Amitrole
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INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 158
AMITROLE
This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization.
Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization
First draft prepared by Dr P.J. Abbott, Department of Health, Housing and
Community Services, Canberra, Australia World Health Orgnization
Geneva, 1994
The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health
Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally
comparable results, and the development of manpower in the field of toxicology. Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents,
coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals.
WHO Library Cataloguing in Publication Data Amitrole.
(Environmental health criteria ; 158)
1.Amitrole - standards 2.Environmental exposure 3.Herbicides I.Series
ISBN 92 4 157158 6 (NLM Classification: WA 240) ISSN 0250-863X
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CONTENTS
1. SUMMARY
1.1. Identity, physical and chemical properties, and analytical methods
1.2. Sources of human and environmental exposure
1.3. Environmental transport, distribution and transformation 1.4. Environmental levels and human exposure
1.5. Kinetics and metabolism in laboratory animals and humans 1.6. Effects on experimental animals and in vitro systems 1.7. Effects on humans
1.8. Effects on other organisms in the laboratory and field 2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES AND ANALYTICAL METHODS
2.1. Identity
2.2. Physical and chemical properties 2.3. Conversion factors
2.4. Analytical methods 2.4.1. Plants 2.4.2. Soil 2.4.3. Water
2.4.4. Formulations 2.4.5. Air
2.4.6. Urine
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1. Natural occurrence
3.2. Anthropogenic sources
3.2.1. Production levels and processes 3.2.2. Uses
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1. Transport and distribution between media
4.1.1. Air 4.1.2. Water 4.1.3. Soil
4.1.3.1 Adsorption 4.1.4. Vegetation and wildlife 4.1.5. Entry into food chain 4.2. Biotransformation
4.2.1. Biodegradation and abiotic degradation 4.2.1.1 Plants
4.2.1.2 Soils 4.2.3. Bioaccumulation 4.3. Ultimate fate following use 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1. Environmental levels 5.1.1. Air
5.1.2. Water 5.1.3. Soil
5.2. General population exposure 5.2.1. Environmental sources 5.2.2. Food
5.3. Occupational exposure during manufacture, formulation or use
6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1. Absorption, distribution and excretion
6.1.1. Mouse 6.1.2. Rat 6.1.3. Human
6.2. Metabolic transformation
7. EFFECTS ON EXPERIMENTAL ANIMALS IN VITRO TEST SYSTEMS 7.1. Single exposure
7.1.1. Oral
7.1.2. Other routes 7.2. Short-term exposure
7.2.1. Oral
7.2.1.1 Dietary
7.2.1.2 Drinking-water 7.2.1.3 Intubation
7.2.2. Inhalational 7.2.3. Intraperitoneal 7.3. Long-term exposure
7.3.1. Oral
7.3.1.1 Mouse 7.3.1.2 Rat
7.3.1.3 Other species 7.3.2. Other routes
7.4. Skin and eye irritation; skin sensitisation
7.5. Reproduction, embryotoxicity and teratogenicity 7.5.1. Reproduction
7.5.2. Embryotoxicity and teratology 7.6. Mutagenicity and related end-points
7.6.1. DNA damage and repair 7.6.2. Mutation
7.6.3. Chromosome damage 7.6.4. Cell transformation 7.6.5. Other end-points 7.7. Carcinogenicity
7.7.1. Mouse 7.7.2. Rats
7.7.3. Other species
7.7.4. Carcinogenicity of amitrole in combination with other agents
7.8. Other special studies
7.8.1. Cataractogenic activity in rabbits 7.8.2. Biochemical effects
7.9. Mechanisms of toxicity - mode of action 8. EFFECTS ON HUMANS
8.1. General population exposure 8.2. Occupational exposure
9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1. Laboratory experiments
9.1.1. Microorganisms 9.1.2. Aquatic organisms 9.1.2.1 Plants
9.1.2.2 Invertebrates 9.1.2.3 Vertebrates
9.1.3. Terrestrial organisms 9.1.3.1 Plants
9.1.3.2 Invertebrates 9.1.3.3 Birds
9.2. Field observations
9.2.1. Terrestrial organisms 9.2.1.1 Plants
9.2.1.2 Invertebrates
10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT
10.1. Evaluation of human health risks
10.2. Evaluation of effects on the environment
11. CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE ENVIRONMENT
11.1. Conclusions
11.2. Recommendations for protection of human health and the environment
12. FURTHER RESEARCH
13. PREVIOUS EVALUATIONS BY NATIONAL AND INTERNATIONAL BODIES REFERENCES
RESUME
RESUMEN
WHO TASK GROUP FOR ENVIRONMENTAL HEALTH CRITERIA FOR AMITROLE
Members
Dr P.J. Abbott, Chemicals Safety Unit, Department of Health, Housing and Community Services, Canberra, Australia
(Rapporteur)
Professor J.F. Borzelleca, School of Basic Health Sciences, Department of Pharmacology, Richmond, Virginia, USAa
Professor V. Burgat-Sacaze, Ecole Nationale Vétérinaire, Toulouse, France
Dr E.M. den Tonkellar, Toxicology Advisory Centre, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands
Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood Station, Abbots Ripton, Huntingdon, Cambridgeshire, United Kingdom
Dr R. Fuchs, Department of Toxicology, Institute for Medical Research and Occupational Health, University of Zagreb, Zagreb, Croatia
Dr D. Kanungo, Division of Medical Toxicology, Central Insecticides Laboratory, Department of Agriculture and
Cooperation, Directorate of Plant Protection, Quarantine and Storage, Faridabad, Haryana, India
Professor M. Kessabi-Mimoun, Institut Agronomique et Vétérinaire Hassan II, Rabat, Morocco
Professor M. Lotti, Università di Padova, Istituto di Medicina del Lavoro, Padua, Italy (Chairman)
Professor A. Rico, Ecole Nationale Vétérinaire, Toulouse, France (Vice-Chairman)
Observers
Mr C. Chelle, CFPI, Gennevilliers, France (GIFAP Representative) Dr L. Diesing, Bayer AG, Institute of Toxicology and Agriculture, Wuppertal, Germany (GIFAP Representative)
a Invited but unable to attend
Dr B. Krauskopf, Bayer AG, Leverkusen-Bayerwerk, Germany (GIFAP Representative)
Dr Rouaud, Agrochemicals Division, CFPI, Gennevilliers, France (GIFAP Representative)
Secretariat
Dr D. McGregor, Unit of Carcinogen Identification and Evaluation, International Agency for Research on Cancer (IARC), Lyon,
France
Dr R. Plestina, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary)
NOTE TO READERS OF THE CRITERIA MONOGRAPHS
Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are kindly requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda.
* * *
A detailed data profile and a legal file can be obtained from the International Register of Potentially Toxic Chemicals, Case postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone No.
9799111).
* * *
This publication was made possible by grant number 5 U01 ES02617-14 from the National Institute of Environmental Health Sciences, National Institutes of Health, USA.
