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The toxicity effects of atmospheres with high content of carbon dioxide with addition of sulphur dioxide on two stored-product pest species: Sitophilus oryzae and Tribolium confusum
Journal of Stored Products Research 57 (2014) 58e62
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The toxicity effects of atmospheres with high content of carbon dioxide with addition of sulphur dioxide on two stored-product pest species: Sitophilus oryzae and Tribolium confusum Jordi Riudavets a, *, Maria José Pons b, Rosa Gabarra a, Cristina Castañé a, Oscar Alomar a, Lourdes F. Vega b, c, Sonia Guri b, c a b c
IRTA, Entomology, Ctra. Cabrils Km 2, E-08348 Cabrils, Barcelona, Spain MatGas-Research Center, Campus UAB, E-08193 Bellaterra, Barcelona, Spain Carburos Metálicos e Air Products Group, Aragón 300, E-08009, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 7 December 2013
Modified atmospheres (MAs) are safe and environmentally friendly alternatives to control pests in stored products. However, to accommodate the requirements of the food industry, the control of insects by a MA can be a too lengthy process. This paper describes the potential of sulphur dioxide as an additive to reduce the long lethal exposure of modified atmospheres (MA) enriched with carbon dioxide for major stored product pests. Specifically, we evaluated whether the addition of SO2 (0e30,000 ppm) to a highCO2 content of 70%e95% MA could enhance its insecticidal effect for the control of Sitophilus oryzae and Tribolium confusum. The addition of 15,000 ppm and 30,000 ppm of SO2 to 95% CO2 enhanced control up to 100% in comparison to CO2 alone for S. oryzae and T. confusum adults when treated for one day in all of the substrates tested. However, the effectiveness of adding SO2 at reduced contents was lower and depended on the substrate and pest species considered. The addition of SO2 also increased the mortality of all of the developmental stages of S. oryzae. The increase in mortality with the addition of SO2 was also observed when included with 70% CO2. Therefore, the addition of SO2 can be considered a feasible means of shortening the length of treatment necessary to achieve the control of these two pests using a highCO2 MA. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Modified atmospheres Carbon dioxide Sulphur dioxide Pest control Sitophilus oryzae Tribolium confusum
1. Introduction Food product commodities can be affected by insect pests during the storage period, and visible contamination due to insect individuals or their remains may be present in the final product. Yet, there are increasing restrictions on the number of active chemical compounds officially registered for pest control and on the maximum residue levels (MRLs) allowed in the final food products. It is, therefore, necessary to implement alternative strategies for the control of pests in stored food products. Modified atmospheres (MAs) based on a high content of carbon dioxide (CO2) offer a suitable, safe and environmentally friendly alternative method for controlling pests that affect food products (Navarro, 2006). MAs can be applied in hermetic structures at different steps of food processing, from the storage of bulk raw and semi-processed materials to the packaging process of the product * Corresponding author. Tel.: þ34 937507511; fax: þ34 937533954. E-mail addresses: [email protected], [email protected] (J. Riudavets). 0022-474X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jspr.2013.12.003
for the end consumer. The main advantages of using a high-CO2 MA are that this method is effective for the control of a wide range of pest species and that it can be used for the treatment of different food commodities without the accumulation of toxic residues after the treatment. However, to accommodate the requirements for marketing in the food industry, the control of insects by means of MAs has the disadvantage of needing a relatively long treatment time. To enhance the use of MAs, it is necessary to find alternatives that allow the reduction of the length of the treatment without compromising the control efficacy. Toxic effects of CO2 are well known and data on the effects of different types of CO2 treatments and dosages are available for many species and stages of storedproduct pests under particular sets of conditions (Banks and Annis, 1990; White et al., 1995; Riudavets et al., 2009; Navarro, 2012). The recommended exposure time to achieve complete pest control at the most appropriate gas content ranges from several days to weeks, depending on the CO2 content, temperature, developmental stage and species (Fleurat Lessard, 1990). For
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example, to control one of the most MA-tolerant pest species, the rice weevil Sitophilus oryzae (L.), the estimated LT95 values using 40%e100% CO2 MA at 25 C range between 1 and 4 days for adults and between 3 and more than 5 days for eggs (Annis and Morton, 1997). Moreover, to achieve full mortality for all of the developmental stages of this species, treatments should last 12 days when using an MA of 90% CO2 at 25 C (Riudavets et al., 2009). With the exception of diapausing Trogoderma granarium Everts larvae, Banks and Annis (1990) reported that, of 10 coleopteran stored-product pests tested, S. oryzae was the species most tolerant to a high-CO2 MA. The beetle Tribolium confusum Jacquelin du Val, one of the most important coleopteran pest present in flour and feed mills, is in comparison less tolerant to MA, but their pupae are also difficult to kill, requiring 8 days with an MA of 90% CO2 at 25 C (Riudavets et al., 2009). Because an MA with high contents of CO2 causes the permanent opening of insect spiracles (Nicolas and Sillans, 1989; Mitcham et al., 2006), a synergistic toxic effect of CO2 when combined with other compounds has been sought. For example, adding CO2 to certain fumigants, such as acrylonitrile, methyl bromide, phosphine, carbon disulfide, ethylene oxide, chloropicrin, methyl formate and hydrogen cyanide, increases their toxicity and permits the reduction of treatment times (Bond and Buckland, 1978; Peters, 1936). Similarly, according to Is¸ikber (2010), the fumigant toxicity of a garlic essential oil was enhanced (more than 4.9-fold) in combination with carbon dioxide (92% CO2) for the control of T. confusum. Sulphur dioxide (SO2) is a gas that is already accepted as food additive (E-220) in a number of countries for several nonperishable food products, such as cereal by-products, nuts and dried fruits (FDA, 2012; The European Commission, 2012). Although SO2 mainly has a long history as a food preservative due to its antimicrobial properties in a range of food products, it has also been used for the control of a number pests (Brieger, 1918), among them the stored product pests Plodia interpunctella (Hübner) and Ephestia spp. (Peters, 1936). More recently, it has been proposed for the control of some insect pest of grapes during storage (Yokoyama et al., 2001). Fumigation with SO2 (routinely applied for fungal control), alone or combined with carbon dioxide, may provide pest control, though this possibility has received little attention (Vota, 1957). Only Vail et al. (1992) reported that SO2 contents comparable to those used in the routine fumigation of grapes killed the key pest Platynota stultana Walshingham (Lepidoptera: Tortricidae) in a laboratory experiment, suggesting that SO2 has the potential to control both fungi and insects. Preliminary work with S. oryzae on rice (Riudavets et al., 2008) indicated the potential of such mixtures for pest control. The objective of this research was to evaluate the extent to which the addition of a range of SO2 contents to a high-CO2 MA could enhance the insecticidal effect of the CO2 MA for the control of two important stored-product pest species, S. oryzae and T. confusum, on different food substrates. Sitophilus oryzae was chosen as a model for cereal grain pests, and T. confusum as a model for cereal by-product pests. In the laboratory, we evaluated the effect of two CO2 MAs (70% CO2 and 95% CO2) combined with different SO2 contents (0e30,000 ppm) on the reduction of emergence of all of the developmental stages of S. oryzae and on the mortality of the adults of T. confusum at a temperature of 25 C. 2. Material and methods The insects used for the experiments originated from the colonies maintained at IRTA (Cabrils, Barcelona). Sitophilus oryzae was reared on brown rice and T. confusum on wheat flour and yeast. The adults, eggs, larvae and pupae of S. oryzae and adults of T. confusum
