From: E.J. Wright, M.C. Webb and E. Highley, ed., Stored grain in Australia 2003. Proceedings of the Australian Postharvest Technical Conference, Canberra, 25–27 June 2003. CSIRO Stored Grain Research Laboratory, Canberra.
Toxicological and regulatory information supporting the registration of VAPORMATETM as a grain fumigant for farm storages
V.S. Haritos,1 K.A. Damcevski and G. Dojchinov Stored Grain Research Laboratory, CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601
Abstract. VAPORMATETM, a BOC Ltd product composed of ethyl formate 16.7% by weight in carbon dioxide, is cur- rently under evaluation as a grain fumigant for farm silos. The research has aimed at devising an application system for the mixture that will maximise the concentration of ethyl formate in the bin but minimise the loss to grain sorption. Also, we are investigating the optimal amount and exposure period to achieve high mortality of a range of tolerant stored grain pests. VAPORMATETM appears very favourable in terms of data to support registration of the product, such as mamma- lian toxicology, residue analysis methods, low risk of environmental impacts, and occupational health and safety aspects of ethyl formate, and its natural occurrence. Ethyl formate is far less acutely toxic to mammals, including humans, than dichlorvos and phosphine, two of the main treatments that are currently available for farm storages. Ethyl formate also has a higher occupational exposure limit (100 ppm versus 0.3 ppm for phosphine and 0.1 ppm for dichlorvos). Ethyl for- mate is a natural component of many foods and the hydrolysis products formed in the human body are formic acid and ethanol which are identical with products formed endogenously. Residue methods for ethyl formate on grains have been established. The high sorption of ethyl formate and its consequent breakdown mean that little is left to ventilate from a bin at the end of fumigation. Nevertheless, ethyl formate has a short half-life in the atmosphere. Ethyl formate poses a low risk of environmental damage, even in the unlikely case of a spill of liquid into waterways or soil. Ahead in the project, we expect to establish a VAPORMATETM application rate and apply for experimental-use permits to enable field trials to be conducted. Also, we are investigating the potential of insects to form resistance to the fumigant.
Introduction • Rapid sorption to grain—ethyl formate has a great affinity for grain and grain has a large capacity to sorb New treatments for stored grains are continuously needed ethyl formate. This reduces the amount of fumigant because the currently used fumigants and protectants are available to control insects, as it is the vapour phase of under threat from either insect resistance, or loss of regis- the fumigant that is thought to be most effective. tration or manufacturer support. On-farm or small-scale • Application system for farm storages—we have tested storers are at a greater disadvantage as phosphine is the ‘forced-flow fumigation’ with VAPORMATETM in a only fumigant in routine use available to them. Phosphine model silo system. In this system, the fumigant is is used on approximately 80% of Australian grain and blown through the grain, using an aeration/drying fan throughout the storage chain and there is a strong need to at a relatively fast flow rate, until the air in the bin is provide an alternative, especially for on-farm use in a replaced by fumigant. The forced flow reduces the res- phosphine resistance management strategy. The avail- idence time of fumigant near grain, thus reducing sorp- ability of ethyl formate as a disinfestant would also benefit tion and leading to an even distribution of fumigant. the farm-scale grain storer as an alternative to dichlorvos • High concentrations of fumigant required—to achieve as a fast-acting treatment. a high level of insect control, relatively high concen- A mixture of ethyl formate in carbon dioxide (16.7% trations of ethyl formate are required. The insect mor- by weight), called VAPORMATETM, has been developed talities obtained for a variety of application rates of by BOC Ltd as a postharvest treatment for horticulture VAPORMATETM are described by Damcevski et al. and stored grains. The main challenges that we see for (these proceedings). VAPORMATETM becoming a fumigant for stored grains • Registration—regulatory approval for a grain fumigant include: still remains one of the largest challenges for any new treatment. While ethyl formate is not a new fumigant 1 Corresponding author:
