1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

yes

Remarks:

; With respect to the properties of the test item (LogPow > 2) 5 % peat instead of 10 % peat were used considering the influence on bioavailability (EPPO 2002)

GLP compliance:

yes (incl. QA statement)

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: own breed
- Strain: Eisenia fetida andrei; strain of Prof Graff, Forschungsanstalt für Landwirtschaft, 38104 Braunschweig, Germany
- Age at test initiation: more than 2 month
- Weight at test initiation: The average weight was 0.33 g.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 ± 2 °C

pH:

6.32 - 6.40

Moisture:

WHCmax (mean): 45.6 (WHCmax is expressed as g water per 100 g dry weight artificial soil.)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1.5 liter preserving jars, covered with glass lids.
- Amount of soil or substrate: 500 g dry weight test soil (equivalent to 580 g wet weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The artificial soil was prepared according to the guideline.

OTHER TEST CONDITIONS
- Photoperiod: constant light
- Light intensity: 400 - 800 lux (integrated luxmeter of the climatic chamber)

EFFECT PARAMETERS MEASURED: mortality and weight

Nominal and measured concentrations:

Nominal concentrations of 100, 178, 316, 562 and 1000 mg test item / kg dry weight soil.

Reference substance (positive control):

yes

Remarks:

Chloroacetamide A.R.

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

316 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: weight

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: LC50 of 11.1 mg Chloroacetamide A.R. / kg dry weight soil

In this study the LC50 (14d) was > 1000 mg test item/kg dry weight soil. The Lowest Observed Effect Concentration (LOEC) of AE 1170437, substance technical to earthworms (Eisenia fetida) was determined to be 562 mg test item /kg dry weight soil. The No Observed Effect Concentration (NOEC) was determined to be 316 mg test item /kg dry weight soil. No morphological and behavioural effects were observed.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jul - 12 Sep 2007

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:

13 Apr 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of test solutions: The test item was mixed into deionised water. The highest test rate was further diluted with deionised water to obtain the desired test item solutions for application. All application solutions were prepared freshly on the day of the test.
- Method of application to soil surface: The spray solutions were applied in a laboratory spraying cabinet with an automatically driven spray broom (calibrated to 800 L/ha ± 10%) in the order of deionised water (control) and lowest to highest test item application rates. Distance for application: approximately 40 cm
- Controls: Deionised water
- Other: After application, the test vessels were left open for at least 1 h.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Forschungsanstalt für Landwirtschaft, Braunschweig, Germany (Prof. Graff)
- Cultivation: In-house for several years at 22 ± 2 °C. Substrate: 55% natural soil, 35% peat, 4.2% straw and 6.8% animal manure (w/w, dry weight in each case). Feeding: Animal manure at 14-d intervals. At the same time, the substrate was also replenished with water.
- Test organisms: To obtain a synchronised culture of earthworms, 100 adult earthworms were kept in a rearing vessel with substrate and food for a period of 4 weeks. After this time, the adult earthworms were picked out of the substrate again and the offspring hatched from the cocoons.
- Age at test initiation: Approximately 8 months old with a well developed clitellum. The age of the worms from the synchronised culture differed by no more than 4 weeks.
- Weight at test initiation: 0.25 - 0.46 g/worm

ACCLIMATION
- Acclimation period: Organisms were acclimated to the artificial soil and test temperature 5 d prior test.
- Acclimation conditions: Same as test

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

The study consisted of 2 parts. Part 1: Exposure of adult earthworms to the test item for 4 weeks (28 d). Part 2: Removal of adults and retention of the cocoons and juvenile earthworms in the test vessels for additional 4 weeks (28 d).

Test temperature:

18 - 22 °C

pH:

5.82 (artificial soil prior test)
6.78 (control at test end)
6.77 - 6.88 (treatments at test end)

Moisture:

17.6% (mean soil moisture prior test)
21.9% (mean soil moisture on Day 0)
21.7% (mean soil moisture on Day 56)

Details on test conditions:

TEST SYSTEM
- Test container: Non-reusable plastic boxes (16.5 cm L x 12 cm W x 6 cm H; area = ca. 200 cm²) covered with transparent and perforated lids that allow air exchange.
- Amount of soil or substrate: 500 g artificial soil (dw) to obtain a depth of approximately 5 cm soil in the test vessel
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- Other: The sequence of inserting the test organisms into the test vessels followed a computer-generated random design. Only healthy animals were used. Within 15 min the earthworms burrowed into the artificial soil.

PROPERTIES OF ARTIFICIAL SOIL
- Composition (% distribution on the basis of dw):
- % Sphagnum peat: 5 (air dried and finely ground)
- % sand: 73.85 (industrial quartz sand, F 36, 0.20 - 0.05 mm = 73.0%)
- % clay: 20 (Kaolinite (Al2Si2O5(OH)4) = 56.0%)
- % Calcium carbonate: 0.15
- % food: 1 (air dried and finely shredded cow manure)
- Preparation of soil: The artificial soil was prepared by mixing the dry component intensely in a laboratory mixer. Then, the soil was moistened with deionised water to reach a water content of 58% of the maximum water holding capacity.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 564 lux (Day 0), 488 lux (Day 28) and 549 lux (Day 56)
- Feeding: Adult earthworms were fed 1x/week during the test period starting on Day 1 (1 d after test item application). Amount of food: 5 g food/vessel. If food consumption was low (determined visually), the additional amount of fresh food was reduced accordingly. The offspring were fed only once at the start of the second 4 week exposure period. The surface of the artificial soil was moistened with deionised water once per week in order to compensate for evaporation.