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were separately evaluated. In the case of S. oryzae, the larvae and pupae were tested together (referred to as ‘larvae-pupae’). All of the laboratory studies were conducted in a climatic chamber at 25 2 C and 70 15% r.h. The experimental arenas consisted of ventilated plastic cages (7.5 cm in diameter and 3 cm high) containing 50 g brown rice, 20 g wheat flour or no food substrate, depending on the experimental condition. The sides of the arenas were painted with FluonÔ to avoid the escape of the insects during the treatment. A total of fifty individuals were added to the arenas, except in the case of the eggs and larvaeepupae of S. oryzae, for which the diet was previously infested with adults (410 adults/kg of brown rice for 7 days) to produce at least 50 individuals in the grains in each arena. The effectiveness of two high-CO2 modified atmospheres (70% CO2 and 95% CO2) combined with different SO2 contents (0e 30,000 ppm) was evaluated. A range of exposure times between 1 day and 5 days were tested according to the species, developmental stage and previous results (Riudavets et al., 2008, 2009). The insects were exposed to each gas mixture in sealed glass desiccators (200 mm in diameter). Three cages of each species and stage were exposed in the same desiccators, with five replications for each gas mixture and exposure time according to each experiment. The gas mixtures were first prepared in cylinders, and the gas content was verified (Carburos Metálicos e Air Products Group). The MAs contained 5% O2, 70% or 95% CO2, and were balanced to 100% with N2 according to the SO2 treatment. The gas mixtures from the cylinders were continuously flushed into the desiccators at a pressure of 2 bars until the desired gas contents were reached. A gas analyser (Abiss model TOM 12) was used to monitor the contents of CO2 and O2 inside the desiccators during the experiment. After the treatment, the desiccators were opened, and the cages were kept in the climatic chamber. To evaluate the mortality of the adults, the number of living individuals in each cage was counted 24 h after the end of the treatment. The percentage mortality was calculated using the initial number of individuals placed in each cage. Sets of control cages were used to determine the percentage of natural mortality. For the eggs and larvaeepupae of S. oryzae, the emerging weevil adults were counted after 9 and 4 weeks, respectively. The number of living adults in each arena was counted, and the percentage of mortality was calculated using the number of individuals that emerged in the control cages. The significant differences in terms of the percentage of mortality for the different stages of the two species tested when exposed to each of the different experimental conditions were analysed using an analysis of Variance (ANOVA) (SAS Institute, 2002). All of the percentage data sets were arcsine transformed to meet the homogeneity and normality assumptions for the analysis of variance. The Tukey Multiple Range test was used to compare the mean values. 3. Results The addition of 15,000 ppm and 30,000 ppm of SO2, respectively to an MA of 95% CO2 ensured the full mortality (100%) of the S. oryzae and T. confusum adults when treated for an exposure time of 1 day using all of the substrates tested (Table 1). However, the effectiveness of adding SO2 at reduced contents was lower and depended on the substrate and pest species (Table 1). For the S. oryzae adults, the presence of the substrate (rice) was reflected in significantly lower mortalities in comparison to the results when no substrate was added. This was already observed without adding SO2, with a lower mortality using rice as a food substrate (81.2%) than when no food substrate was present (99.7%). The addition of 150 ppm of SO2 had no improvement of the effect of CO2 compared to the ‘no-SO2’ treatment (Table 1), and the effect of
J. Riudavets et al. / Journal of Stored Products Research 57 (2014) 58e62
Table 1 Total mortality (%; Mean SEM) of S. oryzae and T. confusum adults exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 1 day (24 h) on different food substrates. Content of SO2
S. oryzae
0 ppm 150 ppm 1500 ppm 4500 ppm 15,000 ppm 30,000 ppm P F df
81.2 1.54 72.6 7.27 29.3 2.76 18.7 1.51 100 a 100 a <0.001 227.12 5; 89
T. confusum
Rice
Wheat flour
No food substrate 99.7 0.16 95.9 0.82 72.7 3.90 98.1 0.65 100 a 100 a <0.001 69.03 5; 89
b b c d
a a b a
43.7 4.58 45.7 0.68 39.4 3.49 95.6 0.65 100 a 100 a <0.001 303.79 5; 89
Rice b b b a
51.5 8.10 b 51.9 8.11 b 93.4 1.40 a 100 a 100 a 100 a <0.001 64.52 5; 89
Means within a given column followed by the same lowercase letter are not significantly different (Tukey test, P < 0.05).
the substrate was maintained. A large decrease in mortality was observed at 1500 ppm and 4500 ppm of SO2 in the presence of rice, reducing the lethal effect to only 29% and 19%, respectively. However, without any food substrate, the decrease in mortality was only significant with the addition of 1500 ppm, whereas a high mortality (98.1%) was obtained at 4500 ppm SO2. Figure 1 is used to highlight this antagonistic effect between CO2 and SO2 at the intermediate range of SO2 tested. When exposed to the MA without SO2, the T. confusum adult mortalities were lower than the mortalities of S. oryzae and slightly but significantly lower on the wheat than on the rice (Table 1, Fig. 2). Adding 150 ppm of SO2 did not increase the mortality of T. confusum. The addition of 1500 ppm or 4500 ppm of SO2 to the rice substrate did not produce the reducing effect concerning the mortality observed with S. oryzae, and the mortality increased up to 93.4% and 100%, respectively. However, when 1500 ppm SO2 were added to the wheat flour substrate, the mortality remained low (39.4%) and only significantly increased with 4500 ppm, which was still significantly lower (95.6%) than on the rice. The addition of SO2 also significantly and progressively increased the mortality of the S. oryzae eggs and larvaeepupae in the three days of exposure to an MA of 95% CO2 (Table 2). The addition of 150 ppm and 1500 ppm SO2 slightly but significantly increased the egg mortality, but the mortality was higher than 99% only with 4500 ppm SO2. In the case of the larvaeepupae, the addition of 150 ppm of SO2 did not increase the mortality, but the addition of 1500 and 4500 ppm SO2 doubled the mortality, though
mortality (%)
100
*
*
*
*
100 mortality (%)
60
*
*
*
80 60 40 20 0 0
150
1,500 4,500 15,000 30,000 ppm
Fig. 2. Total mortality (%; Mean SEM) of T. confusum adults exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 1 day in rice (broken line) and wheat flour (straight line). Significant differences between the substrates for each SO2 content are indicated with an asterisk (P < 0.05).
the mortality only reached an average of 61%. The mortality reached 100% with an addition of 30,000 ppm SO2. An increase in mortality with the addition of SO2 was also observed when the gas was added to an MA with a lower CO2 content of 70% (Table 3). With 30,000 ppm, the mortality increased significantly to 100% for all of the developmental stages and both pest species tested, with the exception of the larvaeepupae of S. oryzae. Nevertheless, in this case, the mortality was also high (82.7%). Although no intermediate contents of SO2 mixed with 70% CO2 were tested, the lowest content tested (150 ppm) did not increase the mortality significantly, except for the eggs of S. oryzae. 4. Discussion The addition of SO2 has been useful to improve the control efficacy of high-CO2 MAs against all of the developmental stages of S. oryzae and the adults of T. confusum. The increase in efficacy would allow a practical reduction of the treatment time. Hence, the addition of 15,000 ppm SO2 to an MA of 95% CO2 reduced the time necessary to reach 100% mortality to only 1 day for S. oryzae and the T. confusum adults at a temperature of 25 C (Table 1). In comparison, without the addition of SO2, the full mortality of S. oryzae and T. confusum adults requires more than 4 days with an MA of 90% CO2 at 25 C (Riudavets et al., 2009). Using a content of 80% CO2, Banks and Annis (1990) reported a treatment time of 3.5 and 2 days to obtain at least 95% mortality of S. oryzae eggs and T. confusum adults, respectively, at 25 C. Annis and Morton (1997) also reported a treatment time of more than 1 day and more than 4 days for the control (99% mortality) of S. oryzae adults and eggs, respectively, when treated with 95% CO2 at 25 C. Table 2 Total mortality (%; Mean SEM) of larvaeepupae and eggs of S. oryzae exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 3 days in rice.
