Fumigation Characteristics of Ozone in Postharvest Treatment of Kabkab Dates (Phoenix dactylifera L.) against Selected Insect Infestation

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1 763 Journal of Food Protection, Vol. 73, No. 4, 2010, Pages Copyright G, International Association for Food Protection Research Note Fumigation Characteristics of Ozone in Postharvest Treatment of Kabkab Dates (Phoenix dactylifera L.) against Selected Insect Infestation MEHRDAD NIAKOUSARI, 1 * ZAHRA ERJAEE, 1 AND SHAHRAM JAVADIAN 2 1 Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, 71345, Iran; and 2 Fars Engineering Research Center, Modares Boulevard, Shiraz, Iran MS : Received 18 November 2008/Accepted 8 November 2009 ABSTRACT Methyl bromide fumigation, the most accepted quarantine treatment for dates and many other dried commodities, will be phased out by 2015 worldwide. As a result, there is a critical need to develop durable alternatives for methyl bromide as postharvest treatments of agricultural commodities. This article presents a new method for postharvest treatment of Kabkab dates (Phoenix dactylifera L.) by application of gaseous ozone to reduce or eliminate all life stages (adults, larvae, and eggs) of Indian meal moth (Plodia interpunctella) and sawtooth grain beetle (Oryzaephilus surinamensis). The effect of the ozonation process on the sugar content of dates was also evaluated. Infested dates were exposed to ozone concentrations of 600, 1,200, 2,000, and 4,000 ppm for 1 and 2 h. As insect eggs are known to be most tolerant to many chemical or physical treatments, they were additionally exposed to an atmosphere of pure carbon dioxide prior to ozonation. Exposing samples to ozone concentrations of.2,000 ppm for 2 h resulted in complete mortality of larvae and adults. Exposure to 4,000 ppm of ozone for 2 h resulted in 80% mortality of eggs, and exposure to CO 2 prior to ozonation did not improve the effect of ozonation on eggs. Ozone did not have any influence on the sugar content of Kabkab dates. The palm tree (Phoenix dactylifera L.) is one of the most sacred and ancient fruit trees known to humans. Its fruit, the date, has played an important role in the history of mankind. Annual production of dates in Iran is about 1 million metric tons (ranked second in the world after Egypt). Kabkab dates, which grow extensively in Fars province, Iran, are a soft fruit with a moisture content of around 20%. Dates may be stored at room temperature for about 8 months following fumigation. It is estimated that between 10 and 20% of Iranian harvested dates are destroyed or are unsuitable for direct human consumption due to poor handling and inappropriate storage conditions (7). Insects such as the Indian meal moth (Plodia interpunctella) and sawtooth grain beetle (Oryzaephilus surinamensis) are believed to contribute significantly to this destruction. To protect dates from pest damage, some means of quarantine and postharvest treatment is desirable. Methyl bromide (MB) has long been the most commonly used form of postharvest treatment. MB, however, has been classified as a class I ozone-depleting substance and is being phased out by 2015 worldwide (32). The loss of MB could have a negative impact on world agriculture, particularly because no available alternatives to MB currently exist for rapid disinfestation of commodities. * Author for correspondence. Tel: z ; Fax: z ; Niakosar@shirazu.ac.ir. Phosphine has been suggested to be an important alternative, having already replaced MB in many countries. However, phosphine is not always appropriate for particular applications as it is almost as dangerous as MB and some insects (particularly their eggs) have developed resistance and require long exposure periods (5 days or more) even at high concentrations of over 140 mg/liter (12, 13, 33). Therefore, phosphine may not be suitable for rapid disinfestations or quarantine fumigations. Other compounds, such as sulfuryl fluoride, have relatively poor ovicidal properties and also require long exposure periods (3 days or more) (31). Unlike other chemical fumigants that are toxic to insects at low concentrations, very high CO 2 concentrations (40 to 100%) and long periods of exposure (2 or more days) are necessary for high mortality rate in insects (6). However, applying a mixture of phosphine and CO 2 is recognized to reduce the resistance of some insects (8). Physical methods such as heat or cold treatments and subzero temperature treatments have also been used for postharvest treatment (16, 20, 24). At 10uC, mortality of the larvae of Indian meal moth reaches 90% after 70 days. Subzero temperatures can also destroy insect eggs, but the method may not be economically feasible. Irradiation is a fairly new technique for disinfestation of dates and eradication of insects and is still being investigated (4, 9). Ozone (O 3 ) is a powerful oxidizing agent usually generated by electrical charges in air. It is generally