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trol in dried fruit, it has not been evaluated by a pesti- 16% and presents a considerable flammability risk when cide regulatory agency to modern standards. A modern used as a grain fumigant. The cylinderised formulation of grain treatment has to be safe for consumers, applica- VAPORMATETM provides a delivery system for ethyl tors, the environment and the commodity, and should formate which substantially reduces handling and poten- not cause issues in the trade of grain. The emphasis of tial exposure to the applicator. No handling of liquid ethyl this paper is on whether ethyl formate can meet these formate is carried out with this formulation. expectations and obtain Australian registration as an insect treatment for stored grains. Although VAPORMATETM presents many challenges, Acute toxicology of ethyl formate it has some distinct advantages that are encouraging for its registration. The main active ingredient, ethyl formate, has The acute toxicity of ethyl formate has been tested in a low mammalian toxicity but rapidly kills grain insect number of animal species following oral and inhalation pests. Ethyl formate occurs naturally in foods and when exposure, as summarised in Table 1. The acute toxicity of applied to grain, particularly warm and wetter grain, the ethyl formate is low by the oral route with an LD50 (lethal residues break down rapidly to natural products. The dose to kill 50% of experimental animals) of around 2 g/ VAPORMATETM formulation has many advantages over kg bodyweight (Table 1). Although the toxicity data are neat liquid ethyl formate. We have shown previously that not as comprehensive for the inhalation route as that for the addition of carbon dioxide from 5% volume/volume the oral route of exposure, it is clear that ethyl formate is and above enhances the toxicity of ethyl formate toward much less acutely toxic than dichlorvos or phosphine, as adults and most juvenile stages of stored product insects demonstrated in Table 2. The 4-hour LC50 (concentration (unpublished data). This means we can reduce the amount of vapour that results in the death of 50% of test rats after of fumigant needed for an efficacious treatment, resulting 4 hours of inhalation exposure) is a measure for in lower residues in the grain after fumigation. comparing the acute toxicity of volatile chemicals. Using The flammability risk of ethyl formate is virtually this measure of toxicity, dichlorvos is approximately 100- eliminated by the presence of high concentrations of fold more acutely toxic and phosphine 1000-fold more carbon dioxide in the VAPORMATETM formulation (Ryan toxic than ethyl formate as shown in Table 2. The much and Bishop, these proceedings). Pure ethyl formate has a lower acute mammalian toxicity of ethyl formate is lower flammability limit of 2.8% and an upper level of reflected in its occupational exposure limit compared with
Table 1. Acute toxicology of ethyl formate in experimental animals.
Species Route Lethal dose or concentrationa Reference
Rat Oral LD50 1850 mg/kg NTIS (1974)
Rabbit Oral LD50 2072 mg/kg Anon. (1978)
Rat Oral LD50 4290 mg/kg Smyth et al. (1954)
Guinea pig Oral LD50 1110 mg/kg NTIS (1974) Rat Inhaled No deaths 24 g/m3, 5 min Smyth et al. (1954) 3 Rat Inhaled LC83 24 g/m , 4 h Smyth et al. (1954) 3 Cat Inhaled LC100 32 g/m , 90 min Von Oettingen (1959) 3 Dog Inhaled LC100 30 g/m , 4 h Anon. (1978) a LD50 (or LC50) = lethal dose (or concentration) that kills 50% of test organisms.
Table 2. Comparison of toxicity and regulatory data for ethyl formate, dichlorvos and phosphine.
Endpoint Ethyl formate Dichlorvos Phosphine a b c d 4 h rat LC50 inhalation Approx. 20 0.2 0.015 toxicity (g/m3) Australian occupational 100 0.1 0.3 exposure limit (ppm)e Acceptable daily intake 3f 0.001g Not set (mg/kg/day)
a LC50 = lethal dose (or concentration) that kills 50% of test organisms. b extrapolated from Smyth et al. (1954); cWHO (1989); dWHO (1988); eNOHSC (1995); fJEFCA (1997); g TGA (2003).