ENVIRONMENTAL HEALTH CRITERIA FOR AMITROLE
A WHO Task Group on Environmental Health Criteria for Amitrole met at the Ecole Nationale Vétérinaire, Toulouse, France, from 18 to 22 May 1993, the meeting being sponsored by the Direction générale de la Santé, Ministère des Affaires sociales, de la Santé et de la Ville, Paris. Professor A. Rico welcomed the participants on behalf of the host institute. Dr R. Plestina, IPCS, opened the meeting and welcomed the participants on behalf of Dr M. Mercier, Director of the IPCS, and the three IPCS cooperating organizations
(UNEP/ILO/WHO).
The first draft was prepared by Dr P.J. Abbott, Department of Health, Housing and Community Services, Canberra, Australia.
Extensive scientific comments were received following circulation of the first draft to the IPCS contact points for Environmental Health Criteria monographs and these comments were incorporated into the second draft by the Secretariat. The Group reviewed and revised the draft document and made an evaluation of the risks for human health from exposure to amitrole.
Professor M. Lotti deserves special thanks for skilfully chairing the meeting and for assistance to the Secretariat in finalizing the monograph. Special thanks are also due to Professor A. Rico for his technical support and exceptional hospitality.
Thanks are also due to Mrs A. Rico and the staff of the Ecole
Nationale Vétérinaire responsible for administrative aspects of the meeting.
The fact that Bayer AG and Union Carbide made available to IPCS and the Task Group proprietary toxicological information on their
products is gratefully acknowledged. This allowed the Task Group to make its evaluation on a more complete data base.
Dr R. Plestina and Dr P.G. Jenkins, both members of the IPCS Central Unit, were responsible for the overall scientific content and technical editing, respectively, of this monograph. The efforts of all who helped in the preparation and finalization of the
monograph are gratefully acknowledged.
ABBREVIATIONS
3-ATAL 3-(3-amino- s-triazole-1-yl)-2-aminopropionic acid ACGIH American Conference of Governmental and Industrial Hygienists
ADI acceptable daily intake DAB 4-dimethylaminobenzene DES diethylstilbestrol
DHPN N-bis(2-hydroxypropyl) nitrosamine DIT diiodotyrosine
EC emulsifiable concentrate GSH-Px glutathione peroxidase
HPLC high performance liquid chromatography IC50 median immobilization concentration MIT monoiodotyrosine
MTD maximum tolerated dose NBU N-nitrosobutylurea
NOAEL no-observed-adverse-effect-level NOEC no-observed-effect concentration NOEL no-observed-effect level
OECD Organisation for Economic Co-operation and Development PBI protein-bound iodine
PHS prostaglandin-H-synthetase T3 L-triiodothyronine
T4 L-thyroxine
TC thin layer chromatography TLV threshold limit value TSH thyreostimulating hormone TWA time-weighted average
WP wettable powder 1. SUMMARY
1.1 Identity, physical and chemical properties, and analytical methods
Amitrole (3-amino-1,2,4-triazole) is a colourless, crystalline powder. It is thermally stable and has a melting point of
156-159 °C. It is readily soluble in water and ethanol and only sparingly soluble in organic solvents such as hexane and toluene.
Chemically, amitrole behaves as a typical aromatic amine as well as an s-triazole. A wide range of analytical methods are available for detection and quantification of amitrole in plants, soil, water, air and urine.
1.2 Sources of human and environmental exposure
Amitrole does not occur naturally. It is manufactured by the condensation of formic acid with aminoguanidine bicarbonate in an inert solvent at 100-200 °C. Amitrole is used as a herbicide with a wide spectrum of activity and appears to act by inhibiting the formation of chlorophyll. It is commonly used around orchard trees, on fallow land, along roadsides and railway lines, or for pond weed control.
1.3 Environmental transport, distribution and transformation
Owing to its low vapour pressure, amitrole does not enter the atmosphere. It is readily soluble in water with a photodegradation half-life in distilled water of more than one year.
Photo-degradation does occur in the presence of the photosensitizer humic acid potassium salt, reducing the half-life to 7.5 h.
Amitrole is adsorbed to soil particles and organic matter by proton association. The binding is reversible and not strong, even in favourable acid conditions. Measured n-octanol/water partition coefficient values classify amitrole as "highly mobile" in soils of pH > 5 and "medium to highly mobile" at lower pH. There is
considerable variation in leaching of the parent compound through experimental soil columns. Generally, movement is most readily seen in sand; increasing the organic matter content reduces mobility.
Degradation in soils is usually fairly rapid but variable with soil type and temperature. Bacteria capable of degrading amitrole have been isolated. The herbicide can act as sole nitrogen source, but not also as sole carbon source, for the bacteria. Microbial degradation is probably the major route of amitrole breakdown;
little or no breakdown has been recorded in studies with sterilized soil. However, abiotic mechanisms, including the action of free radicals, have also been proposed as a means of degradation.
Laboratory studies have indicated degradation to CO2 with a
half-life of between 2 and 30 days. A single field study suggests that the degradation may take longer at lower temperatures and different soil moisture levels; the half-life was about 100 days in a test clay.
Although the parent compound leaches through some soils, degradation products are tightly bound to soil. Since amitrole is degraded rapidly in soil, the high leaching potential of the herbicide does not seem to be realized in practice. Occasional damage to trees reported during the early usage of amitrole has not
been a regular feature of its use.
When applied to vegetation, amitrole is absorbed through the foliage and can be translocated throughout the plant. It is also absorbed through roots and transported in the xylem to shoot tips within a few days.
High water solubility, a very low octanol-water partition coefficient and non-persistence in animals means that there is no possibility for bioaccumulation of amitrole or transport through food chains.
1.4 Environmental levels and human exposure
Particulates containing amitrole may be released from
production plants; atmospheric levels of 0 to 100 mg/m3 have been measured close to one plant.
The use of amitrole in waterways and watersheds has led to transitory water concentrations of up to 150 µg/litre.
Concentrations fall rapidly to non-detectable (<2 µg/litre) levels in running water within 2 h. Application to ponds gave an initial water concentration of 1.3 mg/litre falling to 80 µg/litre after 27 weeks. Close to a production plant, river concentrations ranged from 0.5 to 2 mg/litre.
No residues of amitrole have been detected in food following recommended use. Spraying of ground cover around fruit trees did not lead to residues in apples. Wild growing fruit in the vicinity of control areas can develop residues.
There have been no reports of amitrole in drinking-water.
1.5 Kinetics and metabolism in laboratory animals and humans Following oral administration, amitrole is readily absorbed from the gastrointestinal tract of mammals. It is rapidly excreted from the body, mainly as the parent compound. The main route of excretion in humans and laboratory animals is via the urine, and the majority of excretion takes place during the first 24 h. Metabolic transformation in mammals produces two minor metabolites detectable in the urine of experimental animals. When an amitrole aerosol is inhaled, a similar rapid excretion via the urine takes place.
1.6 Effects on experimental animals and in vitro test systems
Amitrole had low acute toxicity when tested in several species and by various routes of administration (LD50 values were always higher than 2500 mg/kg body weight). It was found to affect the thyroid after single, short-term and long-term exposures. Amitrole is goitrogenic; it causes thyroid hypertrophy and hyperplasia, depletion of colloid and increased vascularity. In long-term experiments these changes precede the development of thyroid neoplasia in rats.