2 764 NIAKOUSARI ET AL. J. Food Prot., Vol. 73, No. 4 TABLE 1. Corrected percent mortality of larvae and adults of Indian meal moth and sawtooth grain beetle placed on the surface of dates and exposed to various ozone concentration time combinations a Mortality (%) SD at ozone concn of: D d D d A a A a C c B b A a A a 0.00 accepted to be an unstable gas with a life span of about 20 min at room temperature; thus, it does not accumulate substantially without continual generation (23). Electrical generation of ozone eliminates the handling, storage, and disposal problems of conventionally used postharvest pesticides. An attractive aspect of ozone is that it decomposes rapidly to molecular oxygen without leaving a residue. These attributes make ozone an attractive candidate for controlling insects and fungi in stored products (22). It is currently used to disinfect fruits, vegetables, and other foodstuffs of microorganisms and viruses; as a means of reducing odor; and for removing taste, color, and environmental pollutants in industrial applications (30). Ozonated water has been successfully used in packing houses and storage rooms to control postharvest diseases of fruit, to retard the production of spores, to sanitize surfaces, and to reduce or eliminate ethylene gas (10, 27, 30). Ozone in its gaseous form has also been considered to have the potential to kill insect pests in commodities. For example, high mortality was achieved for adults of the maize weevil, Sitophilus zeamais (Motschulsky), and of the red flour beetle, Tribolium confusum (Jacqueline du Val), and for the larval stage of the Indian meal moth, P. interpunctella, when exposed to low ozone concentrations ranging from 5 to 45 ppm (17, 21, 25). However, in these studies the time of treatment was 3 days or more, which is allowable for control fumigations but not for quarantine fumigation. In all these studies, the exposure to ozone was on the surface of the products and the degree of penetration of ozone could not be investigated. Other workers used high concentrations of ozone (10,000 ppm) plus carbon dioxide at 5 to 7%, a vacuum of 0.34 bar, and at least 2 h of exposure to kill bean thrips (Caliothrips fasciatus) in navel oranges and the coffee berry borer (Hypothenemus hampei) in coffee beans. They stated that ozone was suitable for killing insects (adults and larvae) but the eggs were the most tolerant (23). The objective of this research was to evaluate the effectiveness of ozone for killing all life stages of insects in Kabkab dates. Furthermore, the toxicity of a combination of ozone and carbon dioxide on insect eggs was also investigated. In addition, the effect of ozonation on the sugar content of Kabkab dates was assessed. MATERIALS AND METHODS Kabkab dates. Untreated Kabkab dates (noninfested and infested) were obtained from TANI producer group, a local date processing and packaging company. Insects. Tests were carried out at various life stages of the Indian meal moth (P. interpunctella) and sawtooth grain beetle (O. surinamensis), the two most common date-infesting insects. Insects (adults, larvae, and eggs) were collected from infested dates and were kept in a container at 28 2uC and 65% 5% relative humidity, on a diet of dates for 2 weeks prior to exposure to ozone. The adult, larvae, and eggs of both insects were chosen at random from the incubation container without being specific on the number of each species. The ozonation process was carried out at all life stages of insects of both species, as they exist in stored dates concurrently. Ozone generator and fumigation chamber. A laboratory corona discharge ozone generator (model OZO-1VTT) was provided by Ehsan Tahghigh Co., Tabriz, Iran. The generator was equipped with a timer and an ozone destruction unit to ensure that the exhaust gas contained no ozone when it was released into the atmosphere. Ozone was generated from purified extra-dry oxygen feed gas. The generator was designed to produce up to 5 g of ozone per h. A 20-liter test chamber was made of stainless steel (ST304) and equipped with four inlet and outlet valves, a small fan to serve as mixer for uniform distribution of gas within the chamber, and a pressure gauge to give pressure indication. The chamber was connected to the generator, ozone analyzer (Ozone Analyzer BMT 964, BMT Messtechnik, Berlin, Germany), and ozone destruction unit by means of silicone tubing. Carbon dioxide gas. Pure carbon dioxide (99.5% 0.5%) was supplied from a gas cylinder. It was measured with a Bedfont gas monitor model 415 equipped with a thermal conductivity detector. Experimental procedure. Four sets of experiments were designed to investigate the efficacy of ozone on infested dates. In the first set, two life stages (larvae and adults) of both insects were placed at random on the surface or in the vicinity of dates and they were exposed to ozone (surface experiment). In the second set, insects (adult and larvae) were placed inside the dates or date paste (thickness,,1 cm) in order to investigate the ozone penetration through dates. For each individual ozonation treatment, in the first and second sets of experiments, 20 adults and 20 larvae (combination of Indian meal moth and sawtooth grain beetle, chosen at random) were used. For the third set of experiments, 50 eggs of both insects were placed on a petri dish covered with date paste and the date paste was exposed to ozone and/or ozone- CO 2. In the latter, samples were exposed to an atmosphere of pure CO 2 in the fumigation chamber for 1 h prior to ozonation. Only ozone at 2,000 or 4,000 ppm was used in this experiment. For all sets of samples, nonozonated insects, eggs, and infested dates were used as controls to estimate the proportion of mortality due to natural death. Four concentrations (approximately 600, 1,200, 2,000, and 4,000 ppm) and two exposure times (1 and 2 h) were used.

