Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

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:

13 April 2005 to 08 June 2005

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:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Preparation of the test substrate: The solubility of the test item in water was not sufficient for the amount of test item. Therefore, the test item was dissolved in acetone and then mixed with finely ground quartz sand. The acetone was removed by evaporation in a fume hood for approximately 30 minutes. The treated sand (10 g sand per test vessel) was then mixed thoroughly by intensive stirring with the artificial soil in a laboratory mixer.

The order of application was as follows:
- control (untreated)
- solvent control
- from the lowest to the highest concentration of the test item
The mixer was cleaned with a brush following application of each test item concentration and with cleaning agents after the last mixing.

- Controls: Two control groups were prepared. One control substrate remained untreated, i.e. was used without the vehicle acetone and contained only the corresponding amount of quartz sand per vessel. The solvent control was prepared (according to the method of the test item application) with acetone in the same amount consistent with that used in the treatments.

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Holding and breeding: Holding and breeding 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 conditions: breeding containers (50 cm x 40 cm x 30 cm); diffuse light; approximately 20 °C; moist soil; approximately pH 7
- Age at test initiation: Adult worms (approximately 4 months old with clitellum)
- Weight at test initiation: 301 - 477 mg/worm

ACCLIMATION
- Acclimation period: Approximately 24 hours in the articial substrate

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

18 – 21 °C

pH:

- Test initiation: 6.1 - 6.2
- Test termination: 6.3 - 6.4

Moisture:

- Test initiation: 35.0% - 35.2% (equivalent to 54.3 — 54.7 % WHC)
- Test termination: 34.7% - 35.7% (equivalent to 53.9 — 55.4 % 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
- Amount of soil or substrate: Approximately 751 g wet weight corresponding to 556 g dry weight of articial soil with approximately 35 % water content
- Type: Closed (closed with perforated transparent lids, which prevented worms from escaping, whilst allowing gaseous exchange between the medium and atmosphere and access of light.)
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per solvent control: 8
The test vessels were set up at random in the test room.

SOURCE AND PROPERTIES OF SUBSTRATE (artificial soil)
- Composition: 10 % Sphagnum peat; 20 % kaolinite clay; 0.4 % calcium carbonate; 69.6 % industrial quartz sand; deonised water
- Maximum water holding capacity: 64.4%
- Pretreatment of soil: Approximately 24 hours before test start, the artificial soil was prepared and deionised water was added to the dry components to adjust the moisture content to approximately 35 % of the dry weight. The selected worms were allowed to acclimatise in a separate batch of the artificial substrate for approximately 24 hours before test initiation.

OTHER TEST CONDITIONS
- Photoperiod: light : dark = 16 : 8 h
- Light intensity: 600 lx
- Feeding: One day after application, 5 g dry and ground horse manure was scattered on the soil surface of each test vessel. The manure on the soil surface was sprinkled with 5 mL deionised water. The feeding interval was weekly during the first four weeks of the test (using horse manure as food).

EFFECT PARAMETERS MEASURED
The number of surviving adults after 4 weeks (adult mortality), the weight change of the adult earthworms after 4 weeks, the number of surviving juveniles after 8 weeks as well as behavioural (including feeding activity) and pathological symptoms were recorded. Behaviour (worms on the soil surface) and feeding activity were assessed during each feeding in weekly intervals. During assessment of the feeding activity the percentage of consumed manure per replicate was determined visually. The water content of the soil was checked weekly by reweighing of the test vessels and the water loss was compensated

VEHICLE CONTROL PERFORMED: Yes

Nominal and measured concentrations:

- Nominal concentrations: 0 (negative control), 0 (solvent control), 0.94, 1.88, 3.75, 7.50, 15, 30, 60 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Benomyl WP 50; 5 and 10 mg product/kg soil dry weight

