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Toxicity to terrestrial arthropods

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Endpoint:
toxicity to terrestrial arthropods: short-term
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from the related substance imazalil base is used to cover this endpoint. The justification for read across is attached in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
17 d
Dose descriptor:
NOEC
Effect conc.:
241.87 mg/kg soil dw
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
reproduction
Endpoint:
toxicity to terrestrial arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-10-2007 to 30-01-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 226 (Predatory Mite (Hypoaspis (Geolaelaps) Aculeifer) Reproduction Test in Soil)
Deviations:
no
GLP compliance:
yes
Application method:
soil
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZR023979G3L431
- Expiration date of the lot/batch: 29/05/2011
- Purity test date:06/09/2005

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature, in closed container, protected from direct daylight
- Solubility and stability of the test substance in the solvent/vehicle: stability in test item solution was not determined, but it was supposed to be sufficient for practical use conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: homogeneity of the test solution was obtained by thorough stirring or mixing immediately before application


Analytical monitoring:
no
Vehicle:
not specified
Details on preparation and application of test substrate:
- preparation of test substrate:
Three crucibles with a frit on the lower part were incubated for 4 hours in a water bath for saturation. Aftrewards the crucibles were weighed and 50 g if drt artificial soil was added. After 24 hours incubation in a water bath the overall weight of the crucibles and the artificial soil was recoarded. The water holding capacity of the substrate used for the test was determined to be 36.73%. In order to achieve a soil water content of approx 50 % of the soil water holding capacity 178.7 g of deionised water had to be added per kg dry artificial soilThree days fore starting the test, the dry artifiical soil was pre-moistened by adding 157.3 g deionized water to 1900 g dry artificial sand and stored in a closed container until use. At the start of the test, the pre-moistened soil was divided into portions of 205.7 g for each test item treatment group and 154.3 g for the control groups. Afterwards the missing amount of sand was added to each soil sample, which was 10 g for each test item group and 7.5 g for the control groups.
- Method of mixing into soil or dung: All test items were prepared by diluting the appropriate amount of Imazalil in 10mL acetone. Two mL of each acetone solution were applied on 10 g quartz sand. After evaporation of the solvent, the sand was mixed with the pre-moistened substrate and moisture content was adjusted by the addition of deionised water. Each batch of soil was mixed thoroughly by hand for approximately 2-3 minutes before being filled into the test vessel.
- Controls: yes
* solvent control (acetone applied on quartz sand)
* non-solvent control (untreated quartz sand)

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: yes
- Volume of test solution applied: 2mL / 10g quartz sand
Test organisms (species):
Hypoaspis aculeifer
Animal group:
Acari (soil-dwelling predatory mite)
Details on test organisms:
TEST ORGANISM
- Common name: soil mite
- Source: the mites were obtained from a healthy laboratory rearing stock maintained at the test site
- Age at test initiation (mean and range, SD): developped females were introduced in the test 28 days after the start of the egg laying period
- Stage at test initiation: developped female
- Disease free: yes


Study type:
laboratory study
Limit test:
no
Total exposure duration:
14 d
Test temperature:
19.5 - 21.5 °C
pH (if soil or dung study):
6.40 - 7.36
Humidity:
16.69 - 18.91 % water content of the test substrate
The inital weight of each vessel was measured to be used as reference for monitoring of soil moisture throughout the test. The water content of the soil substrate in the test vessels was maintained throughout the test by weighing and if necessary re-watering the test vessels twice a week. Losses were replenished as necessary with deionised water.
Photoperiod and lighting:
Long day conditions (16h light / 8h darkness), light intensity of 560-600 lux
Details on test conditions:
TEST SYSTEM
- Test container (material, size): glass vessel (5.5 cm diameter, 5.5 cm height, approx 100 mL volume), covered with a screw lid (with a little hole in the center to guarantee gas exchange closed by a mite impermeable gauze - mesh size 100 µm)
- Amount of soil or dung or substrate: 23.6 g of test substrate (20 g dry mass)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4 (plus 1 additional container for determination of pH and water content)
- No. of replicates per control: 4 (plus 1 additional container for determination of pH and water content)
- No. of replicates per vehicle control: 4 (plus 1 additional container for determination of pH and water content)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil or dung)
- Composition (if artificial substrate):
* 5% sphagnum peat (air dried and finely ground)
* 20% kaolin clay (kaolinite content preferably above 30)
* approx. 75% air-dried industrial sand (predominantly fine sand with more than 50% of the particles between 50 and 200 microns). The study was conducted with a soil batch where the amount of industrial sand was approx. 70%. The missing amount of sand (5%) was added before application of the test solution.
* approx. 0.06% calcium carbonate - precipitated extra pure (the soil pH is adjusted to 6.0 +- 0.5 at the start of the test before the addition of the test item)
- Maximum water holding capacity (in % dry weight): 35.73%
- Pretreatment of soil or dung: The dry components were blended and mixed thoroughly in an electric mixer. For determination of the water holding capacity and the soil pH the missing amount of sand (5%) was added to the soil sample to reach the correct proportion (190 g soil + 10 g sand). In order to achieve a soil water content of approximately 50% of the soil water holding capacity 178.7 g of deionised water had to be added per kg dry artificial soil.
Three days before starting the test, the dry artificial soil was pre-moistened by adding 157.3 g deionized water to 1900 g dry artifical sand and stored in a closed container until use. At the start of the test, the pre-moistened soil was divided into portions of 205.7 g for each test item treatment group and a 154.3 g for the control groups. Afterwards, the missing amount of sand (5%) was added to each soil sample, which was 10g for each test item group and 7.5 g for the control group.