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Toxicological and regulatory information supporting registration of VAPORMATE™ the other two treatments (Table 2). Ethyl formate has the Metabolism and degradation of highest occupational exposure limit of all the current commodity fumigants and of the candidates being consid- ethyl formate ered as alternatives to phosphine or methyl bromide, The most common pathway in the breakdown of ethyl excluding modified atmospheres. formate is hydrolysis to formic acid and ethanol. The rate As part of the registration package for VAPOR- of hydrolysis can be catalysed in acidic and basic condi- MATETM, a comprehensive evaluation of ethyl formate tions. Also, enzymes such as esterases that are present in toxicology is required. A toxicological review has been plants and animals facilitate the breakdown. Formic acid conducted for ethyl formate (Haritos 2000) and much of and ethanol themselves are further metabolised by many the data that can support registration are available in the organisms to be incorporated into cellular components or public domain. used as sources of energy. In water, ethyl formate undergoes slow hydrolysis to Regulatory levels relating to intake of formic acid and ethanol and this process is also thought to occur in grain, but more rapidly. Ethyl formate is rapidly ethyl formate hydrolysed in the body, based on studies that have been conducted with ethyl acetate. This process is catalysed by Humans are constantly exposed to naturally occurring carboxylesterases and other hydrolases (Longland et al. ethyl formate in a wide range of foods but particularly in 1977; Leinweber 1987) in the body but hydrolysis may fruits, in processed foods such as bread and beer, and in also be extensive in gastric or pancreatic juices (Longland cheese and mussels (Desmarchelier 1999). et al. 1977). Hydrolysis of ethyl formate yields ethanol Ethyl formate is considered to have low toxicity to and formate; both chemicals are endogenously produced mammals when exposed chronically through the diet. in humans as part of normal metabolism. The amount of a substance that can be consumed every Formic acid itself can undergo several pathways in the day and cause no adverse effect is referred to as the body: it can be oxidised to carbon dioxide and water, it acceptable daily intake (ADI) by regulatory authorities. can be excreted unchanged or partly metabolised in the Ethyl formate has an ADI of 3 mg/kg body weight/day, tissues, or it can be incorporated into proteins, lipids and set by an expert panel of the World Health Organization nucleic acids and distributed throughout the body (Liesi- (WHO) during its periodic reviews of food additives vuori and Savolainen 1991). The acute and subchronic (JEFCA 1997). This means a 60 kg person can safely toxicity of formic acid has been reviewed in FASEB consume 180 mg ethyl formate per day for a lifetime (1976) and Haritos (2000). Formic acid holds GRAS without any adverse effects. By comparison, dichlorvos status for selected applications to foods in the USA. has an ADI established by the Australian Therapeutic Goods Administration of 0.001 mg/kg/day (TGA 2003); Ethanol is endogenously produced as a metabolite in that is 3000-fold lower than the ADI for ethyl formate, small quantities in the human body. As a metabolite from reflecting its higher toxicity when administered by the low levels of dietary ethyl formate, small quantities of oral route. Phosphine has not had an ADI set either ethanol are not considered to be of toxicological signifi- locally or internationally. cance (JEFCA 1997). An international expert group recently evaluated the intake of ethyl formate to determine the safety margin Natural occurrence of ethyl formate between intake and the ADI (Munro and Kennepohl 2001). The estimated daily intake of ethyl formate from food A very positive fact about ethyl formate is that it is present flavourings was below the threshold amount (<1.8 mg), i.e. naturally in the diet, with grains themselves having a less than 1% of the ADI, and therefore concluded this level reasonable amount of ethyl formate present—the highest was ‘not to be of safety concern’. levels among cereals were found in barley at 1 mg/kg Ethyl formate holds ‘generally regarded as safe’ (Desmarchelier et al. 1999). It is also found in a whole (GRAS) status in the United States of America (USA) for variety of plant and animal products, such as fruits and defined uses in flavouring food. In Australia, the Australian vegetables, beer, wine and spirits, tuna, meat, mussels, Pesticides and Veterinary Medicines Authority (APVMA) cheese and bread (Desmarchelier 1999). Formic acid is has listed ethyl formate in ‘Table 5’, a grouping of chemi- also naturally occurring and there are considerable levels cals for which a maximum residue limit (MRL) is not in cereal grains, in the region of 300 mg/kg (Desmarche- required or for chemicals that are not expected to contami- lier et al. 1999). These high natural levels would over- nate food. There is a current Food Standards Australia and whelm the formic acid arising from ethyl formate New Zealand (FSANZ) MRL of 1 mg/kg for ethyl formate fumigation of grain and its subsequent breakdown to applied to dried fruit. formic acid. Formic acid is also found in fruits and vegeta- bles, wine and spirits, sardines and cheese (Desmarchelier 1999).