EFFECT PARAMETERS MEASURED:
- Mortality and adverse effects (e.g. lack of movement, rigidity, etc.): after 4 weeks (28 d)
- Growth (body weight): Day 0 and Day 28
- Reproduction (n° of surviving juveniles per test vessel): Day 56
- Assessment of food consumption: At each feeding date, the amount of food consumed by the adult earthworms was visually estimated for each test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 1.77
- Range finding study: No

Nominal and measured concentrations:

Control, 10.0, 17.8, 31.6, 56.2, and 100 L test item/ha (nominal)
Control, 43.8, 78.0, 138.4, 246.2, and 438.0 mg test item/kg soil dw (calculated)
Control, 19.10, 34.01, 60.34, 107.34, and 190.97 mg a.s./kg soil dw (calculated)

Reference substance (positive control):

yes

Remarks:

Carbendazim (Derosal flüssig)/fungicide

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

34.01 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

Calculated for 100% test material purity, corresponding to 78 mg test item/kg soil dw and 17.8 L test item/ha

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 190.97 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks on result:

other:

Remarks:

Calculated for 100% test material purity, corresponding to ≥ 438 mg test item/kg soil dw and ≥ 100.0 L test item/ha

Details on results:

- Mortality at end of exposure period: No mortality of adult earthworms was observed after 28 d test duration in the control group and at any application rate, including the highest rate of 100.0 L test item/ha.
- Individual body weight of earthworms at beginning of test: 0.33 - 0.34 g (mean)
- Changes in body weight of live adults (% of initial weight) after 28 d: 73.1% (control) and 66.6 - 78.3% (treatments)
- Mean No. of offspring produced: 322 (control), 294 (10.0 L/ha), 279.8 (17.8 L/ha), 257.5 (31.6 L/ha), 191.3 (56.2 L/ha) and 180.3 (100.0 L/ha)

Results with reference substance (positive control):

- Results with reference substance valid? Yes (performend Jan - Mar 2007)
- Relevant effect levels: No mortality after 28 d. No statistically significant different values for biomass up to the highest application rate. The number of juveniles per test vessel of the application rate of 0.10 and 0.50 kg test item/ha were statistically significant reduced to the control. At the application rate
of 0.25 kg test item/ha no statistically significant reduction of the number of juveniles could be observed (result of a Dunnett´s Multiple t-test, one-sided smaller, α = 0.05).

Reported statistics and error estimates:

Statistical analyses were performed with the statistical software package ToxRatPro Version 2.09. Homogeneity of variance was tested by Cochran's test. Normal distribution was tested by Kolmogorov-Smirnov. Significant differences between control and treatments were tested by means of a Dunnett's multiple t-test (growth) or Williams multiple sequential t-test (reproduction).

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline and is thus considered valid (Table 1).

Table 1. Validity criteria for OECD 222.

Criterion from the guideline to be satisfied in the controls	Outcome	Validity criterion fulfilled
Each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test.	Mean rate of reproduction of juveniles: 322.0 earthworms per control vessel.	Yes
The coefficient of variation of reproduction is ≤ 30%.	The obtained coefficient of variance of reproduction in the control was 18.3%.	Yes
Adult mortality over the initial 4 weeks of the test is ≤ 10%.	Mortality of the adults in the control was 0%.	Yes

 

BIOLOGICAL FINDINGS

Results are summarized in Table 2.

MORTALITY

No mortality of adult earthworms was observed after 28 d in the control group and at any treatment up to the highest application rate of 100.0 L test item/ha (438.0 mg test item/kg soil dw) (Table 2).

 

GROWTH

No statistically significant differences in growth relative to the control were observed up to the highest application rate of 100.0 L test item/ha (438.0 mg test item/kg soil dw) (Table 2).

 

REPRODUCTION

No statistically significant different values for the number of juveniles per test vessel relative to the control were observed at the application rates of 10.0 and 17.8 L test item/ha (43.8 and 78.0 mg test item/kg soil dw, respectively). Statistically significant different values for the number of juveniles per test vessel relative to the control were observed at the application rates of 31.6, 56.2 and 100.0 L test item/ha (138.4, 246.2, and 438.0 mg test item/kg soil dw, respectively) (Table 2).

Table 2. Study outcome.