The carcinogenic effect of amitrole on the thyroid is thought to be related to the continuous stimulation of the gland by
increased thyroid stimulating hormone (TSH) levels, which are caused by the interference of amitrole with thyroid hormone synthesis.
Equivocal results have been reported in some studies on the genotoxic potential of amitrole. In carcinogenicity testing in rats,
amitrole did not induce tumours in organs other than the thyroid.
However, high doses of amitrole caused liver tumours in mice.
Several criteria have been used to assess the early effects of amitrole on the thyroid. The lowest no-observed-adverse-effect level (NAOEL) derived from these studies was 2 mg/kg in the diet of rats and was assessed on the basis of thyroid hyperplasia.
1.7 Effects on humans
A single case of contact dermatitis due to amitrole has been reported. Amitrole did not cause toxic effects when ingested at a dose of 20 mg/kg. In a controlled experiment, 100 mg was found to inhibit iodine uptake by the thyroid at 24 h. Weed control operators exposed dermally to approximately 340 mg amitrole per day for 10 days exhibited no changes in thyroid function.
1.8 Effects on other organisms in the laboratory and field Several studies on the growth of cyanobacteria (blue-green algae) have shown no effect of amitrole at concentrations at or below 4 mg/litre. No consistent adverse effects on nitrogen fixation have been reported. Bacteria from soil were unaffected by
concentrations of 20 mg/litre medium in the case of nitrogen-fixing Rhizobium and 150 mg/kg in the case of cellulolytic bacteria.
There were no effects on nitrification or soil respiration at 100 mg a.i./kg dry soil, 5 times the maximum recommended application rate.
Reduced nodulation in sub-clover was reported at concentrations of up to 20 mg/litre.
Various unicellular algae have been tested for
growth-inhibiting effects. At 0.2 - 0.5 mg amitrole/litre, the growth inhibition of Selenastrum was the most sensitive reported effect.
Most aquatic invertebrates show high tolerance to technical amitrole: LC50 values were > 10 mg/litre for all organisms other than the water flea Daphnia magna, where the acute 48-h EC50 (immobilization) was 1.5 mg/litre. Fish and amphibian larvae are also tolerant to amitrole with LC50 values above 40 mg/litre.
Longer-term studies indicated that young rainbow trout survive an amitrole concentration of 25 mg/litre for 21 days.
Two earthworm species (Eisenia foetida and Allolobophora caliginosa) were unaffected by amitrole (SP50) at 1000 mg/kg
soil and Amitrole-T at 100 mg/kg soil, respectively. Carabid beetles were unaffected after direct spraying with amitrole at rates
equivalent to 30 kg/ha. Effects on nematodes only occurred at high concentrations of amitrole (the LC50 was 184 mg/kg).
Amitrole was reported to be non-hazardous to bees in field trials. Amitrole has low toxicity to birds, all reported dietary LC50 values being above 5000 mg/kg per diet. Acute oral dosing killed no mallard ducks at 2000 mg/kg body weight.
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS
2.1 Identity
Common name: Amitrole
Chemical formula: C2H4N4
Relative molecular mass: 84.08
CAS chemical names: 1 H-1,2,4-triazol-3-amine (9C1) 3-amino- s-triazole (8CI)
IUPAC names: 1 H-1,2,4-triazol-3-ylamine 3-amino-1 H-1,2,4-triazole 3-amino- s-triazole
CAS registry number: 61-82-5 RTECS registry number: XZ3850000
Common synonyms: aminotriazole; 2-aminotriazole;
3-aminotriazole; 3-amino-1,2,4- triazole; 2-amino-1,3,4-triazole;
3-amino-1H-1,2,4-triazole; AT;
3AT; ATA; 3,A-T; ATZ; AT-90;
triazolamine; 1,2,4-triazol-3-amine;
5-amino-1H-1,2,4-triazole.
Common trade names: Amerol; Aminotriazole Weedkiller 90; Aminotriazol Spritzpulver;
Amitril; Amitril T.L.; Amitrol;
Amitrol 90; Amitrol Plus;
Amitrol-T; Amizine; Amizol;
Amizol DP; Amizol F; AT Liquid;
Azaplant; Azolan; Azole; Azaplant Kombi; Campaprim A1544; Cytrol;
Cytrole; Destraclol; Diurol. 5030;
Domatol; Domatol 88; Elmasil;
Emisol; Emisol 50; Emosol F; ENT 25445; Exit; Fenamine; Fenavar;
Fyrbar; Kleer-Lot; Lancer;
Nu-Zinole-AA; Orga 414; Preceed;
Radoxone TL; Ramizol; Sapherb;
Solution Concentree T271; Ustinex;
Vorox; Vorox AA; Vorox AS;
Weedar ADS; Weedar AT; Weedazin;
Weedazin Arginit; Weedazol;
Weedazol GP2; Weedazol Super;
Weedex Granulat; Weedoclor; X-All Liquid.
Technical grade amitrole contains a minimum of 95% active ingredient and is formulated as a solution of 250 g/litre in water, usually with an equimolar concentration of ammonium thiocyanate, or as a 400 g/kg wettable powder, usually in combination with other herbicides.
The major impurities are 3-(N-formylamino)-1,2,4-triazole, 4 H-1,2,4-triazole-3,4-diamine, and
4 H-1,2,4-triazole-3,5-diamine.
2.2 Physical and chemical properties
Some of the physical and chemical properties of amitrole are shown in Table 1.
Amitrole is readily soluble in water, methanol, ethanol and chloroform, sparingly soluble in ethyl acetate, and insoluble in hydrocarbons, acetone and ether. It forms salts with most acids or bases and is a powerful chelating agent. It is corrosive to
aluminium, copper and iron. Chemically, amitrole behaves as an s-triazole and also as an aromatic amine, and hence will diazotize and couple several dyes.
2.3 Conversion factors:
1 mg/kg = 3.43 mg/m3 1 mg/m3 = 0.29 mg/kg
Table 1. Some physical and chemical properties of amitrole
Physical state crystalline Colour colourless Taste bitter Odour none
Thermal stability stable at 20 °Ca Hydrolytic stability (pH 4-9; 90 °C) stableb
Melting point 157-159 °Cc Water solubility (25 °C) 280 g/litrec Water solubility (53 °C) 500 g/litred Ethanol solubility (75 °C) 260 g/litred Solubility in n-hexane (20 °C) < 0.1 g/litred Solubility in dichloromethane (20 °C) 0.1-1 g/litred Solubility in 2-propane 20-50 g/litred Solubility in toluene (20 °C) <0.1 g/litred Vapour pressure (20 °C) 55 nPac
Octanol/water partition coefficient (21 °C) (log Pow) -0.969e
a Klusacek & Krasemann (1986)
b Krohn (1982)
c Worthing & Hance (1991)
d Personal communication from Bayer AG to the IPCS (1993)
e Hazleton Laboratories, USA Report HLA-6001-187 2.4 Analytical methods
2.4.1 Plants
Early methods for the detection of amitrole by paper chromatography or for its quantitative determination by spectrophotometry involved extraction by ethanol or water, diazotization of the 3-amino group and, finally, coupling with either phenol in 20% HCl (Aldrich & McLane, 1957),
N-(1-naphthyl)ethylenediamine dihydrochloride (Storherr & Burke, 1961), H-acid (8-amino-1-naphthol-3,6-disulfonic acid, monosodium salt) (Racusen, 1958; Herrett & Linck, 1961; Agrawal & Margoliash, 1970) or chromotropic acid (Green & Feinstein, 1957). This technique has been used for residue analysis in plants (Aldrich & McLane, 1957; Herrett & Linck, 1961), and vegetable crops (Storherr & Burke, 1961). The detection limit was found by Aldrich & McLane (1957) to be approximately 0.1 µg/spot. The method outlined by Storherr &
Burke (1961) is sensitive to 0.025 mg/kg. Recovery was described by Herrett & Linck (1961) to be close to 100%. Storherr & Onley (1962) found that dry-packed cellulose column chromatography was preferable to paper chromatography for separation of amitrole from some crops.