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Residue analysis tested during planned field trialling of VAPORMATETM. Interferometers, which operate on the principle of the The widespread natural occurrence of ethyl formate leads difference in refractive index of a vapour/air mixture to the question as to whether its residues in grain, arising compared to air alone, will be trialled. Infra-red sensors from fumigation, can be measured. Desmarchelier et al. and photoionisation detectors will also be evaluated. (1999) developed a residue method for ethyl formate which utilises the partitioning between a propanol/water soaking solution, which covers the grain, and the head- Environmental impacts space above in a sealed container. The detection limit for Venting of fumigants to the atmosphere after fumigation is ethyl formate at approximately 0.1 mg/kg is reasonable, becoming increasingly restricted due to potential environ- especially as the analysis suffers interference from many mental impacts or occupational or unacceptable bystander other natural esters present in grain and the solvents used exposure (Wilson 2003, pers. comm.). One advantage of for analysis. The aqueous acetone soaking solution used in the high level of sorption and breakdown of ethyl formate an established multi-residue method for measuring on grain during fumigation is that there is little remaining protectant and fumigant residues interferes with the fumigant after the fumigation period, be it a 3 or 24 hour measurement of ethyl formate and therefore a different fumigation, to vent to the atmosphere. Venting the soaking solution is needed for ethyl formate grain residues quantity remaining is unlikely to cause any issues with (Desmarchelier et al. 1999). Methanol was found to be emission controls or cause violation of the occupational suitable as a soaking solution for grain analysis except exposure levels. Nevertheless, the small amount of ethyl where measurement of low levels of ethyl formate are formate that may be released, or in the event of accidental required as methyl acetate can be generated by reaction release, is acted upon by oxygen free radicals where the between organic acids and the solvent. Methyl acetate co- estimated half-life of ethyl formate in the atmosphere is chromatographs with ethyl formate on several gas chro- 16 days at 25°C (Atkinson 1989). matography columns. For gases and volatile liquids such as ethyl formate, it is In general, ethyl formate hydrolyses on grain. This can unlikely that sufficient quantities would find their way into be a rapid process on warm, moist grain but much slower soil and waterways due to vaporisation of the fumigant. In on cold and dry grain (Y. Ren, pers. comm.). A detailed the unlikely case of a spill of VAPORMATETM where liquid examination of residue breakdown has been conducted ethyl formate may find its way to waterways, it is not under various temperature and grain moisture conditions. expected to have much impact on the environment due to its This information gives an indication of potential with- volatility, low toxicity and hydrolysis to ethanol and formic holding periods which would be required to allow ethyl acid. Ethyl formate is moderately soluble in water (88.3 g/L formate residues to decay to natural levels in grain. Also, at 25°C) and undergoes slow hydrolysis, firstly to ethanol the efficacy of ethyl formate towards stored product and formic acid in water around neutral pH. Under basic insects decreases at temperatures below 15°C, and conditions, the rate of hydrolysis increases rapidly. Ethyl residues take longer to break down at these temperatures. formate is hydrolysed in plants and animals and catalysed Therefore, a minimum temperature for treatment of by a variety of esterases, and is therefore unlikely to accu- infested grain will be considered for addition to the mulate in biota in the environment. Furthermore, the log VAPORMATETM agricultural chemical label. Kow (log octanol–water partition coefficient) for ethyl formate is 0.23 (Hansch et al. 1968) which is well below the fat solubility where potential for bioaccumulation is likely. Occupational health and safety