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doi:10.1016/j.biosystemseng.2005.04.006PA—Precision Agriculture

Biosystems Engineering (2005) 91 (4), 413–419

An Innovative Approach: the Use of Di-nitrogen Tetroxide for Soil Fumigation

Z. Tadmor1; K. Sachs2; I. Chet3; Y. Spiegel4; I. Ravina5; G. Manor5; S. Yannai2

1Department of Chemical Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel2Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel;

e-mail of corresponding author: syannai@tx.technion.ac.il3Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel

4Department of Nematology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel5Department of Agricultural Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel

(Received 17 June ; accepted in revised form 15 April 2005; Published online 24 June 2005)

The effectiveness of di-nitrogen tetroxide (DNTO) as a substitute for methyl bromide in controlling soil-bornenematodes, fungi and bacteria was evaluated. Application of DNTO to soil packed into columns resulted in areduction of the bacterial population by three orders of magnitude within 1 h, and a complete elimination after2 h. The same treatment resulted in the destruction of all fungi and nematodes within 10min. The mosteffective treatment (30min for bacteria elimination and 10min for nematode and fungi elimination) wasachieved in soil with a low moisture level (4%), at a pumping rate of 0�1ml/min DNTO. In a single microplottrial, using 300 g/m2 DNTO, a significant reduction of nematodes was observed. Lack of phytotoxicity ofDNTO was demonstrated using tomato plants. In addition, at the above-mentioned soil moisture and DNTOlevels, the DNTO is readily converted into nitrate. Hence, the use of DNTO can supply a considerable part ofthe nitrogen fertiliser requirement of the treated crops and only a small fraction of it is liberated into theatmosphere.r 2005 Silsoe Research Institute. All rights reserved

Published by Elsevier Ltd

1. Introduction

Current intensive agricultural practice employs fumi-gation of the soil with methyl bromide (MeBr) beforeplanting a wide variety of crops, in order to destroy soil-borne plant pests and pathogens such as nematodes,bacteria, fungi and weeds. Moreover, agriculture pro-duce and storage facilities are also sterilised by MeBrfumigation before transport and export. However, mostof the MeBr injected into the soil is ultimately releasedinto the atmosphere (Jin et al., 1995; Singh andKanakidou, 1993; Yagi et al., 1993), causing unaccep-table environmental damage to the ozone layer. For thisreason, under the Montreal Protocol on Substances thatDeplete the Ozone Layer (2000), MeBr was formallydefined as a chemical that contributes to the depletion ofthe Earth’s ozone layer. Accordingly, the manufactureand import of MeBr will be phased out in developedcountries (which consume virtually all the methylbromide); its use is now banned. In developing

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countries, consumption was frozen in 2002 at 1995–98average levels, and will be followed by a 20% reductionin 2005 and complete phase-out by 2015 (Zurer, 1993).

According to the US Department of Agriculture(USDA), there is no known single alternative fumigantchemical or other technology that can readily substitutefor MeBr in efficiency, low cost, ease of use, wideavailability, worker safety and environmental safetybelow the ozone layer. Research by the USDA indicatesthat multiple alternative control measures will berequired to replace the many essential uses of MeBr.For pre-planting uses, such measures include combina-tions of fungicides, herbicides and insecticides. Otherfumigants and non-chemical alternatives, includingcultural changes in cropping systems, resistant cropsand biological control, are also being employed. Forsome quarantine and export applications, effectivealternatives include irradiation, heat, cold and con-trolled atmosphere treatments. The effective applicationof a single alternative control measure or specific

r 2005 Silsoe Research Institute. All rights reserved

Published by Elsevier Ltd

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combinations is limited to specific crops, the reasonbeing the varying physiological requirements of differentplants, as well as variations in target pests, soil types,climates and state or local regulations.

As a soil fumigant, MeBr is injected into the soil at adepth of 300–400mm before the crop is planted. Thiseffectively sterilises the soil, destroying the vast majorityof detrimental soil organisms. Immediately after theMeBr is injected, the soil is covered with plastic films,which slow down the diffusion of the MeBr from the soilinto the atmosphere. Additional MeBr is emitted intothe atmosphere when the plastic films are removed after24 to 72 h. About 50–95% of the MeBr injected into thesoil can eventually enter the atmosphere. The currentannual world consumption of MeBr is about 67 000 t, ofwhich 80% is used for soil fumigation.

The purpose of this investigation was to explore theusefulness of di-nitrogen tetroxide (DNTO) as analternative soil fumigant for MeBr, as well as for thequarantine treatment of produce and for sterilisation ofbuildings. Moreover, unlike MeBr, this material canalso be used to fumigate the soil of vineyards andbanana groves, because it was found in our experimentsto be non-phytotoxic.

