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Toxicity to soil microorganisms

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toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
no guideline followed
Principles of method if other than guideline:
The influence of imazalil on biochemical transformations in two soil types was studies. Fungicide doses of 0.1 mg/kg soil, i.e. the recommended practical application rate, as well as a tenfold excess were applied.
GLP compliance:
Specific details on test material used for the study:
- Source and lot/batch No.of test material: R27180
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated

- Storage condition of test material: 4°C in the dark

- Treatment of test material prior to testing: Imazalil was used in its sulphate form. A stock solution, containing an equivalent of 100 mg imazlil base (= 133 mg imazalil sulphate) per litre distilled water and a tenfold dilution (10 mg/l) were prepared from synthesis batch B4701. Chemical analysis indicated a purity exceeding 99.5%.
Analytical monitoring:
Details on preparation and application of test substrate:
A solution, containing 200g glucose and 45g (NH4)2SO4 per litre of distilled water was prepared for the soil respiration studies.
For the nitrification study, a 47.2g (NH4)2SO4 per litre of distilled water solution was prepared.
The solutions were passed through a 0.2 µm Gelman Acrodisc disposable filter assembly and stored at 4°C in the dark until needed. Fresh solutions were prepared for each set of experiments.
The given concentrations of the solutions were selected in order to obtain amemdement levels of 2g glucose and 0.45 g (NH4)2SO4 per kg of soil in the respiration studies and of 0.1g NH4-nitrogen per kg soil in the nitrifaction studies, if a 1ml aliquot of the solutions was added to a 100g portion of soil. Lucerne meal was used as obtained from an animal feed supplier.
- Parameters analysed / observed: * Carbon dioxide evolution ofsoil and of soil activated with a (NH4)2SO4-glucose mixture or with lucerne meal was monitored. * Nitrification rates were assayed by determining soluble NH4+, NO2- and NO3- during a 10-week incubation period of soil amended with 100 ppm NH4+-nitrogen. - Short description of test conditions: Respiration: * Biometers: Carbon dioxide production was measured in a biometer-type device: two 100ml glass centrifuge tubes were connected of ground glass joints and silicone tubing; one centrifuge tube contained the test soil (25-50g), whereas the second tube was filled with 50ml of 0.05 N NaOH. At the selected sampling time-points, the tube with base solution was replaced, and the amount of trapped carbon dioxide in the previous solution determined by acid-base titration. * Doses: 0.1 and 1.0 mg imazalil /kg incubated soil * Soil dosing: For ammoniumsulphate-glucose activation: The pre-incubated soil was divided in 3 portions of 250g. To a first portion 2.5ml of distilled water and 2.5ml of (NH4)2SO4-glucose solution were added. The soil was thoroughly mixed. Six 25g portions were transferred to the soil tube of the biometers. Two 25mg portions were extracted with LiCl 0.1 M dry weight fraction (24h, 120°C). To a second 250g portion, 2.5ml of the 10 ppm imazalil solution was added, whereas to the third portion, 2.5ml of the 100 ppm imazalil solution was added. The treatment was continued as above for the soil incubation without fungicide. Incubation was at room temperature (20-23°C). For lucerne meal activation: The pre-incubated soil was divided in two portions of 400g. To a first portion the necessary amount of lucerne meal, i.e. 0.5% on a dry soil basis, was added and the soil thoroughly mixed. The lucerneamended soil was divided in three 110g portions; To a first portion, 1.1ml of distilled water was added, whereas the second and third 110g portions received 1.1ml of the 10ppm imazalil solution and 1.1ml of 100ppm imazalil solution, respectively.