Ethyl formate has low toxicity to fish and to non-target The current occupational exposure standard in Australia invertebrates and therefore is not expected to cause environ- for ethyl formate is 100 ppm (303 mg/m3) (NOHSC 1995) mental impacts as a result of accidental release. The toxicity which is considerably higher than for other fumigants of of ethyl formate to trout has been investigated as a standard commodities. However, ethyl formate concentrations waterborne exposure bioassay. The lethal concentration to inside silos under fumigation could be dangerous. Protec- kill 50% of tested trout during a 96 h exposure (96 h LC50) tive practices and equipment should therefore be available was estimated at 230 mg ethyl formate per litre of water for ethyl formate fumigations, including barriers to entry (McKim et al. 1985), which is much less toxic than dichlo- of the fumigation space and the wearing of gloves, rvos, where 96 h LC50 values range from 0.2 mg/L for overalls and goggles to reduce exposure to ethyl formate cutthroat and lake trout to 12 mg/L for fathead minnow in the case of accidental release of VAPORMATETM. (WHO 1989). Cartridge-type gas masks are available to protect applica- tors from ethyl formate in situations where the occupa- Future plans for the project tional exposure limit is exceeded, however these should only be used as a last resort and not in place of safe fumi- Once an application rate can be determined for VAPOR- gation practice. A range of monitoring equipment, suited MATETM from a current insect efficacy study to either fumigation or ambient concentrations, will be (Damcevski et al., these proceedings), field trialling of
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Toxicological and regulatory information supporting registration of VAPORMATE™
VAPORMATETM in farm storages will be conducted Journal of Physical and Chemical Reference Data, Mono- under experimental permit. Successful field trialling of graph No. 1, 166. the application method for VAPORMATETM should Desmarchelier, J.M. 1999. Ethyl formate and formic acid: occur- demonstrate its potential to replace dichlorvos or phos- rence and environmental fate. Postharvest News and Infor- phine for insect control in farm storages. VAPOR- mation, 10, 7N–12N. MATETM compares very favourably to these other two Desmarchelier, J.M., Allen, S.E., Ren, Y., Moss, R. and Vu, L.T. treatments on the basis of its relatively low acute toxicity 1998. Commercial-scale trials on the application of ethyl by inhalation, very low chronic oral toxicity, high occu- formate, carbonyl sulfide and carbon disulfide to wheat. CSIRO Entomology Technical Report No. 75. Canberra, pational exposure limits, and low impact on the environ- CSIRO, 58 p. ment. Desmarchelier, J.M., Johnston, F.M. and Vu, L.T. 1999. Ethyl The development of resistance to pesticides in stored formate, formic acid and ethanol in air, wheat, barley and grain insects is inevitable but understanding the likelihood sultanas: analysis of natural levels and fumigant residues. and speed of this event is an important aspect of devel- Pesticide Science, 55, 815–824. oping a new grain treatment. As part of the VAPOR- FASEB (Federation of American Societies for Experimental MATETM project, Gaye Weller from the Stored Grain Biology) 1976. Evaluation of the health aspects of formic Research Laboratory has begun inducing mutations in the acid, sodium formate, and ethyl formate as food ingredients. flour beetle (Tribolium castaneum) and challenging these Report prepared for Food and Drug Administration, contract insects with a discriminating dose of ethyl formate to no. FDA 223-75-2004, 21 p. determine whether any of the mutants have acquired a Hansch, C., Quinlan, J.E. and Lawrence, G.L. 1968. Linear free- tolerance of the fumigant. The likelihood of inducing energy relationships between partition coefficients and the resistance to ethyl formate will be compared with aqueous solubility of organic liquids. Journal of Organic inducing resistance to phosphine. Chemistry, 33, 347. Before registering VAPORMATETM, discussions Haritos, V.S. 2000. Review of toxicological studies of carbonyl between stakeholders and