Several gas chromatographic methods have been developed to determine amitrole residues in plants (Jarczyk, 1982a, 1985; Jarczyk & Möllhoff, 1988). The principle of all these methods is similar.
After extraction with an ethanol-water mixture, acetylation with acetic anhydride (conversion of amitrole to the monoacetyl
derivative) and a clean-up step by gel chromatography, the residue is dissolved in acetone or ethanol and determined by a gas
chromatograph equipped with a nitrogen-phosphorus detector.
Weber (1988) developed a method for the determination of
amitrole in plant material by high performance liquid chromatography (HPLC). Amitrole was extracted with an acetone-water mixture and the water phase was extracted with dichloromethane to remove lipophilic compounds. After a further clean-up step with column chromatography on a cation exchange resin and on aluminium oxide, the residues were determined by HPLC with ion pairing reagent and electrochemical detection. In plants the detection limit was 0.01 mg/kg and the recovery was between 91 and 99% in the range 0.01-1.0 mg/kg.
The Codex Committee on Pesticide Residues has recommended the methods of Lokke (1980) and Van der Poll et al. (1988).
The method of Lokke (1980) uses ion-pair HPLC, which in
potatoes or fodder beets had a limit of detection between 0.005 and 0.01 mg/kg.
The method of Van der Poll et al. (1988) is capable of
determining amitrole in plant tissues and sandy soils by capillary gas chromatography with an alkali flame ionization detector. Samples are extracted with ethanol, absorbed on resin and desorbed with ammonia. After acetylation with acetic anhydride and clean-up with a SEP-PAK silica cartridge, the residue is determined by gas
chromatography (GC). The limit of detection is 0.02 mg/kg and average recoveries are 76-81% in the range from 0.05 to 0.2 mg/kg.
2.4.2 Soil
An early method for the determination of residues in soil was developed by Sund (1956) which involved extraction with water followed by colour reaction with nitroprusside in alkaline solutions.
Groves & Chough (1971) developed an improved procedure for the extraction of amitrole from soil using concentrated ammonium
hydroxide and glycol (1:4). Pribyl et al. (1978) investigated the extraction of amitrole from soils and its identification and
quantitation by photometry and thin layer chromatography (TLC). The limit of detection was 0.05 mg/kg. They proposed analysis by TLC after reaction with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansylation), in preference to HPLC. Lokke (1980) suggested that HPLC separation could be used if preceded by clean-up on a polyamide column. Both the GC method (Jarczyk, 1985; Jarczyk & Möllhoff, 1988) and the HPLC method (Weber, 1988) described in section 2.4.1 for plants are also suitable for the determination of amitrole residues in soil.
2.4.3 Water
Marston et al. (1968) and Demint et al. (1970) have used cation ion-exchange column chromatography to extract amitrole from
contaminated creek and canal waters. This is followed by
diazotization and coupling as described by Storherr & Burke (1961).
Alary et al. (1984) have modified these methods to achieve a
spectrophotometric determination of amitrole in waste water in the vicinity of production plants in the presence of interfering amino compounds.
A more recent capillary gas-liquid chromatographic method for determining amitrole in ground water and drinking-water, using an alkali flame ionisation detector, has been described, the reported limit of detection being 0.1 µg/litre (Van der Poll et al., 1988).
Legrand et al. (1991) formed a nitroso derivative of amitrole concentrated from surface and ground waters prior to HPLC analysis.
The nitroso derivative showed an absorption maximum in the near UV spectrum. Aqueous solutions of amitrole in the range of
0.25-0.50 µg/litre were measurable, and the recoveries were 70 ± 8%
(n = 11). The limit of determination was 0.1 µg/litre.
Pachinger et al. (1992) developed an HPLC analytical method with amperometric detection for the determination of amitrole
without derivatization in drinking-water and ground water. Detection limits were 1 mg/litre for directly injected samples and
0.1 µg/litre following an evaporation step to concentrate the samples. Recoveries were close to 100%.
Both the GC method (Jarczyk, 1985; Jarczyk & Möllhoff, 1988) and the HPLC method (Weber, 1988) described in section 2.4.1 for plants are also suitable for the determination of amitrole residues in water.
An immunochemical approach to the detection of amitrole has been recently described by Jung et al. (1991). Development of this rapid and sensitive method is likely to lead to a very effective method for detecting amitrole in waterways.
2.4.4 Formulations
Ashworth et al. (1980) described a potentiometric precipitation titration method using silver nitrate and silver/silver chloride or silver/mercurous sulfate electrode. This method can be used for the
determination of amitrole in its formulations or in the presence of triazines, substituted urea herbicides or plant growth regulators such as bromacil and ammonium thiocyanate. Another method for the determination of amitrole in its formulations has been described by Gentry et al. (1984). This involves dissolving or extracting the sample with dimethylformamide, acidifying by adding 0.5 N HCl and back-titrating the excess acid with 0.5 N sodium hydroxide.
Jacques (1984) has described a simple GC method for the
detection and quantification of amitrole in technical and formulated products.
A TLC method for the routine identification of amitrole in pesticide mixtures has been developed by Ebing (1972).
2.4.5 Air
Alary et al. (1984) have described a method for the analysis of air samples collected on glass-fibre filters followed by
diazotization and coupling to produce a colour reaction. The detection limit was not reported.
2.4.6 Urine
In order to assay amitrole in urine samples, Geldmacher-von Mallinckrodt & Schmidt (1970) separated the amitrole by paper chromatography using phenol saturated with water, or a mixture of
n-butanol:water (15:1) and propionic acid:water (7:6), and identified amitrole by spraying with a solution of
p-dimethyl-aminobenzaldehyde in acetic acid or hydrochloric acid.
In a more recent paper by Archer (1984), a proposed method for biological monitoring of urine samples used HPLC separation with a visible light detector following diazotization and coupling. The detection limit was 200 µg/litre.
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE 3.1 Natural occurrence
Amitrole does not occur naturally.
3.2 Anthropogenic sources
3.2.1 Production levels and processes
The synthesis of amitrole was first reported by Thiele &
Manchot in 1898 and involved the reaction of aminoguanidine with formic acid (Carter, 1975). The current industrial production process, described by Allen & Bell (1946), involves the same reaction, in which an aminoguanidine salt is heated to 100-120 °C with formic acid in an inert solvent (Carter, 1975; Sittig, 1985).