80 60 40 20 0 0
150
1,500 4,500 15,000 30,000 ppm
Fig. 1. Total mortality (%; Mean SEM) of S. oryzae adults exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 1 day in rice (broken line) and without any food substrate (dotted line). Significant differences between the substrates for each SO2 content are indicated with an asterisk (P < 0.05).
Content of SO2
Eggs
0 ppm 150 ppm 1500 ppm 4500 ppm 15,000 ppm 30,000 ppm P F df
60.3 2.07 68.7 1.62 70.8 1.00 99.3 0.14 100 a 100 a <0.001 786.42 5; 89
LarvaeePupae d c c b
25.3 4.08 12.7 2.53 53.1 1.64 60.6 2.18 94.0 0.98 100 a <0.001 198.12 5; 89
d e c c b
Means within a given column followed by the same lowercase letter are not significantly different (Tukey test, P < 0.05).
J. Riudavets et al. / Journal of Stored Products Research 57 (2014) 58e62 Table 3 Total mortality (%; Mean SEM) of adults, larvaeepupae and eggs of S. oryzae and adults of T. confusum exposed to 70% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm and 30,000 ppm) during 1 day (adults) and 3 days (eggs and larvaeepupae) in rice (S. oryzae) and wheat flour (T. confusum). Content of SO2
S. oryzae Eggs
LarvaeePupae
Adults
Adults
0 ppm 150 ppm 30,000 ppm P F df
63.7 1.29 c 97.8 0.4 b 100 a <0.001 900.29 2; 44
30.7 3.01 b 31.4 1.74 b 82.7 1.83 a <0.001 155.67 2; 44
41.7 3.38 b 48.2 8.72 b 100 a <0.001 139.37 2; 44
15.8 1.3 b 12.1 0.94 b 100 a <0.001 1727.38 2; 44
T. confusum
Means within a given column followed by the same lowercase letter are not significantly different (Tukey test, P < 0.05).
Furthermore, the addition of SO2 to a high-CO2 MA would also allow a reduction in the treatment time of the pupae of S. oryzae, the most tolerant developmental stage of this species. In the present experiment, full mortality was observed after 3 days of exposure to a combination of 95% CO2 and 30,000 ppm SO2 (Table 2). In comparison, Annis and Morton (1997) reported a much longer treatment time of 15.3 days of exposure with 95% CO2 only for the control of this developmental stage. According to Riudavets et al. (2009), without the addition of SO2, 8 days would be necessary to achieve 100% mortality of S. oryzae pupae at 90% CO2 and 25 C. Banks and Annis (1990) also reported a long treatment time of 8.5 days to obtain at least 95% mortality using a content of 80% CO2. However, the addition of less than 15,000 ppm of SO2 to a highCO2 MA does not always guarantee an increase in its efficacy. For example, the addition of 1500 ppm SO2 to a 95% CO2 MA significantly increased the mortality of the S. oryzae eggs and larvaee pupae in rice (Table 2). In contrast, the addition of 1500 ppm and 4500 ppm SO2 to rice produced the opposite effect in the adults, and a significant decrease in their mortality was recorded (Table 1). Similarly, the addition of 1500 ppm SO2 did not enhance the mortality of the T. confusum adults when treated in the presence of wheat flour. The addition of 150 ppm of SO2 did not increase the mortality of the adults and pupae of S. oryzae or the adults of T. confusum but did enhance the mortality of eggs of S. oryzae, especially at 70% CO2. This is important because the eggs of S. oryzae usually become the most important key stage to control, as they are able to survive standard industrial processes, such as cereal milling and rice polishing, whereas most of the pupae and adults are killed (Lucas and Riudavets, 2000). The increase in mortality of the S. oryzae eggs obtained with the addition of 150 ppm SO2 was important with 70% CO2 (Table 3) but only produced a slight increase when the CO2 content was higher (95%) (Table 2). It is likely that the increase of mortality induced by SO2 was not as strong with the highest CO2 content tested due to a greater initial shock effect, which is typically produced in insects at high-CO2 atmospheres (Edwards and Batten, 1973; Nicolas and Sillans, 1989). The highest CO2 content might have produced a greater shock and, consequently, a reduced SO2 uptake by the individuals tested. Several authors have also reported comparable antagonistic effects; for example, lower than expected mortality rates were recorded when high-CO2 contents were added to low O2 environments (Ali-Niazee, 1971; Mitcham et al., 1997). Other fumigants combined with CO2 have shown a similar pattern of producing higher mortality with lower CO2 contents (Bond and Buckland, 1978). Enhanced opening of the spiracles may increase mortality by increasing the fumigant uptake. However, a heavy narcotic effect occurs at high-CO2 contents, with metabolic effects, mainly due to changes in the pH, then becoming the more important determinant of mortality (Janmaat et al., 2001).
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The current knowledge cannot explain the antagonist effect between CO2 and SO2 on the S. oryzae adults at the midrange of SO2 contents tested (1500 ppm and 4500 ppm) (Table 1 and Fig. 1). Navarro (1978) also found an antagonistic effect between CO2 and a low-O2 MA on the mortality S. oryzae adults, with less time required for 95% mortality at 1% residual O2 than at 0% or 2% O2. The combination of ethyl formate and CO2 for the control of two pests in harvested strawberries, Frankliniella occidentalis (Pergande) and Tetranychus urticae Koch, also produced an antagonistic effect when the content of CO2 increased to above 40% (Simpson et al., 2004). There is little information available on the mechanism of SO2 toxicity in insects. However, Stratford and Rose (1986) demonstrated that SO2 is able to diffuse freely across the hydrophobic cellular membrane, without facilitation via membrane channels. The toxicity potentially involves systemic oxidative damage caused by free radicals formed during sulphite oxidation (Mathew et al., 2011). However, it is known that elevated CO2 produces metabolic arrest and increases membrane permeability (Bell, 1984; Zhou et al., 2001; Mitcham et al., 2006). Thus, it can be inferred that the mode of action of both gases on insect metabolism is diverse and that a synergistic, additive or antagonistic effect can occur at certain contents. Additionally, the presence of different food substrates or the absence of a food substrate in the experimental cages modified the toxic effect of SO2 on S. oryzae and the T. confusum adults (Fig. 2). This result might be due to the different adsorption capacities of the substrates that would have left different free SO2 contents in the atmosphere (Frickhinger, 1934). Riudavets et al. (2008) obtained different sorption curves when almonds, wheat flour and rice were treated with a combination of high content of CO2 (70%) and 3% SO2. The wheat flour rapidly reached the highest sorption values (up to 3500 ppm after 24 h of treatment), with less adsorption by the almonds and rice (a maximum of 2300 ppm after 7 days of treatment), thus leaving less CO2 in the air. In this study, the lower mortalities recorded on the wheat than on the rice have occurred due to this differential in sorption, and stresses the importance of using more than one substrate when testing the efficacy of MAs. The addition of SO2 can be considered as a feasible alternative to reduce the length of the treatment necessary to achieve the control of two important stored product pests by means of a high-CO2 MA, and then compensate or reduce treatment costs. Although results in this study are promising, the presence of SO2 from sulphites could limit the widespread use of SO2 for disinfestations. The official dosages approved in the EU for different food products range from 50 ppm in cereal by-products to 500 ppm in nuts and 2000 ppm in dried fruits (The European Parliament and the Council of the European Union, 2008). However, according to Riudavets et al. (2008) a high degree of desorption occurs after the proper aeration of the food product treated with a combination of CO2 and SO2. For example, when wheat flour was treated with low SO2 contents (50 ppm and 150 ppm), the initial residual contents of SO2 after the treatment was low, and the residual limits also decreased quickly, even to the minimum detection limit of the technique, after ventilation. When treated with 30,000 ppm SO2, the residual content detected was lower than 500 ppm after 7 days of aeration. The use of SO2 might also produce corrosion, bleaching and changes in pH of treated material (Frickhinger, 1934). However, the SO2 contents tested did not produce changes both in pH and in colour to rice and almonds (results not shown). This is something that needs to be further investigated in the future. Although SO2 is known for its toxic effects when adding quite big amounts, the proposed use is within the range used in the food industry (The European Parliament and the Council of the European Union, 2008), and cylinderized gas formulations of CO2 MAs together with SO2 could be made available for commercial use as they are for other fumigants (Emekci, 2010).