When nitrogen dioxide (NO2), a brown gas at atemperature of over 21 1C, is subjected to pressure, itliquefies and forms the colourless dimer DNTO (N2O4).The physical properties of the strongly oxidising DNTOare available in the literature (Matheson Tri-Gas, 2003).It is an intermediate in the oxidation of ammonia tonitric acid, and the main commercial use is as anoxidising agent for liquid rocket propellants. In addi-tion, DNTO is used as a nitrating agent, catalyst,inhibitor in polymerisation of acrylates and as asterilising agent for surgical cloth. It is expected that,in agricultural applications, much of the DNTO willremain in the soil and actually provide usefulnitrate to the plant roots. Some will, however, ultimatelyleak into the atmosphere; but, this leakage will beinsignificantly small compared to the nitrogen oxidesthat are added to the atmosphere from automobiles,power stations and natural sources. Also, the effect onthe ozone layer is not nearly as severe as for MeBr(Fahey, 2002). Hence, DNTO offers a viable substitutefor MeBr.

2. Experimental details

2.1. Di-nitrogen tetroxide application methods

In order to test the properties of DNTO as a fumigantat a small scale, the following two sets of laboratoryexperiments were performed: (a) DNTO was pumped

into the bottom of stainless-steel columns, packed withsoil infested with nematodes, after which small soilsamples were removed at different time intervals and thepopulation density of the pathogens was assessed (seeSection 2.3), and (b) DNTO was injected into glasscylinders filled with infested soil and the same procedurewas followed (see Section 2.4). All experiments withcolumns and cylinders were performed in the labora-tory, at room temperature.

2.2. Infestation

The nematodes used for infestation were the root-knot nematode Meloidogyne javanica, propagated ontomato (Lycopersicon esculentum Mill cv. Hosen Eilon)in the greenhouse. Eggs were separated from egg masseswith sodium hypochlorite (0�5%, for 1min) and hatchedin twice diluted 0�1M phosphate-buffered saline, pH 7�2(PBS/2), to obtain infective second-stage juveniles (J2).The J2 were used for the experiments with the artificiallyinoculated soils.

The bacteria and fungi investigated were the naturallyoccurring species found in the tested soil, withoutinfestation.

2.3. Soil, pumping arrangement and procedure

The soil used in the column experiments was a moist‘brown Mediterranean clay loam.’ The soil was sievedthrough an American Society for Testing and Materials(ASTM) No.20 sieve (850 mm sieve size aperture), air-dried at room temperature and atmospheric pressure.Portions of 2�5 kg of this soil were packed into 316stainless-steel columns of 75mm internal diameter,510mm length and 2�6 l volume. Disc-shaped end capsand Teflon gaskets were secured with threaded stainless-steel bolts. To support the soil packed into the column, a3mm mesh stainless-steel screen was welded to one endof the cylinder and 80 g of 5mm diameter glass beadswas placed on the screen. The DNTO used wasmanufactured by the Vicksburg Chemical Co. USA,packaged in 1�8 l steel cylinders (containing 2�6 kgDNTO) equipped with stainless-steel Swagelok fittings.

The DNTO in liquid phase was pumped into thebottom of the vertical columns through Teflon tubings.A corrosion-resistant stainless-steel FMI Lab Pump Jr.,model RH (Fluid Metering Inc., Syosset, Long Island,New York) was used. The pressure of the DNTO in thecolumns was approximately 1 atmosphere. Exposuretimes were measured from the first appearance ofbubbles in a vapour trap (5% NaOH) connected tothe top of the soil column.

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2.4. Injection arrangement and procedure

For the injection experiments, seven glass cylinders(70mm internal diameter, 427mm length) were used.The columns, as in the pumping experiments, werepacked with clay loam soil, inoculated with parasiticnematodes and adjusted to a moisture content of 6%.The DNTO was injected with a 220mm long stainless-steel needle through an 8mm diameter glass tubeinserted 200mm deep into the soil.