Amitrole is currently manufactured or formulated in several countries. Its use has declined, particularly in the USA. However, in spite of some recent replacements, amitrole remains a widely used herbicide.
3.2.2 Uses
Amitrole is primarily used as a post-emergent non-selective herbicide and has a very wide spectrum of activity against annual and perennial broad leaf and grass type weeds. Its primary mode of action is unknown but a prominent feature is its inhibition of the formation of chlorophyll, and weeds initially change colour to
white, brown or red, and subsequently die (Carter, 1975). This herbicidal activity is enhanced by the addition of ammonium thiocyanate as a synergist. Amitrole can be used alone as a concentrated solution in water or as a wettable powder in combination with other herbicides. It is primarily used as a
herbicide and as a brush killer. It is also used as a non-selective pre-emergent herbicide on fallow land before planting kale, maize, oilseed rape, potatoes and wheat, and in other non-crop situations (Worthing & Hance, 1991). It is also used along roadsides and railway lines to control weeds. Approved uses of amitrole on soil are either for non-crop land prior to sowing, or for inter-row weed control in tree and vine crops, where contact with food plants is avoided. Amitrole is also used for the control of pond weeds and is an especially effective herbicide in the control of water hyacinth
(Eichhornia crassipes).
Amitrole has also been used as a cotton defoliant in some countries (Hassall, 1969).
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION 4.1 Transport and distribution between media
4.1.1 Air
The very low vapour pressure of amitrole (Table 1) means that it will not enter the atmosphere.
4.1.2 Water
Amitrole is readily soluble in water (280 g/litre at 25 °C) and has a half-life of more than one year at 22 °C and pH 4-9 (Worthing & Hance, 1991). Although no direct photolysis occurred in doubly distilled water, the photodegradation rate increased in the presence of humic acid, potassium salt (100 mg/litre), a natural
"photosensitizer", resulting in a half life of 7.5 h (Jensen-Korte et al., 1987).
4.1.3 Soil
4.1.3.1 Adsorption
Amitrole is adsorbed to soil particles and organic matter by proton association. The adsorbed aminotriazolium cation will enter into cationic exchange reactions (Nearpass, 1969). Binding is strongly pH dependent, and the cation is adsorbed to a greater extent in acid conditions. The aminotriazolium cation is bound more strongly than sodium but is displaced by calcium ions. The binding is reversible and not strong, even in favourable acid conditions.
The binding capacity of soils at pH 5 or more is limited (Nearpass, 1970). Anderson & Hellpointner (1989) determined the Koc values for amitrole in four soils i.e. silty clay, sandy loam, sand and silt, to be 112, 30, 20 and 52, respectively. Adsorption increased at lower pHs; adjustment of pH to a constant 4.5 resulted in Koc
values ranging from 77 to 356. The authors classified amitrole as highly mobile in the soils at their equilibrium pH values of 5.6 to 7.4 and medium to highly mobile with the pH adjusted from 4.2 to 4.5.
There is considerable variation in the literature, both old and recent, in reported adsorption and leachability of amitrole. Sund (1956) described adsorption to soil as strong. He demonstrated that amitrole could be efficiently removed from aqueous solution with a resin cation exchanger and argued that soil would also bind the
compound efficiently. A correlation between the base exchange
capacity of soil and binding of amitrole was postulated. This agrees with the theoretical and experimental work of Nearpass (1969, 1970), although the latter author does not support the strength of
adsorption proposed by Sund (1956). Day et al. (1961) investigated leaching of amitrole through 400-g, 4-cm diameter columns of three different soils from a citrus growing area of California following occasional reports of damage to trees after application of high rates of amitrole for the control of perennial weeds. Amitrole moved readily with the leaching water for all soil types (two sandy loams and one silt loam) and most readily through quartz sand. Zandvoort et al. (1981) supported this conclusion, suggesting that the high water solubility of amitrole could result in leaching from sandy soils. Weller (1987) investigated leaching of amitrole through 27-cm, 5-cm diameter columns of two soils, a sandy "standard soil 2.1" and a second soil with substantially higher organic content.
Immediately after incorporation of the 14C-amitrole to give 2 mg on the surface area of the column, leaching with deionized water began, 393 ml being pumped onto the soil column over 2 days. The leachate was collected in two fractions: 175-191 ml and 185-200 ml.
Duplicate experiments showed 24 and 31% of the initial radioactivity in the leachate (entirely in fraction II) in the sandy soil, with 11%, 16% and 46%, respectively, remaining in the upper, middle and lower third of the soil column. The second soil leached markedly less of the added radioactivity (1.4 and 1.8% for the duplicate columns); this also appeared in fraction II. The radioactivity in the leachate was unchanged amitrole.
Since amitrole is degraded rapidly in soil (section 4.2.1), the high potential of amitrole to leach through sandy soils does not seem to be realized in practice. The occasional damage to trees reported in the study by Day et al. (1961) has not been a regular feature of the use of amitrole. Degradation products of amitrole do not leach significantly through soil (section 4.2.1).
4.1.4 Vegetation and wildlife
When applied directly to vegetation as a herbicide, amitrole is absorbed through the foliage and can be translocated throughout the plant. Translocation occurs in the photosynthetic stream and is dependent on light. When applied to soil, amitrole can be adsorbed through the roots and transported in the xylem, within a few days, to the tips of the shoots (Carter, 1975).
4.1.5 Entry into food chain
Amitrole is not to be used on food crops and therefore food residues should not occur. Grazing animals could consume amitrole as surface residues on vegetation after application or as residues within the plant. Amitrole is not persistent in animals and would not be expected to pass through the food chain.
4.2 Biotransformation
4.2.1 Biodegradation and abiotic degradation 4.2.1.1 Plants
Racusen (1958) reported the first comprehensive studies of amitrole metabolism in plants. Two major metabolites were isolated, neither of which were as phytotoxic as amitrole. These results were supported by studies by Carter & Naylor (1960). One metabolite was identified as the product of the reaction of amitrole with serine,
namely, 3-(3-amino- s-triazole-1-yl)-2-aminopropionic acid (3-ATAL). Formation of 3-ATAL is considered to represent
detoxification since ammonium thiocyanate, which synergizes the action of amitrole, inhibits the formation of 3-ATAL (Smith et al.
1969). Other products of amitrole metabolism in plants have not been identified. Fang et al. (1967) found that metabolism of amitrole in leaves was exponential, with half-lives in sugar beet, corn and bean leaves being 18.7, 28.0 and 23.2 h, respectively. A review of the degradation of amitrole in plants has been presented by Carter (1975).
The soluble metabolites of [3,5-14C]-amitrole in apples were examined by Schneider et al. (1992) following soil application.
Significant proportions of the radioactivity were found as bound residues, but 69-90% were extractable with acetonitrile. In addition to 3-ATAL, 3-(1,2,4-triazole-1-yl)-2-aminopropionic acid
(3-aminotriazolylalanine) was also identified, in both the free form and as conjugates. This was the major metabolite in apple cell
cultures treated with amitrole (Stock et al., 1991).