Test organisms (inoculum):
Total exposure duration:
10 wk
Test temperature:
20-23°C (respiration test); 21°C (nitrogen test)
60% of soil water capacity (nitrogen test)
Details on test conditions:
- Testing facility: Janssen Pharmaceutica, B-2340 Beerse, Belgium
- Test container (type, material, size): two 100ml glass centrifuge tubes connected by means of ground glass joints and silicone tubin with one of the tubes containing the test soil (respiration test); 250 ml polyethylene bottles (nitrogen test)
- Amount of soil: 25-50g (respiration test); 50g (nitrogen test)
- No. of replicates per concentration: 1
- No. of replicates per control: 1

- Method: not indicated

- Geographical reference of sampling site (latitude, longitude): Two soils were investigated: an agricultural sand [USDA classification] from Lichtaart, Belgium, and a silt loam from Haacht, Belgium
- History of site: Samples were collected from a field receiving normal agricultural practice

- Soil characteristics:
* Texture: 87.1% sand, 9.4% loam, 3.5% clay
* organic matter: 4.7%
* pH (H2O): 5.7
* pH (KCl): 4.5
* Cation exchange capacity: 15.9 meq/100 g
* water capacity: 50.1 g/100g

* Texture: 24.2% sand, 62.2% loam, 13.5% clay
* organic matter: 4.7%
* pH (H2O): 6.5
* pH (KCl): 5.8
* cation exchange cpacity: 22.2 meq/100 g
* water capacity: 44.5 g/100g

- Pretreatment of soil: The soils were air-dried at 21°C by spreading on a coarse filter paper until sieving over a 2-mm sceen became feasible. The sieved soils were transferred to 2-l polyethylene bottles and rewetted with distilled water until 60% of the water holding capacity was approximated: 29.8g H2O/100g for Lichtaart and 25.0g H2O/100g for Haacht
- Storage (condition, duration): The storage bottles were loosely capped and stored at 21°C in the dark until the start of the respiration and nitrification experiments

DETAILS OF PREINCUBATION OF SOIL (if any): not indicated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
* Respiration (CO2 production), with intervals of 0-2, 2-5, 5-7, 7-9, 9-12 and 12-14 days for soils without amendment and after amendment with 0.5% lucerne meal
* Respiration (CO2 production), with intervals of 0-7, 0-16, 0-24, 7-31, 16-40, 31-48, 24-48 days for soils activated with a (NH4)2SO4-glucose mixture
* Nitrification, with intervals of à, 0.5, 1, 2, 4, 6, 10 days

Nominal and measured concentrations:
Nominal concentrations: 0.0, 0.1, 1.0 ppm imazalil
Reference substance (positive control):
Key result
10 wk
Dose descriptor:
Effect conc.:
1 other: ppm
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
respiration rate
Details on results:
Haacht soil:
Imazalil did not influence the CO2 production of unamended soils.
No significant effects of the fungicide on respiration rates could be discerned, whereas the nitrogen transformations occured fastly in both treated and untreated soils, with a tendency towards decreasing NO2- contents and increasing NO3- concentrations.

Lichtaart soil:
The Lichtaart soil was stressed to significatn extents by the fungicide. A decrease in respiration rates was observed, which was stronger for the low treatment level of imazalil. The NO3- concentration was reversibly affected after the 0.1 ppm treatment. The inverse relationship between treatment dose and observed effects was probably related to the ecological balance between various groups of sensitive and non-sensitive microorganisms and the availability of nutrients.

Only Lichtaart respiration data showed significant deficits, never exceeding 15 to 20%. The effects were shown to be reversible within 14 days. Therefore a negligible influence of imazalil on the respiration rate of Lichtaart soil, i.e. apparently the most stressed case, could be deduce from a duration versus effect diagram.
Validity criteria fulfilled:
not applicable
Imazalil exerted no significant effect on the respiration or on the nitrification of a biologically active soil when applied at 0.1 and 1.0 ppm. A less active soil was stressed by the application of Imazalil to a measurable extent, though negligible from an environmental view-point, since the effects were both limited and reversible. Applied at these doses, it is concluded that imazalil contitutes no hazard for principal biological processes in the soil.

Description of key information

The study of Van Leemput (1983), investigating the toxicity of Imazalil on soil respiration and soil nitrification, was considered as the key study for endpoint coverage. When applied at 0.1 and 1.0 ppm imazalil, no hazard was identified for principal biological processes in the soil.

Key value for chemical safety assessment

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