3 J. Food Prot., Vol. 73, No. 4 APPLICATION OF OZONE TO ELIMINATE INSECTS IN STORED KABKAB DATES 765 TABLE 2. Corrected mortality of larvae and adults of Indian meal moth and sawtooth grain beetle placed inside the dates or paste for various ozone concentration time combinations (penetration experiment) a Corrected mortality (%) SD at ozone concn of: D d D d A a A a C c B b A a A a 0.00 After each ozone treatment, the adults and larvae were kept for 5 days at a temperature of 28 2uC and relative humidity of 65% 5% on a diet of dates until examined for mortality. Eggs were kept for 7 days under the same conditions until transformation (egg hatch). In the presentation of mortality results, no distinction is made between mortality of larvae and adults of each species; however, as eggs were more tolerant to fumigation, their mortality results are tabulated separately. All data obtained from the treatments were corrected using Abbott s formula (1), which recognizes that a proportion of the insects (larvae, adults, and eggs) may die from natural causes (control sample) during the trial even when they are not exposed to ozone. In the fourth set of experiments 200 g of fresh, clean, unspoiled, and noninfested dates was exposed to ozone in order to investigate the effect of ozonation on the sugar content of dates. The control and ozonated samples were stored in polyethylene terephthalate (PET) containers at room temperature (28 2uC) prior to further analysis for evaluation of total and invert sugars (AOAC and ) (18). All experiments were carried out in triplicate. Data from three replications for each experiment were analyzed by analysis of variance using the General Linear Models procedure of SAS. Significance of differences between means was determined (P, 0.05) using the least significant difference procedure (29). RESULTS AND DISCUSSION Table 1 shows the percent mortality of larvae and adults of both insects for four ozone concentration time combinations. The mortality was extremely low (about 15%) when adults and larvae were exposed to ozone concentrations of,2,000 ppm for 1 h, rendering these concentration-time combinations inadequate for fumigation in the commercial facilities. Mortality exceeded 94% when the larvae and adults of both insects were exposed to concentrations of ozone in excess of 2,000 ppm for 1 h, while mortality reached 100% at 4,000 ppm. Mortality exceeded the 50% mark when larvae and adults were fumigated with 1,200 ppm of ozone for 2 h. The results show that ozone, particularly at low concentrations, required much longer exposure times to be effective. This could be related to the unstable nature of ozone, its short half-life, its high reactivity, or its conversion to O 2 during the ozonation process. The results obtained in the present study are in agreement with those reported by others, whereby exposure durations of at least 7 h or even days were required to achieve high mortality rates (21, 25). When treatments were carried out at higher concentrations (2,000 and 4,000 ppm), the effect of duration of exposure to ozone was not significant (P, 0.05). The present results are comparable with several studies on the efficacy of ozone to control insect pests of stored grain. The results obtained by Kells et al. (21) showed that high mortality was achieved for adults of the maize weevil and the red flour beetle and for the larval stage of the Indian meal moth when exposed to lowconcentration ozone (about 100 ppm) for 3 days. A laboratory scale study carried out by Manson et al. (25) showed that exposing adult sawtooth grain beetle and confused flour beetle to an atmosphere containing 5 ppm of ozone for 5 days resulted in 100% mortality of insects. Laboratory studies by Leesch (23) showed high mortalities in pupae of Indian meal moths exposed to 300 ppm of ozone for 4 h, while Isikber et al. (19) reported 100% mortality of Ephestia kuehniella (larvae) when exposed to the same concentration of ozone for 2 h. The exposure times for these studies would be acceptable for control fumigations but not for quarantine fumigation. FIGURE 1. Percentage of eradication of eggs of Indian meal moth and sawtooth grain beetle exposed to various ozone concentrations and times. Means followed by different letters are significantly different (P, 0.05).