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2.5. Microplot field tests

Seventeen 1m2 microplots (1000 l containers buried inan open field) were prepared for the experiments. Thepreparation included infestation of the soil with ca.2500M. javanica nematodes per kg soil and subse-quently turning the soil over to prepare the plots forfumigation. At this stage it was visually observed thatthe deeper soil layers were still wet. Since this wasundesirable, a last effort was made to induce fasterdrying by digging ditches around the plots. Then, 12 outof 17 plots were covered with polyethylene sheets, andthe plots were fumigated by injections of DNTO,through the plastic sheets, 10–15 cm deep into the soil.Into each plot, 7 or 8 injections were made at pre-marked locations. Four plots were fumigated with 150 g/m2 (i.e., three times the standard MeBr dosage).Another plot was fumigated with 300mg/m2 (six timesthe MeBr dosage). In addition, reference-control plotswere employed; four were covered with polyethylenesheets and four were left uncovered. One week later,tomato seedlings were planted into each of the micro-plots. To evaluate the nematode inoculation level at zerotime, a soil sample was removed from each plot andtransferred to a 750ml pot, into which a tomato seedlingwas planted. The pots were kept in a temperature-controlled greenhouse. Thirty days later, the gallingindices (on a 1 to 5 scale) were recorded. Eight weekslater, the plants in the microplots were removed toevaluate the damage caused by nematodes. Nematocidalactivity was evaluated by indexing the galls on the roots.

0

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CFU

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Time, h

Fig. 1. Bacteria (~) and fungi (m) counts in soil columns aftertreatment with di-nitrogen tetroxide (DNTO) at 0�5 ml/min as a

function of treatment time; CFU, colony-forming units

3. Results

3.1. Pumping into columns

In the initial experiments, a flow rate of 0�5ml/minwas used. Samples, each weighing 20 g, were removedfrom the middle layer of the soil after 0�5, 1, 2 and 4 hand compared for bacteria and fungi counts with theuntreated samples. The bacteria included gram-negative

species, such as Pseudomonas spp., and spore-forminggram-positive bacteria such as Bacillus spp., as well asMicrococcus spp. The fungi included Penicillium spp.,Aspergillus spp., Rhizoctonia solan, Sclerotium rolfsii,Trichoderma spp. and Fusarium spp. The results areshown in Fig. 1.

Clearly, a treatment duration of 1 h reduced theconcentration of bacteria by three orders of magnitude,and it took 2 h to destroy all of them.

Fungi were completely eliminated within 30min. Nocounting was made for the population density of thenematodes in soil samples tested in the first set ofexperiments, since these pathogens are known to bemore sensitive to chemical agents than bacteria andfungi, and it was expected that these would also bedestroyed. The same sequence of sensitivities is alsoevident from the results of our later experiments.

A second set of experiments was carried out at a lowerDNTO pumping rate of 0�1ml/min and shorter DNTOexposure times. Figure 2 shows that even with this lowerdosage a complete elimination of all fungi was achievedafter 20min. However, the results were much lesssatisfactory in the case of bacteria. As expected, in thesurviving bacteria we found a high proportion ofBacillus spp., which are very resistant, spore-formingbacteria.

The effect of soil moisture concentration was testedusing samples with 4%, 8% or 14% moisture. Themaximum exposure time was, again, 30min. The resultsare given in Fig. 3 for the bacteria count, and in Fig. 4

for the fungi count. The results indicate a completeelimination of the bacterial population within 30min at4% and 8% moisture levels, and a considerably lesserdecrease in bacteria at the 14% level. As can be seen inFig. 4, the fungi were destroyed in 10min at the lowestmoisture level and in 20min at the two higher moisturelevels used.

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atod

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soil]

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atod

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Fig. 5. Nematode counts per 100 g soil sample treated with di-nitrogen tetroxide (DNTO) at 0�1 ml/min flow rate, at twomoisture concentrations, 4% moisture (~) and 8% moisture(m): (a) parasitic nematodes; and (b) saprophytic nematodes

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Fig. 2. Bacteria (~) and fungi (m) counts in soil columns aftertreatment with di-nitrogen tetroxide (DNTO) at 0�1 ml/min as a

function of treatment time; CFU, colony-forming units

0 10 20 30Time, min

0

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106

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CFU

Fig. 3. Bacteria counts in soil columns after treatment with di-nitrogen tetroxide (DNTO) at 0�1 ml/min as a function oftreatment time, at different soil moisture levels: ~, 4% moisture;m, 8% moisture; ’, 14% moisture; CFU, colony-forming units

0 10 20 30 Time, min

0

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104

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CF

U

Fig. 4. Fungi counts in soil columns after treatment with di-nitrogen tetroxide (DNTO) at 0�1 ml/min as a function oftreatment time, at different soil moisture levels: ~, 4% moisture;m, 8% moisture; ’, 14% moisture; CFU, colony-forming units

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Finally, nematodes in the treated soil sampleswere tested at 4% and 8% moisture. Results are shownin Fig. 5, indicating that both plant-parasitic andsaprophytic nematodes (living on decaying organicmatter) were controlled within 10min of exposure toDNTO, under the conditions described for the latterthree experiments.