4.2.1.2 Soils
There is general agreement that degradation of amitrole in soil is usually fairly rapid and variable with soil type and temperature.
However, there is no clear consensus on the relative roles of biotic and abiotic processes in the breakdown of the compound.
Day et al. (1961) measured amitrole colorimetrically in 55 different soils of 5 main types from California and estimated the depletion after 2 weeks of incubation. The results were very variable, 26 soils having no measurable amitrole after 2 weeks, 6 soils showing traces and the remaining 23 soils having higher quantities, in some cases comparable to initial levels. Four soils had more than half of the original amitrole after the 2-week
incubation. It was not possible to correlate depletion of amitrole to soil type. The authors classified the soils according to general type and ranked them in terms of "heaviness"; the four soils
retaining most amitrole ranked 7, 23, 30 and 54 in the list. There was a geographical correlation with reported incidents of non-target effects of the herbicide. Some specific characteristic of a variety of soils from a single location had led to movement of the herbicide and its retention longer than in apparently comparable soils
elsewhere. Decomposition rates in steam-sterilized soils were much lower than in unsterilized soils, which led the authors to conclude that breakdown was principally due to microorganisms. Decomposition was optimal at temperatures between 20 and 30 °C and at medium to high soil moisture content. Breakdown was not well correlated with soil classification, texture, base-exchanged capacity or adsorption capacity for amitrole. Differences in microbial populations were cited as the most likely explanation for the variation.
Kaufman et al. (1968) also found that sterilization of soil reduced the breakdown of amitrole. Within 20 days, 69% of the radioactivity of 14C-labelled amitrole was released as 14CO2
in unsterilized soil. Soil treated with sodium azide or ethylene oxide released 46% and 35%, respectively, whilst autoclaved soil released only 25%. Reinoculation of soil with microorganisms isolated from the original soil failed to restore the capacity to degrade amitrole. Amending the soil with other organic compounds reduced amitrole degradation. The authors concluded that degradation of amitrole was largely a chemical process and that microbial action was indirect. Free radicals (such as HO.) were proposed as agents for oxidation of the amitrole nucleus. Plimmer et al. (1967) studied
the degradation of amitrole by free-radical generating systems. They demonstrated that riboflavin (and light) or an ascorbate-copper reagent (Fenton's reagent) promotes oxidation of amitrole, resulting in ring cleavage, loss of CO2 and production of urea, cyanamide and possibly molecular nitrogen. Riepma (1962) observed a lag-phase which he considered typical of microbial breakdown. Carter (1975) concluded that "whatever the mechanism, triazole ring opening occurs rapidly in soils and the resulting products ... should be readily metabolized by soil microorganisms".
Campacci et al. (1977) reported the isolation of bacteria capable of degrading amitrole, strengthening the argument for microbial involvement. Only one of three media tested succeeded in growing organisms that could degrade amitrole. Of 36 isolates from this culture, 10 were found to be capable of degrading amitrole.
Nine of these were gram-positive rods (Bacillus spp. and Corynebacterium spp.) and one was identified as a Pseudomonas sp. The growth of these bacteria was roughly proportional to amitrole concentration up to 256 mg/litre. The organisms could
degrade amitrole as sole nitrogen source but not also as sole carbon source; the medium was enriched with sucrose. This explained
previous failures to isolate organisms capable of degrading the herbicide.
Various studies have quantified the degradation of amitrole in soil. Scholz (1988) observed the release of 48% of applied
radioactivity (14C-amitrole) as 14CO2 after 5 days. In
degradation studies in the laboratory, half-lives of between 2.4 and 9.6 days were observed in different soils. DT90 (the time required for degradation of 90% of the amitrole) values were in the range of 13 to 22 days (LUFA, 1977; Jarczyk, 1982b,c,d). Hawkins et al.
(1982b) measured 70-80% degradation to CO2 in standard soil and 40-50% in English clay soil within 28 days. There was no release of
14CO2 from autoclaved soil. Hawkins et al. (1982a) measured
decomposition in the same English clay in the field. Here 53% of the applied radioactivity remained after 112 days. The slower rate of breakdown in the field was ascribed to the temperature and soil moisture content. Schneider et al. (1992) suggested that amitrole can be deaminated in soil to give triazole.
A study by Weller (1987) examined the leaching of "aged"
residues of amitrole. Soils, with 14C-amitrole incorporated as
described in section 4.1.3.1, were incubated for 30 and 92 days, in duplicate experiments, and then used in leaching tests as for the initial soils. For both the "standard soil 2.1" and the second soil, between 50 and 73% of initial radioactivity was lost as 14CO2
during incubation. Of the remaining radioactive material, negligible amounts leached through the soil column in tests after 30 and 92 days. Almost all of the activity remained in the upper third of the soil column. After 30 days 4% or less of the activity was unchanged amitrole. The breakdown products of amitrole (not characterized except for traces of urea) were tightly bound to the soil and were not leachable or easily extractable.
4.2.3 Bioaccumulation
Flow-through studies on fish using 14C-amitrole indicated that the bioaccumulation of amitrole in bluegill sunfish (Lepomis
macrochirus) and in channel catfish (Ictalurus punctatus), exposed to 1 mg/litre, was only slight after 21 days of exposure (approximately 1.7-3.0 times the amitrole concentration in the
water). When the fish were returned to untreated water, the amitrole
concentration in their organs fell rapidly (Iwan et al., 1978).
Bioaccumulation of amitrole by aquatic organisms would not be expected because of its high water solubility and very low
octanol-water partition coefficient (Table 1).
4.3 Ultimate fate following use
MacCarthy & Djebbar (1986) described a method using chemically modified peat to decontaminate eluant from chemical production plants before it enters major waterways. When converted to a granular product suitable for column chromatography, the peat can act as an efficient ion-exchange material for the removal of amitrole and other basic chemicals.
Amitrole is resistant to hydrolysis and the action of oxidizing agents. Burning the compound with polyethylene is reported to result in > 99% decomposition (Sittig, 1985).
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 5.1 Environmental levels
5.1.1 Air
Amitrole-containing particles are released from the stack of production plants during dry crushing and, to a lesser extent, bagging operations. Atmospheric levels in the range of 0 to 100 µg/m3 were measured in the vicinity of such a plant (Alary et al., 1984). Severe chlorosis and defoliation was noted following atmospheric fallout in the vicinity of the plant.
5.1.2 Water
Grzenda et al. (1966) studied the persistence of amitrole and three other herbicides in pond water following an aquatic weed control programme. The initial level on day one of 1.34 mg/kg decreased gradually to 1.03 mg/kg on day 11, 0.73 mg/kg on day 25, 0.49 mg/kg at 9.5 weeks and 0.08 mg/kg at 27 weeks.
In a study by Marston et al. (1968) in which 100 acres of a watershed in Oregon was sprayed, the levels of amitrole in water samples were measured on the downstream edge of the sprayed area. A maximum concentration of 155 µg/litre was found 30 min after
application began, but this decreased to 26 µg/litre by the end of the application. No amitrole could be detected 6 days after
spraying. The detection limit was 2 µg/litre.