4 766 NIAKOUSARI ET AL. J. Food Prot., Vol. 73, No. 4 FIGURE 2. Percent mortality of eggs of Indian meal moth and sawtooth grain beetle treated with ozone and combined ozone and carbon dioxide. Means followed by different letters are significantly different (P, 0.05). The results in Table 2 illustrate the mortality of larvae and adults when they were placed inside the dates or in the paste. Exposing adults and larvae to an ozone concentration of 4,000 ppm for 1 h resulted in killing all adults and larvae of both insects. The mortality rate, however, was reduced to about 84% when the ozone concentration was decreased to 2,000 ppm. The mortality of larvae and adults was,15% when the concentration was 600 ppm. Based on these results, concentrations of,2,000 ppm for short exposure times of 1 and 2 h cannot be recommended for fumigation. The eradication of eggs was, however, more challenging, as eggs were strongly resistant to most known chemical or physical treatments. Figure 1 represents the results obtained for fumigation of eggs with concentrations of ozone from 600 to 4,000 ppm for 1 and 2 h. Fumigating eggs with an ozone concentration of 4,000 ppm for 1 h resulted in killing only about 20% of eggs. The mortality was even lower at concentrations of,4,000 ppm. Fumigating eggs for 2 h with 2,000 and 4,000 ppm of ozone resulted in killing 50 and 80% of eggs, respectively. These results show that ozone has an initial problem in being able to penetrate through the egg, reducing its ability to kill eggs completely at deeper levels. Results presented by others show that eggs cannot be completely eradicated (11, 14). Leesch (23) reported that high concentrations of ozone (10,000 ppm) did not kill 100% of eggs of Indian meal moths. These results were confirmed by information provided to authors by the commercial facilities in Iran (7). To study the effect of exposure of eggs to a CO 2 environment prior to ozonation, eggs were pretreated for 1 h with 100% CO 2. Other investigators have suggested that combinations of two or more treatments have produced synergistic effects (8). Figure 2 shows the mortality of eggs exposed to CO 2 followed by 2 h of treatment with ozone at concentrations of 2,000 and 4,000 ppm. The levels of eradication of eggs were 44 and 70% at 2,000 and 4,000 ppm, respectively. Exposing eggs to 1 h of 100% CO 2 and 2 h of ozonation did not enhance the mortality of eggs. It might be argued that low O 2 concentrations did not influence the potentiating effect of CO 2. Our finding is in agreement with results reported by Isikber et al. (19), who exposed eggs of E. kuehniella to a combination of CO 2 and ozone. Dates have a high sugar content (mostly glucose and fructose), ranging from 44 to 70% (5). Tables 3 and 4 summarize the variation of total and invert sugar contents of the control and ozonated dates. The total and invert sugar contents in the control and ozone-fumigated dates did not differ significantly (P, 0.05). The total sugar content of dates for controls was about 63 mg for every 100 g of pitted fruit. Fumigating dates with 4,000 ppm of ozone for 2 h hardly changed the sugar content. Dates are known to be good sources of vitamins, antioxidants, anthocyanins, carotenoids, and phenolic compounds (3, 26). It is likely that ozone preferentially was reduced by low-molecularweight components such as antioxidants within dates, leaving the sugar content unaffected. The result is comparable to those reported by Aguayo et al. (2). These authors did not observe any significant difference between the monosaccharide levels of ozonated tomato and those of the nonozonated samples. However, results reported by Perez et al. (28) on ozonation of strawberries showed a TABLE 3. Effect of ozone treatment on total sugar content of Kabkab dates a Mean sugar content (mg/100 g of dates) SD at ozone concn of: 0 (control) A a A a A a A a A a A a A a A a A a A a A a A a 0.69

5 J. Food Prot., Vol. 73, No. 4 APPLICATION OF OZONE TO ELIMINATE INSECTS IN STORED KABKAB DATES 767 TABLE 4. Effect of ozone treatment on invert sugar content of Kabkab dates a Mean invert sugar content (mg/100 g of dates) SD at ozone concn of: 0 (control) A a A a A a A a A a A a A a A a A a A a A a A a 0.70 decrease in the sugar content of the fruit from 19.9 to 8 mg/ g of fresh weight. They also reported an increase in the concentration of vitamin C from 0.07 to 0.14 mg/g. They related this increase in vitamin C to high oxidative capacity and ability of ozone to generate toxic molecular species, acting as a potent phytotoxic agent. They further suggested that the sugar may change to vitamin C in the ozonation process. It is reported by other investigators that in aqueous solutions it is likely that ozone may react with glucose to produce gluconic acid (15). In the present study, only the sugar content was evaluated and no analysis on other components of dates was performed. 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