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As found in the present work, the different developmental stages show different degrees of sensitivity to the mixture of gases; in all probability, different species would also have variable sensitivities. Therefore, it would be necessary to determine the most effective dosage for each potential pest species to obtain the most effective level for use in control. Furthermore, as with other fumigants, temperatures below 15 C tend to limit the efficacy of highCO2 MAs treatments (Mann et al., 1999; Sauer and Shelton, 2002; Conyers and Bell, 2006). In comparison, SO2 in combination with a low storage temperature was considered a potential alternative to such chemical fumigants as methyl bromide to control pests of regulatory concern in exported table grapes, including F. occidentalis, Pseudococcus maritimus (Ehrhorn), P. stultana, Tetranychus pacificus McGregor and T. urticae (Yokoyama et al., 1999, Yokoyama et al., 2001). The performance of the combined use of SO2 and CO2 at low temperatures for the control of stored-product pests should be investigated in the future. Acknowledgements This work was partially financed by Carburos Metálicos-Air Porducts Group and by CDTI, Ministerio de Ciencia e Innovación, Spanish Government, under the project CENIT SOST-CO2 (CEN2008-1027), a CENIT project belonging to the Ingenio 2010 program, and a grant from the Instituto Nacional de Investigación Agraria y Alimentaria RTA 2011-00025-C02-01 (FEDER). References Ali-Niazee, M.T., 1971. Effect of carbon dioxide gas on respiration of the confused flour beetle. J. Econ. Entomol. 64, 1304e1305. Annis, P.C., Morton, R., 1997. The acute mortality effects of carbon dioxide on various life stages of Sitophilus oryzae. J. Stored Prod. Res. 33, 115e124. Banks, H.J., Annis, P.C., 1990. Comparative advantages of high CO2 and low O2 types of controlled atmospheres for grain storage. In: Calderon, M., Barkai-Golan, R. (Eds.), Food Preservation by Modified Atmosphere. CRC Press, Inc, Boca Raton, Florida, USA, pp. 93e122. Bell, C.H., 1984. Effects of oxygen on the toxicity of carbon dioxide to storage insects. In: Ripp, B.E. (Ed.), Controlled Atmosphere and Fumigation of Grain Storages. Elsevier, Amsterdam, pp. 67e74. Bond, E.J., Buckland, C.T., 1978. Control of insects with fumigants at low temperatures: toxicity of fumigants in atmospheres of carbon dioxide. J. Econ. Entomol. 71, 307e309. Brieger, W., 1918. Gasbäder mit Schwefeldioxyd [Gas baths with SO2]. Naturwissenschaften 50, 739. Conyers, S.T., Bell, C.H., 2006. A novel use of modified atmospheres: storage insect population control. J. Stored Prod. Res. 43, 367e374. Edwards, L.J., Batten II, R.W., 1973. Oxygen consumption in carbon dioxide anesthetized house flies, Musca domestica Linn. (Diptera: Muscidae). Comp. Biochem. Physiol. 44A, 1163e1167. Emekci, M., 2010. Quo Vadis the fumigants? Jul. Kühn Arch. 425, 303e313. FDA, 2012. Food Additives, U.S. Food and Drug Administration [Online]. Available: (accessed 11. 06. 12.). http://www.fda.gov/Food/FoodIngredientsPackaging/ FoodAdditives/default.htm. Fleurat Lessard, F., 1990. Effect of modified atmospheres on insects and mites infesting stored products. In: Calderon, M., Barkai-Golan, R. (Eds.), Food Preservation by Modified Atmospheres. CRC Press, Inc, Boca Raton, Florida, USA, pp. 93e122. Frickhinger, H.W., 1934. Gase in der Schädlingsbekämpfung: eine Zusammenfassung für Amtsärzte, Desinfektoren, Hygieniker, Kommunalverwaltungen usw [Fumigants for pest control: a summary for medical officers, disinfectors, hygienists, local governments, etc.]. Anz. Schädl. 10, 49. Is¸ikber, A.A., 2010. Fumigant toxicity of garlic essential oil in combination with carbon dioxide (CO2) against stored-product insects. Jul. Kühn Arch. 425, 371e376.
Janmaat, A.F., de Kogel, W.J., Woltering, E.J., 2001. Enhanced fumigant toxicity of pcymene against Frankliniella occidentalis by simultaneous application of elevated levels of carbon dioxide. Pest Manag. Sci. 58, 167e173. Lucas, E., Riudavets, J., 2000. Lethal and sublethal effects of rice polishing process on Sitophilus oryzae (Coleoptera: Curculionidae). J. Econ. Entomol. 93, 1837e1841. Mann, D.D., Jayas, D.S., White, N.D.G., Muir, W.E., 1999. Mortality of adult Cryptolestes ferrugineus (Stephens) exposed to changing CO2 concentrations. J. Stored Prod. Res. 35, 385e395. Mathew, N.D., Schlipalius, D.I., Ebert, P.R., 2011. Sulfurous gases as biological messengers and toxins: comparative genetics of their metabolism in model organisms. J. Toxicol. Article ID 394970, doi:10.1155/2011/394970. Mitcham, E.J., Zhou, S., Bikoba, V., 1997. Controlled atmosphere for quarantine control of three pests of table grape. J. Econ. Entomol. 90, 1360e1370. Mitcham, E., Martin, T., Zhou, S., 2006. The mode of action of insecticidal controlled atmospheres. Bull. Entomol. Res. 96, 213e222. Navarro, S., 1978. The effects of low oxygen tensions on three stored-product insect pests. Phytoparasitica 6, 51e58. Navarro, S., 2006. Modified atmospheres for the control of stored product insects and mites. In: Heaps, J.W. (Ed.), Insect Management for Food Storage and Processing. AACC International, St. Paul, Minnesota, USA, pp. 105e145. Navarro, S., 2012. The use of modified and controlled atmospheres for the disinfestation of stored products. J. Pest Sci. 85, 301e322. Nicolas, G., Sillans, D., 1989. Immediate and latent effects of carbon dioxide on insects. Annu. Rev. Entomol. 34, 97e116. Peters, G., 1936. Sammlung Chemischer und Chemisch-Technischer Vorträge, Neue Folge, Heft 31. Chemie und Toxikologie der Schädlingsbekämpfung [Chemistry and toxicology of pest control]. Verlag von Ferdinand Enke, Stuttgart, Germany. Riudavets, J., Gabarra, R., Pons, M.J., Castañé, C., Alomar, O., Guri, S., 2008. Toxicity effects of high carbon dioxide modified atmospheres in combination with sulphur dioxide on the rice weevil Sitophilus oryzae. In: Daolin, G., Navarro, S., Jian, Y., Cheng, T., Zuxum, Yue, L., Yang, L., Haipeng, W. (Eds.), Proceedings of the International Conference on Controlled Atmosphere and Fumigation in Stored Products. Sichuan Publishing Group, Sichuan Publishing House of Science & Technology, China, pp. 21e26. Riudavets, J., Castañé, C., Alomar, O., Pons, M.J., Gabarra, R., 2009. Modified atmosphere packaging (MAP) as an alternative measure for controlling ten pests that attack processed food products. J. Stored Prod. Res. 45, 91e96. SAS Institute, 2002. Enterprise Guide v2.0. SAS Institute, Cary, NC. Sauer, J.A., Shelton, M.D., 2002. High-temperature controlled atmosphere for postharvest control of Indian meal moth (Lepidoptera: Pyralidae) on preserved flowers. J. Econ. Entomol. 95, 1074e1078. Simpson, T., Bikoba, V., Mitcham, E.J., 2004. Effects of ethyl formate on fruit quality and target pests mortality for harvested strawberries. Postharvest Biol. Technol. 34, 313e319. Stratford, M., Rose, A.H., 1986. Transport of sulphur dioxide by Saccharomyces cerevisiae. J. Gen. Microbiol. 