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

60 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

mortality and growth

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

> 60 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

mortality 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'.
The results indicate that the test item caused no mortality at any tested concentration during 4 weeks. No mortality (0 %) occurred in the two control groups either. No effects on behaviour (including feeding activity) of the worms were observed during the test.
The test item caused no statistically significant change in worm growth (change in weight after 4 weeks relative to initial fresh weight) relative to the pooled control treatment at any concentration tested; i.e. a weight increase of 20.8, 22.3, 22.1, 21.0, 20.5, 19.9, 21.8 and 21.5 % was recorded in the pooled control group and at concentrations 0.94, 1.88, 3.75, 7.5, 15, 30 and 60 mg/kg d.w., respectively.
No statistically significant effects (DUNNET T-Test, p > 0.05) on the number of juveniles compared to the control group were recorded at all concentrations tested. The NOEC was determined to be 60 mg/kg d.w., the highest concentration tested. The EC50 could not be calculated but it can be concluded that the EC50 is greater than 60 mg/kg d.w.

Results with reference substance (positive control):

To verify the sensitivity of the test system, the reference item Benomyl is routinely tested at concentrations of 5 and 10 mg product/kg dry weight of soil. In the most recent test, dated 18.11.2004, the number of juveniles was reduced by 63 % and 100 % in comparison with the control group.

Reported statistics and error estimates:

The statistical analysis was performed with the software ToxRat Professional 2.07. The T-test was used to compare the solvent with the non-solvent control. Because the controls were not statistically significantly different from each other they were pooled (combined) and subsequent comparisons with test item treatments were against the pooled control.
The DUNNE T-test was used to compare the pooled control with the independent test item groups. For statistical evaluation of the body weight change, the changed mean fresh weight of surviving worms per replicate was used.

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

	Test substance (mg/kg soil dw)
Treatments	Control	Solvent control	0.94	1.88	3.75	7.50	15	30	60
Replicate	Initial fresh weight / worm [mg] (mean per replicate)
1	353.2	373.7	373.0	366.1	372.2	367.0	356.6	369.4	373.3
2	379.6	375.5	380.3	378.8	377.5	379.4	380.3	381.0	382.4
3	387.6	387.8	382.6	393.2	386.8	393.3	385.8	386.4	384.1
4	406.3	399.6	401.1	401.0	405.9	398.1	406.4	399.3	395.9
5	375.5	363.5
6	378.5	381.4
7	383.4	388.3
8	403.4	396.1
mean	383.4	383.2	384.3	384.8	385.6	384.5	382.3	384.0	383.9
SD	16.7	12.1	12.0	15.5	14.8	14.1	20.5	12.4	9.3
cv %	4.4	3.2	3.1	4.0	3.8	3.7	5.4	3.2	2.4
Fresh weight / worm [mg ] after 4 weeks (mean per replicate)
1	428.7	450.1	481.9	466.1	444.4	457.2	458.8	450.0	476.9
2	447.2	453.6	449.0	467.7	473.0	467.0	446.0	449.0	451.7
3	472.1	493.2	458.0	462.7	475.7	465.8	447.3	477.9	472.6
4	473.6	464.9	490.9	480.7	472.9	462.1	478.7	494.5	463.7
5	480.8	453.7
6	446.4	450.0
7	480.2	472.2
8	477.7	462.8
mean	463.3	462.6	470.0	469.3	466.5	463.0	457.7	467.9	466.2
SD	19.7	14.7	19.7	7.9	14.8	4.4	15.1	22.2	11.1
cv %	4.3	3.2	4.2	1.7	3.2	1.0	3.3	4.8	2.4
Biomass change (change in fresh weight after 4 weeks relative to initial weight) / worm [mg] (mean per replicate)
1	75.5	76.4	108.9	100.0	72.2	90.2	102.2	80.6	103.6
2	67.6	78.1	68.7	88.9	95.5	87.6	65.7	68.0	69.3
3	84.5	105.4	75.4	69.5	88.9	72.5	61.5	91.5	88.5
4	67.3	65.3	89.8	79.7	67.0	64.0	72.3	95.2	67.8
5	105.3	90.2
6	67.9	68.6
7	96.8	83.9
8	74.3	66.7
mean	79.9	79.3	85.7	84.5	80.9	78.6	75.4	83.8	82.3
SD	14.4	13.6	17.8	13.0	13.5	12.5	18.4	12.2	17.0
Biomass change (change in fresh after 4 weeks relative to fresh weight) [%] (mean per replicate)
1	21.4	20.4	29.2	27.3	19.4	24.6	28.7	21.8	27.8
2	17.8	20.8	18.1	23.5	25.3	23.1	17.3	17.8	18.1
3	21.8	27.2	19.7	17.7	23.0	18.4	15.9	23.7	23.0
4	16.6	16.3	22.4	19.9	16.5	16.1	17.8	23.8	17.1
5	28.0	24.8
6	17.9	18.0
7	25.2	21.6
8	18.4	16.8
mean	20.9	20.8	22.3	22.1	21.0	20.5	19.9	21.8	21.5

