dry soil groups. The mortality was 100% in 500 and 1000 mg/kg dry soil treatment. No abnormal symptoms were detected in live worms. Body weights of all live worms were lower at the end of the study than at the beginning. When compared with the control (Tween 80), no influence on the average body weights of worms up to a concentration of 125 mg/kg was observed. At the 250 mg ai/kg test concentration, body weight was reduced by 34.7% on average. No statistics were provided to determine the significance of this reduction. However, this decrease is not considered critical since body weight losses in this order of magnitude are often observed in controls. Such effects is likely to be attributable mainly to adaptation problems for worms transferred from a nutrient-rich, wet soil to a test medium poor in organic nutrients and with lower humidity. Based on the mortality findings, the 14-day LC50 was determined to be 335 mg/kg soil dw with 95% confidence limit of 304.5 - 368.5 mg/kg of dry soil.

Chronic toxicity test (Friedrich 2003)

Adult earthworms of the species Eisenia fetida andrei (about 6 months old, with clitellum) were exposed to different rates of the test substance in formulation applied onto the soil surface of an artificial soil in a single application (automatic track sprayer; applied water volume: 600 L/ha deionised water). This study was in accordance with ISO 11268-2 test guideline and was in compliance with GLP criteria. Four replicates, each with 10 earthworms per treated group (413 - 433 mg body weight), were exposed to nominal levels of 87, 171, 283, 564 and 1129 g a.i./ha (equivalent to 978, 1922, 3181, 6339 and 12690 g test substance/ha or equivalent to 0.12, 0.23, 0.38, 0.75, 1.5 mg a.i./kg soil dw, considering soil depth and density of 5 cm and 1.5 g/cm3, respectively). One additional group was used as control (water only). The number of surviving adults (adult mortality) and the weight loss / increase of the adult earthworms was assessed after exposure under the test conditions for 4 weeks and the number of living juveniles per test vessel recorded after a total of 8 weeks. Behaviour (including feeding activity) and pathological symptoms in the worms were recorded throughout the 8-week test period. The temperature (in the test room), pH and moisture were remained in a range of 19.0 - 23.5 °C, 6.2 - 6.3 and 35.0% – 35.8%, respectively. To demonstrate the sensitivity of the test system, the reference item benomyl was tested under the study conditions at an application rate of 1.25 kg product/ha two days prior to the start of this test substance study.

In the systems treated with the reference item, the number of juveniles was reduced to 8.8% of the control number after 8 weeks. Therefore, the test system was shown to be capable of determining sublethal effects. No effects of the test item on adult worm survival were observed at rates of 87, 171, 283, 564 and 1129 g ai/ha in 600 L water/ha during 4 weeks. The test item caused no statistically significant decrease in worm growth relative to the control treatment at any rate tested. No statistically significant effects on number of juveniles compared to the control group were recorded at all rates tested. No effects on behaviour and morphology of the adult worms and on feeding activity were observed during the first four weeks of the test. Based on the findings, the 56-day NOEC of the test substance for mortality, growth and reproduction is 1129 g a.i./ha, the highest rate tested. This rate corresponds to a concentration of 1.5 mg a.i./kg soil dw, assuming homogeneous distribution in the top 5 cm of the soil of density of 1.5 g/cm3.