132, 1e6. The European Commission, 2012. Food Safety: From the Farm to the Fork [Online]. Available: (accessed 11 June 2012). http://ec.europa.eu/food/fs/sfp/flav_index_ en.html. The European Parliament and the Council of the European Union, 2008. Regulation (EC) no. 1333/2008 of the European Parliament and of the council of 16 December 2008 on food additives. Off. J. Eur. Un. L 354, 16e33. Vail, P.V., Tebbets, J.S., Smilanick, J., 1992. Sulfur dioxide control of omnivorous leafroller in the laboratory. In: Burditt Jr., A.K. (Ed.), Insecticide & Acaricide Tests: 1992. Entomological Society of America, Lanham, MD, pp. 371e372. Vota, M., 1957. Preliminary study on storage of Emperor grapes in controlled atmospheres with and without sulphur dioxide fumigation. Proc. Am. Soc. Hortic. Sci. 69, 250e253. White, N.D.G., Jayas, D.S., Muir, W.E., 1995. Toxicity of carbon dioxide at biologically producible levels to stored product beetles. Environ. Entomol. 24, 640e647. Yokoyama, V.Y., Miller, G.T., Crisosto, C.H., 1999. Low temperature storage combined with sulphur dioxide slow release pads for quarantine control of omnivorous leafroller Platynota stultana (Lepidoptera: Tortricidae). J. Econ. Entomol. 92, 235e238. Yokoyama, V.Y., Miller, G.T., Crisosto, C.H., 2001. Pest response in packed table grapes to low temperature storage combined with slow-release sulfur dioxide pads in basic and large-scale tests. J. Econ. Entomol. 94, 984e988. Zhou, S., Criddle, R.S., Mitcham, E.J., 2001. Metabolic response of Platynota stultana pupae under and after extended treatment with elevated CO2 and reduced O2 concentrations. J. Insect Physiol. 47, 401e409.
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Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
The toxicity effects of atmospheres with high content of carbon dioxide with addition of sulphur dioxide on two stored-product pest species: Sitophilus oryzae and Tribolium confusum Jordi Riudavets a, *, Maria José Pons b, Rosa Gabarra a, Cristina Castañé a, Oscar Alomar a, Lourdes F. Vega b, c, Sonia Guri b, c a b c
IRTA, Entomology, Ctra. Cabrils Km 2, E-08348 Cabrils, Barcelona, Spain MatGas-Research Center, Campus UAB, E-08193 Bellaterra, Barcelona, Spain Carburos Metálicos e Air Products Group, Aragón 300, E-08009, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 7 December 2013
Modified atmospheres (MAs) are safe and environmentally friendly alternatives to control pests in stored products. However, to accommodate the requirements of the food industry, the control of insects by a MA can be a too lengthy process. This paper describes the potential of sulphur dioxide as an additive to reduce the long lethal exposure of modified atmospheres (MA) enriched with carbon dioxide for major stored product pests. Specifically, we evaluated whether the addition of SO2 (0e30,000 ppm) to a highCO2 content of 70%e95% MA could enhance its insecticidal effect for the control of Sitophilus oryzae and Tribolium confusum. The addition of 15,000 ppm and 30,000 ppm of SO2 to 95% CO2 enhanced control up to 100% in comparison to CO2 alone for S. oryzae and T. confusum adults when treated for one day in all of the substrates tested. However, the effectiveness of adding SO2 at reduced contents was lower and depended on the substrate and pest species considered. The addition of SO2 also increased the mortality of all of the developmental stages of S. oryzae. The increase in mortality with the addition of SO2 was also observed when included with 70% CO2. Therefore, the addition of SO2 can be considered a feasible means of shortening the length of treatment necessary to achieve the control of these two pests using a highCO2 MA. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Modified atmospheres Carbon dioxide Sulphur dioxide Pest control Sitophilus oryzae Tribolium confusum
1. Introduction Food product commodities can be affected by insect pests during the storage period, and visible contamination due to insect individuals or their remains may be present in the final product. Yet, there are increasing restrictions on the number of active chemical compounds officially registered for pest control and on the maximum residue levels (MRLs) allowed in the final food products. It is, therefore, necessary to implement alternative strategies for the control of pests in stored food products. Modified atmospheres (MAs) based on a high content of carbon dioxide (CO2) offer a suitable, safe and environmentally friendly alternative method for controlling pests that affect food products (Navarro, 2006). MAs can be applied in hermetic structures at different steps of food processing, from the storage of bulk raw and semi-processed materials to the packaging process of the product * Corresponding author. Tel.: þ34 937507511; fax: þ34 937533954. E-mail addresses: [email protected], [email protected] (J. Riudavets). 0022-474X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jspr.2013.12.003
for the end consumer. The main advantages of using a high-CO2 MA are that this method is effective for the control of a wide range of pest species and that it can be used for the treatment of different food commodities without the accumulation of toxic residues after the treatment. However, to accommodate the requirements for marketing in the food industry, the control of insects by means of MAs has the disadvantage of needing a relatively long treatment time. To enhance the use of MAs, it is necessary to find alternatives that allow the reduction of the length of the treatment without compromising the control efficacy. Toxic effects of CO2 are well known and data on the effects of different types of CO2 treatments and dosages are available for many species and stages of storedproduct pests under particular sets of conditions (Banks and Annis, 1990; White et al., 1995; Riudavets et al., 2009; Navarro, 2012). The recommended exposure time to achieve complete pest control at the most appropriate gas content ranges from several days to weeks, depending on the CO2 content, temperature, developmental stage and species (Fleurat Lessard, 1990). For
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example, to control one of the most MA-tolerant pest species, the rice weevil Sitophilus oryzae (L.), the estimated LT95 values using 40%e100% CO2 MA at 25 C range between 1 and 4 days for adults and between 3 and more than 5 days for eggs (Annis and Morton, 1997). Moreover, to achieve full mortality for all of the developmental stages of this species, treatments should last 12 days when using an MA of 90% CO2 at 25 C (Riudavets et al., 2009). With the exception of diapausing Trogoderma granarium Everts larvae, Banks and Annis (1990) reported that, of 10 coleopteran stored-product pests tested, S. oryzae was the species most tolerant to a high-CO2 MA. The beetle Tribolium confusum Jacquelin du Val, one of the most important coleopteran pest present in flour and feed mills, is in comparison less tolerant to MA, but their pupae are also difficult to kill, requiring 8 days with an MA of 90% CO2 at 25 C (Riudavets et al., 2009). Because an MA with high contents of CO2 causes the permanent opening of insect spiracles (Nicolas and Sillans, 1989; Mitcham et al., 2006), a synergistic toxic effect of CO2 when combined with other compounds has been sought. For example, adding CO2 to certain fumigants, such as acrylonitrile, methyl bromide, phosphine, carbon disulfide, ethylene oxide, chloropicrin, methyl formate and hydrogen