Demint et al. (1970) measured the amitrole concentration in two flowing water canals following treatment of a single ditchbank of each canal with amitrole at the normal treatment rate. Samples taken at stations up to 7.2 km downstream indicated rapid decreases in amitrole levels following passage of the initial amitrole-bearing water, the levels having declined to 1 µg/litre within 2 h. In a preliminary environmental survey conducted in 1984 in Japan,
amitrole was not detected (detection limit 4 µg/litre) in any of 24 water samples nor was it detected in any of the 24 bottom sediments, the detection limit being 5-20 µg/kg (Environment Agency Japan, 1987).
Alary et al. (1984) measured the level of amitrole in water samples collected in a river downstream from the discharge of an aeration pond in the vicinity of a production plant. The levels were in the range of 0.5 to 2 mg/litre while the concentration in the
water of the aeration pond was in the range of 50 to 200 mg/litre.
Legrand et al. (1991) tried to detect 38 compounds including amitrole in different areas of France (13 sampling points) with a detection limit of 0.1 µg/litre, but no amitrole was found.
5.1.3 Soil
As discussed in chapter 4, amitrole, when applied to soil, is readily degraded or adsorbed to the soil particles.
5.2 General population exposure
5.2.1 Environmental sources
No exposure would be expected from environmental sources.
5.2.2 Food
Amitrole is not to be used on food crops, and food residues should therefore not occur. Using a limit of determination of 0.05 mg/kg, amitrole was not detectable in a wide range of food crops (Duggan et al., 1966, 1967; Corneliussen, 1969, 1970). This was confirmed by several studies (Bayer AG, 1993a,b).
Experimental studies in West Virginia, USA, indicated that residues of amitrole on whole apples could not be detected 3 months after ground cover application, but could be detected when either fruit or foliage or both were directly treated with amitrole (Schubert, 1964). The analyses were conducted using the method of Storherr & Burke (1961) with a detection level of 0.025 mg/kg.
Similarly, residue trials conducted in Tasmania and New South Wales on apples did not reveal amitrole at a detection limit of 0.01 mg/kg following ground cover application (Moore, 1968, 1969, 1970). A slight modification of the method of Storherr & Burke (1961) was used.
In one study, residues of amitrole were found in blackberries growing very near a railway line that was sprayed by amitrole in the normal way by the railway authorities. Thirteen days after spraying at a dose 3,5 kg a.i./ha, blackberries were picked close to the railway at two different locations. The mean residues found at the two locations were 0.67 (0.2-1.4) mg/kg and 2.0 (0.1-3.8) mg/kg. The places where the blackberries were picked was prohibited to the general public. This study shows that spraying of amitrole on blackberries results in considerable residues (Dornseiffen &
Verwaal, 1988).
5.3 Occupational exposure during manufacture, formulation or use The potential for toxicity via the dermal or inhalational routes appears to be low. A threshold limit value (TLV) of
0.2 mg/m3, as an 8-h time-weighted average (TWA), has been set for amitrole by the American Conference of Governmental & Industrial Hygienists (ACGIH, 1991-1992). Amitrole is a mild skin and eye irritant.
6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS 6.1 Absorption, distribution and excretion
6.1.1 Mouse
The distribution of [5-14C]-radiolabelled amitrole has been
examined in non-pregnant C57BL strain female mice (Tjalve, 1975) and in the fetuses of pregnant NMRI strain mice (Tjalve, 1974). In each case, the mice received amitrole (5 µCi) either intravenously or orally, and the distribution of radioactivity was determined by whole-body autoradiography of the adult or fetus at intervals of 5 min to 5 days after administration. In the non-pregnant animals, further analysis of the distribution of radioactivity was performed by microautoradiography of the spleen and thymus, and by subcellular fractionation of the liver, spleen and thymus. The highest
radioactivity was found in tissues with rapid cell turnover, e.g., bone marrow, spleen, thymus and gastrointestinal mucosa. Only a moderate level of radioactivity was found in the thyroid. The level of radioactivity in the tissues was the same whether the treatment was intravenous or oral. In all cases, there was a significant decrease over the 5-day period. Microautoradiography indicated amitrole was located mostly in the cytoplasm. 14C-labelled
amitrole crossed the placental barrier and could be detected in fetal tissues 4 and 8 h after administration to the dams by intravenous injection or gavage.
Following intravenous administration of 14C-amitrole (3.4 mg/kg body weight), adult ICR mice were killed at given
intervals (5 min, 30 min, 8 h and 24 h) and submitted to whole-body autoradiography and microautoradiography. The liver had the highest accumulation of radioactivity and two distribution patterns were observed: a homogenous distribution up to 8 h following injection, and a subsequent heterogenous one. Liver sections were extracted with trichloroacetic acid and methanol, but considerable amounts of radioactivity remained non-extractable. A microauto-radiography of the liver 8 h after 14C-amitrole injection revealed that the
radioactivity was localized in the centrolobular areas (Fujii et al., 1984).
6.1.2 Rat
Kinetic studies on amitrole in rats were performed by Fang et al. (1964). Groups of Wistar rats were administered 1 mg
14C-amitrole by gavage and the distribution of radioactivity was analysed at various time intervals between 30 min and 6 days. High levels of radioactivity (70-95% of the administered radioactivity) were found in the urine during the first 24 h, but only low levels in the faeces, indicating rapid and almost complete absorption from the gastrointestinal tract followed by rapid excretion. Tissue levels were very low after 3 days, and significant amounts were found only in the liver. In a second experiment (Fang et al., 1966),
14C-amitrole was administered at various dose levels (1-200 mg/kg body weight). The average total radioactivity found in urine and faeces (as a percentage of the administered dose) was comparable for all the doses applied. The difference in average half-time for
clearing of radioactivity from various organs was considered to be insignificant between dosages of 1 and 200 mg/kg. The fate of two unidentified plant metabolites of amitrole, i.e. 14C-metabolite 1 and 14C-metabolite 3 (isolated from bean plants), was also
examined by Fang et al. (1966). Radioactivity from metabolite-1 was excreted rapidly in the urine in the first 48 h and identified as unchanged metabolite-1. Elimination of metabolite-3 was mainly in the faeces. In a study by Grunow et al. (1975), 14C-amitrole was administered to rats by gavage at a dose level of 50 mg/kg, and the urine and faeces were examined over 3 days. The major route of excretion of radioactivity was the urine, the majority of the radioactivity being excreted in the first 24 h.
Two groups of five male and five female Sprague-Dawley rats weighing 200-250 g were exposed (either nose only or whole body) to atmospheres of 5-14C-amitrole (radiochemical purity > 97%) in
water aerosols at concentrations in air of 49.2 µg/litre
(2.6 µCi/litre) or 25.8 µg/litre (1.4 µCi/litre), respectively, for 1 h, and then observed for 120 h (MacDonald & Pullinger, 1976). The particle size distribution of the aerosols was not reported. The calculated elimination half-life of radioactivity was approximately 21 h for both exposures; approximately 75% of the radioactivity was eliminated in the urine within 12 h.
6.1.3 Human
Urinary excretion of unchanged amitrole has been reported in a woman who ingested approximately 20 mg/kg of the herbicide
(Geldmacher-von Mallinckrodt & Schmidt, 1970).