SD: Standard deviation; CV%:Coefficient of variation

 

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

	Test substance (mg/kg soil dw)
Treatments	Control	Solvent control	0.94	1.88	3.75	7.50	15	30	60
Replicate	Number of surviving adultworms per replicate(4 weeks aftertestinitiation)
1	10	10	10	10	10	10	10	10	10
2	10	10	10	10	10	10	10	10	10
3	10	10	10	10	10	10	10	10	10
4	10	10	10	10	10	10	10	10	10
5	10	10
6	10	10
7	10	10
8	10	10
mean	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0
SD	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
cv %	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Mortality (%)
mean	0	0	0	0	0	0	0	0	0
Number juvenile worms per replicate
l	169	137	193	160	197	142	160	134	113
2	131	171	190	144	148	144	164	176	198
3	203	164	157	175	170	171	141	166	179
4	173	150	155	151	126	153	133	145	144
5	129	199
6	138	144
7	186	210
8	165	111
mean	161.8	160.8	173.8	157.5	160.3	152.5	149.5	155.3	158.5
SD	26.9	32.6	20.5	13.4	30.4	13.2	14.9	19.2	37.7
cv %	16.6	20.3	11.8	8.5	19.0	8.7	10.0	12.3	23.8
Reduction of reproduction [%]
% to control	0	0.6	-7.4	2.6	0.9	5.7	7.6	4.0	2.0

Validity of the study:

The study is considered valid because the following criteria were met:

- adult mortality: ≤ 10 % (being 0 % 4 weeks)

- number of juveniles: ≥ 30 (being 169, 131, 203, 173, 129, 138, 168 for replicate 1, 2, 3, 4, 5, 6, 7 and 8).

- coefficient of variation of reproduction: ≤ 30 % (being 16.6%)

Validity criteria fulfilled:

yes

Remarks:

See validity of the study in 'Any other information on results incl. tables'

Conclusions:

In this earthworm reproduction study with Eisenia fetida, performed in accordance with OECD TG 222, the 8-week NOEC was determined to be 60 mg/kg soil d.w. for reproduction. No effects were observed on mortality, growth, behaviour, or number of juveniles.

Executive summary:

The toxicity of the test substance to earthworms (Eisenia fetida) was studied according to OECD TG 222 and in compliance with GLP criteria. Four replicates of 10 earthworms per treated group were exposed in artificial soil to nominal concentrations corresponding to 0.94, 1.88, 3.75, 7.50, 15, 30 and 60 mg/kg dw soil for 8 weeks. An untreated group and an acetone treated group were included as negative control and solvent control, respectively (10 worms/replicate; 8 replicates). The toxic reference, a Benomyl formulation, was applied at concentrations of 5 and 10 mg product/kg soil dry weight in a separate experiment. The test adult earthworm (about 4 months old with clitellum; weighted 301 – 477 mg) were acclimatised in an artificial soil for 24 hours before exposure. One day after application, 5 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. The number of surviving worms and the bodyweight of worms per vessel was determined. The juvenile worms remained in the test vessels for a further 4 weeks. When the test was terminated, the number of survival, behavioural and pathological symptoms of the juvenile worms were recorded. The test conditions were maintained at 18 - 21 ˚C; 16-hour light and 8-hour darkness (light intensity: 600 lux), and pH 6.1 – 6.4.