cyanide, increases their toxicity and permits the reduction of treatment times (Bond and Buckland, 1978; Peters, 1936). Similarly, according to Is¸ikber (2010), the fumigant toxicity of a garlic essential oil was enhanced (more than 4.9-fold) in combination with carbon dioxide (92% CO2) for the control of T. confusum. Sulphur dioxide (SO2) is a gas that is already accepted as food additive (E-220) in a number of countries for several nonperishable food products, such as cereal by-products, nuts and dried fruits (FDA, 2012; The European Commission, 2012). Although SO2 mainly has a long history as a food preservative due to its antimicrobial properties in a range of food products, it has also been used for the control of a number pests (Brieger, 1918), among them the stored product pests Plodia interpunctella (Hübner) and Ephestia spp. (Peters, 1936). More recently, it has been proposed for the control of some insect pest of grapes during storage (Yokoyama et al., 2001). Fumigation with SO2 (routinely applied for fungal control), alone or combined with carbon dioxide, may provide pest control, though this possibility has received little attention (Vota, 1957). Only Vail et al. (1992) reported that SO2 contents comparable to those used in the routine fumigation of grapes killed the key pest Platynota stultana Walshingham (Lepidoptera: Tortricidae) in a laboratory experiment, suggesting that SO2 has the potential to control both fungi and insects. Preliminary work with S. oryzae on rice (Riudavets et al., 2008) indicated the potential of such mixtures for pest control. The objective of this research was to evaluate the extent to which the addition of a range of SO2 contents to a high-CO2 MA could enhance the insecticidal effect of the CO2 MA for the control of two important stored-product pest species, S. oryzae and T. confusum, on different food substrates. Sitophilus oryzae was chosen as a model for cereal grain pests, and T. confusum as a model for cereal by-product pests. In the laboratory, we evaluated the effect of two CO2 MAs (70% CO2 and 95% CO2) combined with different SO2 contents (0e30,000 ppm) on the reduction of emergence of all of the developmental stages of S. oryzae and on the mortality of the adults of T. confusum at a temperature of 25 C. 2. Material and methods The insects used for the experiments originated from the colonies maintained at IRTA (Cabrils, Barcelona). Sitophilus oryzae was reared on brown rice and T. confusum on wheat flour and yeast. The adults, eggs, larvae and pupae of S. oryzae and adults of T. confusum
59
were separately evaluated. In the case of S. oryzae, the larvae and pupae were tested together (referred to as ‘larvae-pupae’). All of the laboratory studies were conducted in a climatic chamber at 25 2 C and 70 15% r.h. The experimental arenas consisted of ventilated plastic cages (7.5 cm in diameter and 3 cm high) containing 50 g brown rice, 20 g wheat flour or no food substrate, depending on the experimental condition. The sides of the arenas were painted with FluonÔ to avoid the escape of the insects during the treatment. A total of fifty individuals were added to the arenas, except in the case of the eggs and larvaeepupae of S. oryzae, for which the diet was previously infested with adults (410 adults/kg of brown rice for 7 days) to produce at least 50 individuals in the grains in each arena. The effectiveness of two high-CO2 modified atmospheres (70% CO2 and 95% CO2) combined with different SO2 contents (0e 30,000 ppm) was evaluated. A range of exposure times between 1 day and 5 days were tested according to the species, developmental stage and previous results (Riudavets et al., 2008, 2009). The insects were exposed to each gas mixture in sealed glass desiccators (200 mm in diameter). Three cages of each species and stage were exposed in the same desiccators, with five replications for each gas mixture and exposure time according to each experiment. The gas mixtures were first prepared in cylinders, and the gas content was verified (Carburos Metálicos e Air Products Group). The MAs contained 5% O2, 70% or 95% CO2, and were balanced to 100% with N2 according to the SO2 treatment. The gas mixtures from the cylinders were continuously flushed into the desiccators at a pressure of 2 bars until the desired gas contents were reached. A gas analyser (Abiss model TOM 12) was used to monitor the contents of CO2 and O2 inside the desiccators during the experiment. After the treatment, the desiccators were opened, and the cages were kept in the climatic chamber. To evaluate the mortality of the adults, the number of living individuals in each cage was counted 24 h after the end of the treatment. The percentage mortality was calculated using the initial number of individuals placed in each cage. Sets of control cages were used to determine the percentage of natural mortality. For the eggs and larvaeepupae of S. oryzae, the emerging weevil adults were counted after 9 and 4 weeks, respectively. The number of living adults in each arena was counted, and the percentage of mortality was calculated using the number of individuals that emerged in the control cages. The significant differences in terms of the percentage of mortality for the different stages of the two species tested when exposed to each of the different experimental conditions were analysed using an analysis of Variance (ANOVA) (SAS Institute, 2002). All of the percentage data sets were arcsine transformed to meet the homogeneity and normality assumptions for the analysis of variance. The Tukey Multiple Range test was used to compare the mean values. 3. Results The addition of 15,000 ppm and 30,000 ppm of SO2, respectively to an MA of 95% CO2 ensured the full mortality (100%) of the S. oryzae and T. confusum adults when treated for an exposure time of 1 day using all of the substrates tested (Table 1). However, the effectiveness of adding SO2 at reduced contents was lower and depended on the substrate and pest species (Table 1). For the S. oryzae adults, the presence of the substrate (rice) was reflected in significantly lower mortalities in comparison to the results when no substrate was added. This was already observed without adding SO2, with a lower mortality using rice as a food substrate (81.2%) than when no food substrate was present (99.7%). The addition of 150 ppm of SO2 had no improvement of the effect of CO2 compared to the ‘no-SO2’ treatment (Table 1), and the effect of
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Table 1 Total mortality (%; Mean SEM) of S. oryzae and T. confusum adults exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 1 day (24 h) on different food substrates. Content of SO2
S. oryzae
0 ppm 150 ppm 1500 ppm 4500 ppm 15,000 ppm 30,000 ppm P F df
81.2 1.54 72.6 7.27 29.3 2.76 18.7 1.51 100 a 100 a <0.001 227.12 5; 89
T. confusum
Rice
Wheat flour
No food substrate 99.7 0.16 95.9 0.82 72.7 3.90 98.1 0.65 100 a 100 a <0.001 69.03 5; 89
b b c d
a a b a
43.7 4.58 45.7 0.68 39.4 3.49 95.6 0.65 100 a 100 a <0.001 303.79 5; 89
Rice b b b a
51.5 8.10 b 51.9 8.11 b 93.4 1.40 a 100 a 100 a 100 a <0.001 64.52 5; 89
Means within a given column followed by the same lowercase letter are not significantly different (Tukey test, P < 0.05).