6.2 Metabolic transformation
The limited data available indicates that little metabolic transformation of amitrole occurs in mammalian species. In the mouse, tissue residues were identified by TLC as mainly unchanged amitrole (84% of the detected radioactivity) when measured 8 h after exposure (Tjalve, 1975). Similarly, paper chromatographic analysis of rat liver residues following oral administration revealed
unchanged amitrole plus one unidentified metabolite (Fang et al., 1964). In the urine of rats, the majority of the radioactivity was also unchanged amitrole; one unidentified metabolite was isolated which represented approximately 20% of the total radioactivity. The liver was the site of the unidentified metabolite-1 formation and the rate of elimination of this metabolite from liver and kidney was much slower (Fang et al., 1964).
In a more extensive analysis of urinary metabolites in the rat by Grunow et al. (1975), the major part of the radioactivity
identified by paper chromatography corresponded to unchanged amitrole. Two urinary metabolites were identified as
3-amino-5-mercapto-1,2,4-triazole and
3-amino-1,2,4-triazolyl-(5)-mercapturic acid, which together amounted to approximately 6% of the administered dose.
In a metabolic study (Turner & Gilbert, 1976), which was
supplementary to the inhalation exposure experiment and is described in section 6.1.2 (MacDonald & Pullinger, 1976), it was found that approximately 60% of the urinary radioactivity chromatographed on silica gel 60 TLC in methanol: 880 ammonia (100: 1.5, s/s) as amitrole, 15-20% remained at the origin and 5-8% migrated faster than amitrole. Treatment with ß-glucuronidase had no effect upon this TLC distribution.
7. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS 7.1 Single exposure
7.1.1 Oral
The acute oral toxicity data for amitrole when administered as an aqueous suspension are presented in Table 2.
Table 2. Acute oral toxicity of amitrole
Species LD50 (mg/kg Reference
body weight)a
Rat > 4080 (m and f) Gaines et al. (1973) > 4200 Seidenberg & Gee 24 600 (m) Bagdon et al.(1956) > 10 000 Hecht (1954)
> 2500 Kimmerle (1968) > 5000 (m) Thyssen (1974) > 5000 (m) Heimann (1982) Mouse 11 000 Hapke (1967) 14 700 (m) Fogleman (1954) Cat > 5000 (m and f) Bagdon et al. (1956)
a m = males; f = females
In cats and dogs the general signs of toxicity were dyspnoea, ataxia, and diarrhoea with vomiting. Coma and death appeared to be associated with profound respiratory depression. Gastro-intestinal irritation and haemorrhage were the only treatment-related findings.
The toxicity of a mixture of amitrole and ammonium thiocyanate (1:1), referred to as Amitrol-T, appeared to be slightly higher than that of amitrole itself, but was still very low. LD50 values
obtained following oral administration in rats were 3500 mg/kg and 10.5 ml/kg of the commercial product (DeProspo & Fogleman, 1973;
Field, 1979).
The possibility that amitrole might form a Schiff's base with glucose was investigated by Shaffer et al. (1956). An
amitrole-glucose adduct was prepared and administered orally to rats and mice (10 mg/kg), intraperitoneally to mice (10 mg/kg), and
intravenously to mice (1.6 mg/kg). There were no deaths or signs of toxicity following treatment.
7.1.2 Other routes
The acute toxicity of amitrole by other routes of administration is very low, as shown in Table 3.
Table 3. Acute, dermal, intraperitoneal and intravenous toxicity of amitrole
Species Route LD50 (mg/kg bw) Reference
Rat dermal > 2500 (m and f) Gaines et al. (1 intraperitoneal > 4000 (m) Shaffer et al. ( Mouse intravenous > 1600 (m) Shaffer et al. ( intraperitoneal > 10 000 Shaffer et al. ( intraperitoneal 5470 (m) Nomiyama et al.
subcutaneous 5540 (m) Nomiyama et al.
Rabbit dermal > 10 000 Elsea (1954) Dog intravenous > 1800 (m) Fogleman (1954) Cat intravenous > 1750 (m and f) Shaffer et al. (
a m = males; f = females
Amitrole applied in water formulations to the unabraded skin of
rabbits for 24 h caused a very mild and reversible erythema (Elsea, 1954). Intraperitoneal administration in mice and rats and
intravenous administration in either mice, dogs or cats produced no signs of toxicity (Fogleman, 1954).
No toxicity was observed in rats after inhalation of amitrole following either head-only (approximately 50 µg/litre) or whole-body (approximately 25 µg/litre) exposure for a period of one hour
(MacDonald & Pullinger, 1976).
7.2 Short-term exposure
7.2.1 Oral 7.2.1.1 Dietary
When groups of Carworth Farm male and female rats (five per group) were administered amitrole in the diet at dose levels of 0, 100, 1000 or 10 000 mg/kg for 63 days, reduced body weight gain for both males and females was observed at the two highest dose levels, this being accompanied by reduced food consumption. There were no deaths or clinical signs of toxicity. Histo-pathological examination of the liver, kidney, portions of the small intestine, spleen, and testes revealed increased vacuolization of the liver cells around the central vein and steatosis at the two highest dose levels (Fogleman, 1954).
Mayberry (1968) studied the effects on the thyroid of a dietary level of 1000 mg amitrole/kg in rats during 83 days and compared this to the effects of other anti-thyroid chemicals,
propylthiouracil (1000 mg/kg) and potassium chlorate (1000 mg/kg).
At various time intervals, starting with 3 days, the relative thyroid weight and total iodine content of the thyroid were measured. An increase in thyroid weight and a decrease of total iodine were observed within the amitrole group; this was already observable after 3 days and becoming more pronounced during the course of the experiment. The effects of propylthiouracil were comparable, but, in the case of potassium chlorate, the weight increase was less pronounced and the iodine content was lower than with amitrole. In another experiment, uptake and release of
radioactive iodine was measured after a single 131I injection to a control group of rats and to a group simultaneously receiving 10 mg amitrole subcutaneously. Animals were killed after 1, 2, 3, 4, 5 or 6 days. The t´ for 131I in the thyroid was 4.9 and 1.3 days for control and amitrole-treated animals, respectively. Separation by paper chromatography of 131I-containing thyroid fractions showed that levels of monoiodotyrosine (MIT) were increased, diiodotyrosine (DIT) were constant and T3 and T4 were markedly reduced. The
author concluded that amitrole not only interferes with organification of iodine but also inhibits the coupling of iodotyrosines to form iodothyronines (Mayberry, 1968).
The effect of amitrole on thyroid hormones was studied by giving groups of male Sprague-Dawley rats (20 per dose level) amitrole (94.6% pure) in the diet at dose levels of 0, 30, 100 or 300 mg/kg during 28 days, followed by a recovery period of 28 days (Babish, 1977). The assessment of thyroid function was performed by measuring T3 and T4 in blood by a radioimmuno-assay. On days 3, 7, 14, 21 and 28 of the treatment period and on days 19, 21 and 28 of the post-treatment period, blood samples were collected from two animals which were then killed for autopsy. There were no adverse effects on the general health of the rats during the treatment or post-treatment period. Consumption of 100 or 300 mg amitrole/kg diet