Endpoint	Control	Treatments
Application rates [L/ha]	--	10.0	17.8	31.6	56.2	100.0
Calculated soil concentration [mg test item/kg soil dw]	--	43.8	78.0	138.4	246.2	438.0
Mortality of adult earthworms [%]	0	0	0	0	0	0
Mean change of body weight of the adults from Day 0 to Day 28 [%]	+ 73.1	+ 78.3	+ 70.4	+ 75.9	+ 68.2	+66.6
Standard Deviation	± 9.4	± 14.6	± 13.8	± 8.3	± 8.9	± 4.3
Statistical comparison to the control**	--	n.s.	n.s.	n.s.	n.s.	n.s.
Mean number of offspring per test vessel after 56 d	322.0	294.0	279.8	257.5	191.3	180.3
Standard Deviation	± 59.0	± 73.6	± 32.3	± 50.1	± 6.9	± 23.8
Statistical comparison to the control***	--	n.s.	n.s.	s.	s.	s.

* Conversion according to the condition of the test (500 g dry weight artificial soil and 200 cm² surface area per test vessel; the density of the test item was taken into account)

** Result of a Dunnett´s Multiple t-test, two-sided, α = 0.05

*** Result of a Williams Multiple Sequential t-test, one-sided smaller, α = 0.05

n.s.: mean value not statistically significantly different compared to the control (p ≥ 0.05)

s.: mean value statistically significantly different compared to the control (p < 0.05)

 

EFFECT VALUES

The overall NOEC (56 d) for reproduction is 17.8 L test item/ha, corresponding to 78.0 mg test item/kg soil dw. The re-calculated NOEC (56 d) for a test material purity of 100% is 34.01 mg a.s./kg soil dw.

The overall LOEC (56 d) is 31.6 L test item/ha, corresponding to 138.4 mg test item/kg soil dw The re-calculated LOEC (56 d) for a test material pruity of 100% is 60.34 mg a.s./kg soil dw.

Effect values are summarized in Table 3.

 

Table 3. Summary of effect values.

Effect value	Endpoint	Result
NOEC (28 d)	Growth	≥ 100 L test item/ha corresponding to ≥ 438.0 mg test item/kg soil dw and ≥ 190.97 mg a.s./kg soil dw
LOEC (28 d)	Growth	> 100.0 L test item/ha corresponding to > 438.0 mg test item/kg soil dw and > 190.97 mg a.s./kg soil dw
NOEC (56 d)	Reproduction	17.8 L test item/ha corresponding to 78.0 mg test item/kg soil dw and 34.01 mg a.s./kg soil dw
LOEC (56 d)	Reproduction	31.6 L test item/ha corresponding to 138.4 mg test item/kg soil dw and 60.34 mg a.s./kg soil dw

Validity criteria fulfilled:

yes

Remarks:

For details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (56 d) = 34.01 mg a.s./kg soil dw (re-calculated to 100% test material purity, nominal, OECD 222, E. fetida)

Key value for chemical safety assessment

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

34.01 mg/kg soil dw

Additional information

Two studies are available, in which the short-term and long-term toxicity of Indaziflam (CAS 950782-86-2) to soil macroorganisms was assessed according to OECD guidelines and GLP.

In the short-term study (M-284352-01-2), according to OECD 207 a 14-day test with artificial soil was performed with the earthworm Eisenia fetida with concentrations ranging from 100 to 1000 mg test item/kg soil dw. The 14-day LC50 was > 1000 mg a.s./kg test item as no death occurred at this limit dose. The NOEC was 316 mg a.s./kg dry weight soil based on earthworm growth (weight). No morphological and behavioural effects were observed.

In the long-term study (M-295080-01-1), a 56 d test with artificial soil according to OECD 222 was performed with the earthworm Eisenia fetia and five test item application rates of 10.0, 17.8, 31.6, 56.2, and 100 L test item/ha, corresponding to 19.10, 34.01, 60.34, 107.34 and 190.97 mg a.s./kg soil dw, which were applied to the soil by spray. The test consisted of two parts. In part 1, adult earthworms were exposed to the test item for 4 weeks (28 d). After a period of 4 weeks (28 d) the adult earthworms were removed from the test vessels and the survivors were counted and their fresh weight was measured. From these data mortality and biomass effects were determined. In part 2, adults were removed from the test vessels and the cocoons and juvenile earthworms retained in the test vessels for additional 4 weeks (28 d). The cocoons and juvenile earthworms remained in the vessels for additional 4 weeks (28 d). From these the reproduction performance was determined by counting the number of offspring hatched.

After 28 d, no mortality of adult earthworms was observed in any treatment and no statistically significant differences in growth relative to the control were observed up to the highest application rate of 100.0 L test item/ha (190.97 mg a.s./kg soil dw).

After 56 d, no statistically significant different values for the number of juveniles per test vessel compared to the control were observed at the application rates of 10.0 and 17.8 L test item/ha (19.10 and 34.01 mg a.s./kg soil dw, respectively). Statistically significant different values for the number of juveniles per test vessel compared to the control were observed at application rates of 31.6, 56.2 and 100.0 L test item/ha (60.34, 107.34 and 190.97 mg a.s./kg soil dw, respectively). Thus, the overall NOEC (56 d) for reproduction is 17.8 L test item/ha, corresponding to 34.01 mg a.s./kg soil dw.