No effects of the test item on adult worm survival were observed in any of the test concentrations during the 4 weeks of exposure. No effects on the behaviour (including feeding activity) of the worm were observed either. The biomass has no significant changes in comparison with the pooled control was recorded at any of the test concentrations, i.e. a weight increase of 20.8, 22.3, 22.1, 21.0, 20.5, 19.9, 21.8 and 21.5 % was recorded in the pooled control group and at concentrations 0.94, 1.88, 3.75, 7.5, 15, 30 and 60 mg/kg d.w., respectively. There are no significant effects on the number of juveniles compared to the control group either. Based on the findings, the 8-week NOEC was determined to be 60 mg/kg soil d.w. for reproduction, mortality and growth, based on the nominal concentrations.

Description of key information

All available data was assessed. The study representing the worst-case effects was included here and its effect value was used as the key value. Other studies are included as supporting information.

8-wk, NOEC = 60 mg/kg soil dw, Eisenia fetida, mortality, growth and reproduction, OECD TG 222, Friedrich 2005

Key value for chemical safety assessment

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

60 mg/kg soil dw

Additional information

Two acute and two chronic toxicity study of the test substance on earthworm (Eisenia fetida), are available. All of them are standard guideline followed and GLP compliant studies. In one of the 56 days chronic toxicity tests, the earthworm (4 replicates per treatment) was exposed to the test substance at concentrations of 0.94, 1.88, 3.75, 7.5, 15, 30 and 60 mg/kg soil dw. An untreated control, a solvent control (acetone, 8 replicates per control) and a toxic reference treatment of Benomyl WP 50 (5 and 10 mg product/kg soil dw) were studied as well. The toxic reference test was carried out in a separate test run. The test conditions were maintained at 18 - 21 ˚C; 16 hour light and 8 hour darkness (light intensity: 600 lux), and pH 6.1 – 6.4. No significant changes in biomass or effect on the number of juveniles compared to the control were recorded at any of the concentration tested. The 56-d NOEC was determined to be 60 mg/kg soil dw for mortality, growth and reproduction (Friedrich 2005a). This study represents the worst-case with regards to toxicity to soil macroorganisms except arthropods of the substance and was selected as the key study.

In another chronic study earthworms were exposed to the test substance at concentrations of 7.5, 15, 30, 60 and 120 mg/kg soil dw. The same controls and toxic reference treatment were used as in the first chronic toxicity test. The 56-d NOEC was determined to be 120 mg/kg soil dw for mortality, growth and reproduction (Friedrich 2007a).

In the 14-d acute toxicity test, the earthworm was exposed to the test substance at concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dw. In addition, a control (quartz sand) group and toxic reference groups (2-chloroacetamide; 14.1, 18.3, 23.8, 31.0 and 40.4 mg/kg soil dw) were included. The 14-d LC50 was determined to be > 1000 mg/kg soil dw for mortality and biomass (Friedrich 2007 b). In another 14-d acute toxicity test, the earthworm was exposed to the test substance at concentrations of 62.5, 125, 250, 500 and 1 000 mg/kg soil dw. Two control treatments and a toxic reference treatment of 2-chloroacetamide applied at 14.1, 18.3, 23.8, 31.0 and 40.4 mg/kg soil were included as well. The toxic reference test was carried out in a separate test run. The 14-d EC50 was determined to be > 1000 mg/kg soil dw for mortality and biomass (Friedrich 2005b).