the substrate was maintained. A large decrease in mortality was observed at 1500 ppm and 4500 ppm of SO2 in the presence of rice, reducing the lethal effect to only 29% and 19%, respectively. However, without any food substrate, the decrease in mortality was only significant with the addition of 1500 ppm, whereas a high mortality (98.1%) was obtained at 4500 ppm SO2. Figure 1 is used to highlight this antagonistic effect between CO2 and SO2 at the intermediate range of SO2 tested. When exposed to the MA without SO2, the T. confusum adult mortalities were lower than the mortalities of S. oryzae and slightly but significantly lower on the wheat than on the rice (Table 1, Fig. 2). Adding 150 ppm of SO2 did not increase the mortality of T. confusum. The addition of 1500 ppm or 4500 ppm of SO2 to the rice substrate did not produce the reducing effect concerning the mortality observed with S. oryzae, and the mortality increased up to 93.4% and 100%, respectively. However, when 1500 ppm SO2 were added to the wheat flour substrate, the mortality remained low (39.4%) and only significantly increased with 4500 ppm, which was still significantly lower (95.6%) than on the rice. The addition of SO2 also significantly and progressively increased the mortality of the S. oryzae eggs and larvaeepupae in the three days of exposure to an MA of 95% CO2 (Table 2). The addition of 150 ppm and 1500 ppm SO2 slightly but significantly increased the egg mortality, but the mortality was higher than 99% only with 4500 ppm SO2. In the case of the larvaeepupae, the addition of 150 ppm of SO2 did not increase the mortality, but the addition of 1500 and 4500 ppm SO2 doubled the mortality, though
mortality (%)
100
*
*
*
*
100 mortality (%)
60
*
*
*
80 60 40 20 0 0
150
1,500 4,500 15,000 30,000 ppm
Fig. 2. Total mortality (%; Mean SEM) of T. confusum adults exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 1 day in rice (broken line) and wheat flour (straight line). Significant differences between the substrates for each SO2 content are indicated with an asterisk (P < 0.05).
the mortality only reached an average of 61%. The mortality reached 100% with an addition of 30,000 ppm SO2. An increase in mortality with the addition of SO2 was also observed when the gas was added to an MA with a lower CO2 content of 70% (Table 3). With 30,000 ppm, the mortality increased significantly to 100% for all of the developmental stages and both pest species tested, with the exception of the larvaeepupae of S. oryzae. Nevertheless, in this case, the mortality was also high (82.7%). Although no intermediate contents of SO2 mixed with 70% CO2 were tested, the lowest content tested (150 ppm) did not increase the mortality significantly, except for the eggs of S. oryzae. 4. Discussion The addition of SO2 has been useful to improve the control efficacy of high-CO2 MAs against all of the developmental stages of S. oryzae and the adults of T. confusum. The increase in efficacy would allow a practical reduction of the treatment time. Hence, the addition of 15,000 ppm SO2 to an MA of 95% CO2 reduced the time necessary to reach 100% mortality to only 1 day for S. oryzae and the T. confusum adults at a temperature of 25 C (Table 1). In comparison, without the addition of SO2, the full mortality of S. oryzae and T. confusum adults requires more than 4 days with an MA of 90% CO2 at 25 C (Riudavets et al., 2009). Using a content of 80% CO2, Banks and Annis (1990) reported a treatment time of 3.5 and 2 days to obtain at least 95% mortality of S. oryzae eggs and T. confusum adults, respectively, at 25 C. Annis and Morton (1997) also reported a treatment time of more than 1 day and more than 4 days for the control (99% mortality) of S. oryzae adults and eggs, respectively, when treated with 95% CO2 at 25 C. Table 2 Total mortality (%; Mean SEM) of larvaeepupae and eggs of S. oryzae exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 3 days in rice.
80 60 40 20 0 0
150
1,500 4,500 15,000 30,000 ppm
Fig. 1. Total mortality (%; Mean SEM) of S. oryzae adults exposed to 95% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm, 1500 ppm, 4500 ppm, 15,000 ppm and 30,000 ppm) for 1 day in rice (broken line) and without any food substrate (dotted line). Significant differences between the substrates for each SO2 content are indicated with an asterisk (P < 0.05).
Content of SO2
Eggs
0 ppm 150 ppm 1500 ppm 4500 ppm 15,000 ppm 30,000 ppm P F df
60.3 2.07 68.7 1.62 70.8 1.00 99.3 0.14 100 a 100 a <0.001 786.42 5; 89
LarvaeePupae d c c b
25.3 4.08 12.7 2.53 53.1 1.64 60.6 2.18 94.0 0.98 100 a <0.001 198.12 5; 89
d e c c b
Means within a given column followed by the same lowercase letter are not significantly different (Tukey test, P < 0.05).
J. Riudavets et al. / Journal of Stored Products Research 57 (2014) 58e62 Table 3 Total mortality (%; Mean SEM) of adults, larvaeepupae and eggs of S. oryzae and adults of T. confusum exposed to 70% CO2 at 25 C in combination with different SO2 contents (0 ppm, 150 ppm and 30,000 ppm) during 1 day (adults) and 3 days (eggs and larvaeepupae) in rice (S. oryzae) and wheat flour (T. confusum). Content of SO2
S. oryzae Eggs
LarvaeePupae
Adults
Adults
0 ppm 150 ppm 30,000 ppm P F df
63.7 1.29 c 97.8 0.4 b 100 a <0.001 900.29 2; 44
30.7 3.01 b 31.4 1.74 b 82.7 1.83 a <0.001 155.67 2; 44
41.7 3.38 b 48.2 8.72 b 100 a <0.001 139.37 2; 44
15.8 1.3 b 12.1 0.94 b 100 a <0.001 1727.38 2; 44
T. confusum
Means within a given column followed by the same lowercase letter are not significantly different (Tukey test, P < 0.05).
Furthermore, the addition of SO2 to a high-CO2 MA would also allow a reduction in the treatment time of the pupae of S. oryzae, the most tolerant developmental stage of this species. In the present experiment, full mortality was observed after 3 days of exposure to a combination of 95% CO2 and 30,000 ppm SO2 (Table 2). In comparison, Annis and Morton (1997) reported a much longer treatment time of 15.3 days of exposure with 95% CO2 only for the control of this developmental stage. According to Riudavets et al. (2009), without the addition of SO2, 8 days would be necessary to achieve 100% mortality of S. oryzae pupae at 90% CO2 and 25 C. Banks and Annis (1990) also reported a long treatment time of 8.5 days to obtain at least 95% mortality using a content of 80% CO2. However, the addition of less than 15,000 ppm of SO2 to a highCO2 MA does not always guarantee an increase in its efficacy. For example, the addition of 1500 ppm SO2 to a 95% CO2 MA significantly increased the mortality of the S. oryzae eggs and larvaee pupae in rice (Table 2). In contrast, the addition of 1500 ppm and 4500 ppm SO2 to rice produced the opposite effect in the adults, and a significant decrease in their mortality was recorded (Table 1). Similarly, the addition of 1500 ppm SO2 did not enhance the mortality of the T. confusum adults when treated in the presence of wheat flour. The addition of 150 ppm of SO2 did not increase the mortality of the adults and pupae of S. oryzae or the adults of T. confusum but did enhance the mortality of eggs of S. oryzae, especially at 70% CO2. This is important because the eggs of S. oryzae usually become the most important key stage to control, as they are able to survive standard industrial processes, such as cereal milling and rice polishing, whereas most of the pupae and adults are killed (Lucas and Riudavets, 2000). The increase in mortality of the S. oryzae eggs obtained with the addition of 150 ppm SO2 was important with 70% CO2 (Table 3) but only produced a slight increase when the CO2 content was higher (95%) (Table 2). It is likely that the increase of mortality induced by SO2 was not as strong with the highest CO2 content tested due to a greater initial shock effect, which is typically produced in insects at high-CO2 atmospheres (Edwards and Batten, 1973; Nicolas and Sillans, 1989). The highest CO2 content might have produced a greater shock and, consequently, a reduced SO2 uptake by the individuals tested. Several authors have also reported comparable antagonistic effects; for example, lower than expected mortality rates were recorded when high-CO2 contents were added to low O2 environments (Ali-Niazee, 1971; Mitcham et al., 1997). Other fumigants combined with CO2 have shown a similar pattern of producing higher mortality with lower CO2 contents (Bond and Buckland, 1978). Enhanced opening of the spiracles may increase mortality by increasing the fumigant uptake. However, a heavy narcotic effect occurs at high-CO2 contents, with metabolic effects, mainly due to changes in the pH, then becoming the more important determinant of mortality (Janmaat et al., 2001).