the registrant will need to be sulfide, ethyl formate and carbon disulfide. CSIRO Entomol- ogy Technical Report No. 87, August 2000, 36 p. conducted on aspects such as the ‘Table 5’ status of ethyl JEFCA (Joint FAO/WHO Expert Committee on Food Additives) formate. This is a regulatory position that ethyl formate 1997. Evaluation of certain food additives and contaminants. holds with the APVMA where ‘no maximum residue limit Forty-sixth report of the joint FAO/WHO Expert Committee is required’. Suitable withholding periods may need to be on Food Additives. WHO Technical Report Series, 868, 21– introduced to maintain the Table 5 status of ethyl formate 23. which will ensure residues arising from treatment reduce Leinweber, F.-J. 1987. Possible physiological roles of carboxylic to natural levels. In addition, the necessity for further acid hydrolases. Drug Metabolism Reviews, 18, 379–439. downstream processing studies to be conducted with Liesivuori, J. and Savolainen, H. 1991. Methanol and formic VAPORMATETM-treated wheat will need to be discussed acid toxicity: biochemical mechanisms. Pharmacology & with the relevant groups once an application rate is deter- Toxicology, 69, 157–163. mined. Wheat treated with ethyl formate applied in water Longland, R.C., Shilling, W.H. and Gangolli, S.D. 1977. The at the rate of 90 g ethyl formate per tonne grain in earlier hydrolysis of flavouring esters by artificial gastrointestinal field trials was assessed in a milling and baking trial. The juices and rat tissue preparations. Toxicology, 8, 197–204. ethyl formate treatment did not cause detrimental effects McKim, J., Schmieder, P. and Veith, G. 1985. Absorption to the milling and baking qualities of wheat nor were ethyl dynamics of organic chemical transport across trout gills formate residues detected above background levels in related to octanol–water partition coefficients. Toxicology whole or processed fractions (Desmarchelier et al. 1998). and Applied Pharmacology, 77, 1–10. Munro, I.C. and Kennepohl, E. 2001. Comparison of estimated daily per capita intakes of flavouring substances with no- Acknowledgments observed-effect levels from animal studies. Food and Chemi- cal Toxicology, 39, 331–354. The Grains Research and Development Corporation and NOHSC (National Occupational Health and Safety Commis- Partners of Stored Grain Agreement are thanked for their sion) 1995. Exposure standards for atmospheric contami- financial support of CSE0009 and ethyl formate projects. nants in the occupational environment: guidance note on the James Darby and Gaye Weller are thanked for their assis- interpretation of exposure standards for atmospheric con- tance in the project and BOC Ltd for support of VAPOR- taminants in the occupational environment (NOHSC: 3008). MATETM. Canberra, NOHSC, 3rd edition. NTIS (National Technical Information Service) 1974. Scientific literature reviews on generally regarded as safe (GRAS) food References ingredients—formic acid and derivatives. Report prepared for United States Food and Drug Administration by Tracor-Jitco Anon. 1978. Ethyl formate. Monographs on fragrance raw mate- Inc, No. FDABF-GRAS-204 (PB-228 558), 74 p. rials. Food and Chemical Toxicology, 16, 737–739. Smyth, H.F. Jr, Carpenter C.P., Weil, C.S. and Pozzani, U.C. Atkinson, R. 1989. Kinetics and mechanism of the gas-phase 1954. Range-finding toxicity data. List V. AMA Archives of reactions of the hydroxyl radical with organic compounds. Industrial Hygiene and Occupational Medicine, 10, 61–68.
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TGA (Therapeutic Goods Administration) 2003. Acceptable WHO (World Health Organization) 1988. Phosphine and daily intakes for agricultural and veterinary chemicals. Com- selected metal phosphides. Environmental Health Criteria monwealth Department of Health and Ageing, 39 p. No. 73. Geneva, WHO, 64–67. Von Oettingen, W.F. 1959. The aliphatic acids and their esters— WHO (World Health Organization) 1989. Dichlorvos. Environ- toxicity and potential dangers. A.M.A. Archives of Industrial mental Health Criteria No. 79. Geneva, WHO, 157 p. Health, 20, 517–531.
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