OTHER TEST CONDITIONS
- Photoperiod: long day conditions (16h light / 8h darkness)
- Light intensity: 560-600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality, behavioural effects and reproduction after 14 days.

On day 14 after introduction of the test organisms the survivign mites were extracted from the soil via heat / light extraction using a Tullgren-type extracting device. the principle of the heat extraction is to make conditions for the test organisms gradually worse in the soil samples (heating from above), so that they will leave the substrate and fall in a collecting container. The soil samples were transferred carefully into extraction containers (volume: 150 mL) with a gauze at the bottom. The test containers were checked for remaining mites, which were counted and afterwards added to the number of extracted mites. For the heating of the soil samples light bulbs (25 and 40 watt) were used, which were fixed on a board. The increase in temperature was caused by using light bulbs with higher voltage and reducing the distance between the light bulbs and the soil samples.

After one day of exposition to light the test organism (adults and juveniles) which have been driven out were deep frozen and counted later. The number of juveniles (larvae, protonymphs, deutonymphs) and adults were counted separately using a stereomicroscope. Any adult mites not found at this time were recorded as dead, assuming that such mites have died and decomposed prior to the assessment.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study : yes
- Test concentrations: 0.1, 1.0, 10, 100, 1000 mg test item/kg soil dry weight
- Results used to determine the conditions for the definitive study: yes

Nominal and measured concentrations:
Nominal concentrations: 19.9, 34.8, 60.9, 106.6, 186.6, 326.5, 571.4, 1000 mg test item / kg soil dry weight
Reference substance (positive control):
no
Key result
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
281 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% confidence limits: 248.8-313.1 mg/kg soil dry weight
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
181.9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality at end of exposure period: Imazalil caused no statistically significant effects on mortality of the soil mites up to and including 1000 mg/kg soil dry weight (Bonferroni U-Test, one-tailed, a = 0.05)
Treatment rate (mg/kg sdw) Mean mortality (%) Corrected mortality (%)
Controls* 2.5 -
19.9 7.5 5.1
34.8 2.5 0
60.9 2.5 0
106.6 2.5 0
186.6 7.5 5.1
326.5 2.5 0
571.4 10 7.7
1000 5 2.6

* As no difference between solvent control and non-solvent control was observed the mortality data of both control groups were combined.

- No. of offspring produced: Statistically significant effects on the reproduction of the mites ware observed at the three highest rates of 326.5, 571.4 and 1000 mg/kg soil dry weight
Treatment rate (mg/kg sdw) Mean mnumber of juveniles per replicate Reduction to controls (%)
Controls* 336.6 -
19.9 311.5 7.5
34.8 332.0 1.4
60.9 288.5 14.3
106.6 326.5 3.0
186.6 284.5 15.5
326.5 126.5 62.4
571.4 53.0 84.3
1000 15.3 95.5

* A mean number of 336.6 juveniles was calculated for the combined control groups

- Morphological abnormalities: none observed
- Behavioural abnormalities: non observed

Reported statistics and error estimates:
Data were tested for normality and homscedasticity using shapiro-Wilk's test and residual analysis.

For comparison of reproduction data of solvent control and water control a t-test (pooled, two-tailed, a = 0.05) was used. As no significant difference between the controls occured control data were combined in accordance with OECD (2006). Arcsine-transformed mortality data of the test item did not meet normality and homoscedasticity criteria. Therefore, mortality data were analysed for significant differences between the test item treatment groups and the control group using Bonferroni U-Test (Holms corrected, one-tailed, a = 0.05). Reproduction data did not meet homoscedasticity criteria. Thus, Welch Test (Holms corrected, one-tailed, a = 0.05) was conducted to detect significant differences between reproduction data of the test item treatment groups and the control groups.

The statistical software program SAS release 9.1.3 was used for the statistical analysis.
Validity criteria fulfilled:
yes
Conclusions:
The toxicity of Imazalil to soil mites (Hypoaspis (Geolaelaps) aculeifer) was determined in a 14 day study according to OECD guideline 226. With respect to the test results, it can be concluded that Imazalil caused no statistically significant effects on mortality of Hypoaspis (Geolaelaps) aculeifer up to and including 1000 mg/kg soil dry weight. The NOEC concerning reproduction was determined as 186.6 mg/kg soil dry weight, corresponding to 181.9 mg a.s./kg soil dry weight. The EC50 was determined as 288.3 mg/kg soil dry weight (95% confidence limits: 255.3 - 321.3 mg/kg soil dry weight), corresponding to 281.0 mg a.s./kg soil dry weight (95% confidence limits: 248.8 - 313.1 mg a.s./kg soil dry weight). The results of the test can be considered reliable without restriction.

Description of key information

The study of Adelberger (2008), investigating the toxicity of Imazalil to soil mites (Hypoaspis (Geolaelaps) aculeifer) according to the OECD guideline 226, was considered as the key study for endpoint coverage. No statistically significant effect was observed on mortality up to and including 1000 mg/kg sdw. Based on reproduction, a NOEC value was determined as 181.9 mg/kg dw, which corresponds to 241.87 mg imazalil sulphate /kg soil dw.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:
241.87 mg/kg soil dw

Additional information