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The current knowledge cannot explain the antagonist effect between CO2 and SO2 on the S. oryzae adults at the midrange of SO2 contents tested (1500 ppm and 4500 ppm) (Table 1 and Fig. 1). Navarro (1978) also found an antagonistic effect between CO2 and a low-O2 MA on the mortality S. oryzae adults, with less time required for 95% mortality at 1% residual O2 than at 0% or 2% O2. The combination of ethyl formate and CO2 for the control of two pests in harvested strawberries, Frankliniella occidentalis (Pergande) and Tetranychus urticae Koch, also produced an antagonistic effect when the content of CO2 increased to above 40% (Simpson et al., 2004). There is little information available on the mechanism of SO2 toxicity in insects. However, Stratford and Rose (1986) demonstrated that SO2 is able to diffuse freely across the hydrophobic cellular membrane, without facilitation via membrane channels. The toxicity potentially involves systemic oxidative damage caused by free radicals formed during sulphite oxidation (Mathew et al., 2011). However, it is known that elevated CO2 produces metabolic arrest and increases membrane permeability (Bell, 1984; Zhou et al., 2001; Mitcham et al., 2006). Thus, it can be inferred that the mode of action of both gases on insect metabolism is diverse and that a synergistic, additive or antagonistic effect can occur at certain contents. Additionally, the presence of different food substrates or the absence of a food substrate in the experimental cages modified the toxic effect of SO2 on S. oryzae and the T. confusum adults (Fig. 2). This result might be due to the different adsorption capacities of the substrates that would have left different free SO2 contents in the atmosphere (Frickhinger, 1934). Riudavets et al. (2008) obtained different sorption curves when almonds, wheat flour and rice were treated with a combination of high content of CO2 (70%) and 3% SO2. The wheat flour rapidly reached the highest sorption values (up to 3500 ppm after 24 h of treatment), with less adsorption by the almonds and rice (a maximum of 2300 ppm after 7 days of treatment), thus leaving less CO2 in the air. In this study, the lower mortalities recorded on the wheat than on the rice have occurred due to this differential in sorption, and stresses the importance of using more than one substrate when testing the efficacy of MAs. The addition of SO2 can be considered as a feasible alternative to reduce the length of the treatment necessary to achieve the control of two important stored product pests by means of a high-CO2 MA, and then compensate or reduce treatment costs. Although results in this study are promising, the presence of SO2 from sulphites could limit the widespread use of SO2 for disinfestations. The official dosages approved in the EU for different food products range from 50 ppm in cereal by-products to 500 ppm in nuts and 2000 ppm in dried fruits (The European Parliament and the Council of the European Union, 2008). However, according to Riudavets et al. (2008) a high degree of desorption occurs after the proper aeration of the food product treated with a combination of CO2 and SO2. For example, when wheat flour was treated with low SO2 contents (50 ppm and 150 ppm), the initial residual contents of SO2 after the treatment was low, and the residual limits also decreased quickly, even to the minimum detection limit of the technique, after ventilation. When treated with 30,000 ppm SO2, the residual content detected was lower than 500 ppm after 7 days of aeration. The use of SO2 might also produce corrosion, bleaching and changes in pH of treated material (Frickhinger, 1934). However, the SO2 contents tested did not produce changes both in pH and in colour to rice and almonds (results not shown). This is something that needs to be further investigated in the future. Although SO2 is known for its toxic effects when adding quite big amounts, the proposed use is within the range used in the food industry (The European Parliament and the Council of the European Union, 2008), and cylinderized gas formulations of CO2 MAs together with SO2 could be made available for commercial use as they are for other fumigants (Emekci, 2010).
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As found in the present work, the different developmental stages show different degrees of sensitivity to the mixture of gases; in all probability, different species would also have variable sensitivities. Therefore, it would be necessary to determine the most effective dosage for each potential pest species to obtain the most effective level for use in control. Furthermore, as with other fumigants, temperatures below 15 C tend to limit the efficacy of highCO2 MAs treatments (Mann et al., 1999; Sauer and Shelton, 2002; Conyers and Bell, 2006). In comparison, SO2 in combination with a low storage temperature was considered a potential alternative to such chemical fumigants as methyl bromide to control pests of regulatory concern in exported table grapes, including F. occidentalis, Pseudococcus maritimus (Ehrhorn), P. stultana, Tetranychus pacificus McGregor and T. urticae (Yokoyama et al., 1999, Yokoyama et al., 2001). The performance of the combined use of SO2 and CO2 at low temperatures for the control of stored-product pests should be investigated in the future. Acknowledgements This work was partially financed by Carburos Metálicos-Air Porducts Group and by CDTI, Ministerio de Ciencia e Innovación, Spanish Government, under the project CENIT SOST-CO2 (CEN2008-1027), a CENIT project belonging to the Ingenio 2010 program, and a grant from the Instituto Nacional de Investigación Agraria y Alimentaria RTA 2011-00025-C02-01 (FEDER). References Ali-Niazee, M.T., 1971. Effect of carbon dioxide gas on respiration of the confused flour beetle. J. Econ. Entomol. 64, 1304e1305. Annis, P.C., Morton, R., 1997. The acute mortality effects of carbon dioxide on various life stages of Sitophilus oryzae. J. Stored Prod. Res. 33, 115e124. Banks, H.J., Annis, P.C., 1990. Comparative advantages of high CO2 and low O2 types of controlled atmospheres for grain storage. In: Calderon, M., Barkai-Golan, R. (Eds.), Food Preservation by Modified Atmosphere. CRC Press, Inc, Boca Raton, Florida, USA, pp. 93e122. Bell, C.H., 1984. Effects of oxygen on the toxicity of carbon dioxide to storage insects. In: Ripp, B.E. (Ed.), Controlled Atmosphere and Fumigation of Grain Storages. Elsevier, Amsterdam, pp. 67e74. Bond, E.J., Buckland, C.T., 1978. Control of insects with fumigants at low temperatures: toxicity of fumigants in atmospheres of carbon dioxide. J. Econ. Entomol. 71, 307e309. Brieger, W., 1918. Gasbäder mit Schwefeldioxyd [Gas baths with SO2]. Naturwissenschaften 50, 739. Conyers, S.T., Bell, C.H., 2006. A novel use of modified atmospheres: storage insect population control. J. Stored Prod. Res. 43, 367e374. Edwards, L.J., Batten II, R.W., 1973. Oxygen consumption in carbon dioxide anesthetized house flies, Musca domestica Linn. (Diptera: Muscidae). Comp. Biochem. Physiol. 44A, 1163e1167. Emekci, M., 2010. Quo Vadis the fumigants? Jul. Kühn Arch. 425, 303e313. FDA, 2012. Food Additives, U.S. Food and Drug Administration [Online]. Available: (accessed 11. 06. 12.). http://www.fda.gov/Food/FoodIngredientsPackaging/ FoodAdditives/default.htm. Fleurat Lessard, F., 1990. Effect of modified atmospheres on insects and mites infesting stored products. In: Calderon, M., Barkai-Golan, R. (Eds.), Food Preservation by Modified Atmospheres. CRC Press, Inc, Boca Raton, Florida, USA, pp. 93e122. Frickhinger, H.W., 1934. Gase in der Schädlingsbekämpfung: eine Zusammenfassung für Amtsärzte, Desinfektoren, Hygieniker, Kommunalverwaltungen usw [Fumigants for pest control: a summary for medical officers, disinfectors, hygienists, local governments, etc.]. Anz. Schädl. 10, 49. Is¸ikber, A.A., 2010. Fumigant toxicity of garlic essential oil in combination with carbon dioxide (CO2) against stored-product insects. Jul. Kühn Arch. 425, 371e376.
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