3.2. Injection into glass cylinders as a model

The DNTO was injected at four different levels: 0�2,0�5, 1�0 and 2�0ml per column. These correspond to anequivalent MeBr field dosage of one, two, four and eighttimes the standard MeBr dosage of 50 g/m2, respectively.After injection, the cylinders were sealed with 0�04mmthick polyethylene film. After 24 h, the sealed cylinderswere uncovered and kept in an exhaust hood for 3 h.Subsequently, 8 cm thick soil samples were removedfrom the cylinders and tested.

Five sets of experiments were carried out, in duplicate,with five countings per set. The dosage levels were asfollows: Sets A and B with the standard MeBr dosage(50 g/m2), C and D with twice standard MeBr dosage, Eand F with four times standard MeBr dosage, and Gand H with eight times standard MeBr dosage; I and J

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0-8 8-16 16-24 24-32 32-40

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atod

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Fig. 6. Surviving nematodes after treatment with variousdosages of di-nitrogen tetroxide (DNTO): set A (~) and B(m), standard MeBr dosage of 50 g/m2; sets C (’) and D (�)2�MeBr dosage; sets E (� ) and F (}) 4�MeBr dosage;sets G (n) and H (&) 8�MeBr dosage; sets I (J) and J ( )

untreated controls

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Fig. 7. Galling index of nematode-infected tomato plants grownin pots on soil samples collected from the microplots, subjected todi-nitrogen tetroxide (DNTO) dosages equivalent to thestandard MeBr dosage (50 g/m2), or to multiples of this dosage;the pots were kept in a temperature-controlled greenhouse;results were analysed according to the Tukey test (probabilitya ¼ 0�05). The galling index measures the level of nematodeinfestation of the plant roots by means of a scale from zero tofive, a value below two implying that the plant will be able to

grow and develop properly

USE OF DI-NITROGEN TETROXIDE FOR SOIL FUMIGATION 417

were untreated control samples. The results are given inFig. 6.

The results indicate that, in order to fumigate the soil,a standard MeBr dosage with DNTO is not effective,although the nematode count was significantly reducedin most samples. At twice the standard dosage, however,DNTO appears to be very effective. At higher dosages,no viable nematodes were detected at all.

3.3. Injection into microplot in field tests

The results are represented in terms of averages andsummarised in Fig. 7. The galling indices of the controland the three times standard MeBr equivalent DNTOdosage are statistically insignificant. Yet, the six timesstandard MeBr equivalent DNTO dosage reduced thegalling index to an acceptable value of 1, and the MeBrcomparison reduced it to zero.

3.4. Phytotoxicity experiments

During the growth period of the tomatoes, it wasnoticed that the three times MeBr equivalent DNTO-treated plots were less developed than the MeBr-treatedplots. This raised concern about the possible phytotoxiceffect on the tomato plants by DNTO. The fact that thesix times MeBr equivalent DNTO-treated plot grew aswell as the MeBr plots should have put these concerns torest. However, since there was only one high-dosage

DNTO plot, it was decided to conduct a separate seriesof phytotoxicity tests.

Nine 25 l pots were filled with nematode-free soil,three of which were control samples, three more wereaggressively fumigated by DNTO and the remainingthree were first aggressively fumigated with DNTO andthen thoroughly washed with water. The expectationwas that the unwashed samples might demonstrate anyphytotoxic effects. Tomatoes were then planted in allpots and placed in the open air. Plant growth wasmonitored until fruiting. The results indicated nodifferences in growth among all pots, indicating thatDNTO has no phytotoxic effect on tomatoes.

4. Discussion

The laboratory experiments with DNTO permeatingthrough a dry soil-packed column show that nematodesare effectively destroyed in 10min, fungi after 10–20minand bacteria can be eliminated in 30min. The effective-ness of DNTO as fumigant increases with decreasing soilmoisture. However, our laboratory injection tests withDNTO indicate that fumigating the soil with an amountequivalent to a standard MeBr dosage (50 g/m2) isinsufficient for effective results, although the nematodecount was significantly reduced in most samples. Yet,with twice that amount, DNTO was very effective ineliminating nematodes.

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The results observed in the microplot experimentcontradict those of the laboratory injection experiments(Fig. 6), which indicated that, at three times the standardMeBr dosage equivalent level, a significant reduction ofnematode population could be expected. In fact, in thelaboratory experiments, even when the amount ofDNTO applied was at the standard MeBr dosageequivalent level, an average of two-fold nematodepopulation reduction was observed, and at twice theMeBr dosage level a ten-fold decrease in nematodepopulation was observed.

There are two possible explanations for the observedresults. Firstly, we have the insufficient drying of themicroplot and the relatively high moisture level ob-served in the lower layers of the microplot. The moisturelevels in the laboratory experiments were kept low, andadditional laboratory experiments have shown that,with increasing moisture, the efficiency of the DNTOfumigation decreases (see Section 3.1 above) sinceDNTO reacts with moisture to produce nitrate (whichis not toxic to soil microorganisms). The other possiblereason is poor dispersion of the DNTO in the soil. Thelaboratory experiments were done using small samplescompared to the microplots, and although precautionwas exercised by injecting the DNTO at seven or eightdifferent locations, still the dispersion may not havebeen sufficient. In the conventional field procedure ofapplying fumigant to the soil, the fumigant is injectedalong a line and not at single, scattered points. Efficientoperational use requires injecting DNTO diluted withlarge amounts of dry compressed air. This will greatlyimprove the efficiency of the dispersion of the fumigantin the soil, provided the application is done when the soilis dry.

The microplot field experiments indicated that DNTOcan be safely used as a fumigant for tomato plantswithout any visible damage to the plants, but soilfumigation was effective in these experiments only at thehigh, six times the MeBr-equivalent DNTO dosage level,at which the results were comparable to those observedusing MeBr. This conclusion is based on one microplottrial. The DNTO fumigation with three times the MeBr-equivalent DNTO dosage was ineffective, whereas theselevels in the laboratory experiments were effective. Thepossible reasons for these contradictory results are thehigh moisture in the microplots, and insufficientdispersion of the fumigant in the soil. Therefore, basedon microplot condition and prior laboratory experi-ments, it is reasonable to expect that in microplots orfields with drier soil and with good dispersion (withpossible dilution of DNTO with compressed dry air), theefficacy of DNTO fumigation would be greatly im-proved. Results further indicate that more attentionshould be given to the preparation of the soil prior to the

application of DNTO. Specifically, it was suggested thatthe field should be treated with a subsoiler at up to 60 cmdepth, followed by fine break-up of the upper 15–20 cmlayer and leaving the soil to dry adequately.

Moreover, the application of DNTO as a fumigantfor non-seasonal plants (such as bananas, apricots,almonds and grapes), which are very sensitive tonematode infestation and damage, should be studied.For these applications MeBr has a distinct disadvantageover DNTO, because of the phytotoxic effect of MeBr.For this reason, MeBr is normally applied at a certaindistance from the roots. On the other hand, DNTOcould effectively be applied directly around the roots. Inplants that show toxic effects after exposure to DNTO,procedures will have to be optimised so that damage isreduced to an acceptable level.

Pressurised DNTO is a liquid handled in largecommercial quantities and can be applied with standardequipment. It is expected, therefore, that conventionalMeBr delivery systems and soil injection equipment beused for DNTO, with only minor modifications. DNTOis non-corrosive to most metals at normal temperature;however, it does corrode copper and copper alloys.Teflon is the preferred material for all equipmentgaskets. Metal parts coming into contact with wetDNTO (for example, soil injectors) will have a longerservice life if they are fabricated from 316 or 306stainless steel.

Of the different types of polymer films tested for theirusefulness as a DNTO evaporation barrier—low-densitypolyethylene, polyester, cellophane and low-densitylinear amide—low-density polyethylene proved to bethe best choice. Furthermore, it is also one of thecheapest polymers in use.

The additional merit of employing this fumigant,namely its action as a nitrogenous fertiliser for cropsgrown in DNTO-treated soils, will make it moreattractive compared to alternative chemicals.

5. Conclusions

Laboratory and preliminary field experiments with di-nitrogen tetroxide (DNTO) as a soil fumigant stronglysuggest that this may be a viable alternative to MeBrand, most importantly, its use will not result inenvironmental damage caused by MeBr, provided thatthe emission of N2O4 to the atmosphere will remainsufficiently small. This can be accomplished by adequatemoistening of the upper soil layer (to approximately4%) sometime after the DNTO application, as well asby covering it with plastic films. Retaining some of themoisture in the soil will also provide part of the nitrogenfertiliser, which otherwise will have to be added.

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Acknowledgements

This study was generously supported by a grant fromVicksburg Chemical Company of the USA, a subsidiaryof Trans Resources Inc. of New York. Special thanksare due to John Miles of Vicksburg Chemicals for hisadvice and help and to Ady Langham of HaifaChemicals for his help with the microplot experimenta-tion, as well as to Ms. Dana Friesem for her help inmicrobiological countings.

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