# Effect of temperature on insecticidal efficiency of hypercarbic atmospheres against three insect species of packaged foodstuffs

Francis F. Fleurat-Lessard, J.M. Le Torc'H, Gisele Marchegay

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Francis F. Fleurat-Lessard, J.M. Le Torc'H, Gisele Marchegay. Effect of temperature on insecticidal efficiency of hypercarbic atmospheres against three insect species of packaged foodstuffs. 7. International Working Conference, Oct 1998, Beijing, China. hal-02770256

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# Effect of temperature on insecticidal efficiency of hypercarbic atmospheres against three insect species of packaged foodstuffs

Francis Fleurat-Lessard, Jean-Marc Le Torc'h and Gisèle Marchegay<fnref n="1" />

## Abstract

Eggs, larvae and adults of the most noxious insect species for packaged cereal foods entering the marketing channel, 1. e. *Tribolium confusum*, *Dermestes maculatus* and *Plodia interpunctella* (except adult stage), were exposed to hypercarbic modified atmospheres (40, 60 and 80% CO₂) at three temperature levels (20, 27.5 and 35°C) in hermetic enclosures

The exposure time giving 50 or 99% kill was determined. For *T. confusum*, the most tolerant stage was the larval one. The TL 99 was in a range of 1.8 to more than 9 days at the extreme conditions i e 80% CO₂ at 35°C and 40% CO₂ at 20°C, respectively. There was not a significant difference between stages for *D. maculatus* and only a limited enhancement of temperature rise for the reduction of TL 99. Nevertheless, the effect of CO₂ concentration rise from 40 to 80% decreased the survival time by a three fold factor. Larvae of *P. interpunctella* were much more tolerant than eggs and the enhanced killing effect consecutive to CO₂ concentration increase was also more than three fold at 35°C

The combination of CO₂ modified atmospheres packaging with a moderately high temperature (35°C) maintained during five days at least could be considered as an effective preventive measure for the eradication of main insect pests in cereal foods products.

## Introduction

Insect-proof packaging of dry foodstuffs can greatly reduce the infestation by insect pests when the product is in the commercial channels. Such an advantage can be lost if the package protection is broken when the product is still in infested areas of storage where infested goods can neighbour

sound ones or on the shelves in commercial shops. In order to avoid part of the inconvenience of the discovery of an infestation by the consumer, the manufacturer can fumigate packed foodstuffs but this is realistic only when two conditions are satisfied: the packaging enclosure must not be airtight and the time allowed for the fumigation must be as short as possible. These two conditions are fulfilled for fumigation with methyl bromide (CH₃Br) or by high-pressure carbon dioxide (CO₂) treatment (Le Torc'h and Fleurat-Lessard 1990; Prozell and Reichmuth 1990; Reichmuth and Wohlgemuth 1994). Every of these two solutions in agreement with the requirements of the users need alternatives because CH₃Br will be banned from year 2002 and high-pressure CO₂ equipment implies a very expensive investment and liquid CO₂ costs are too high to be included in the market price of a majority of food products. However, most of the previous works on modified atmospheres (MA) enriched with CO₂ (also called hypercarbic atmospheres) were restricted to the use of CO₂ in the same manner as a fumigant with high concentrations maintained during a short time in a moderately airtight enclosure. This process is in all cases more expensive than classical fumigation with methyl bromide for two main reasons:

1. The MA has to replace a volume of air equal to the whole capacity of the fumigation enclosure while a classical fumigation needs only a small amount of fumigant (e. g. 32 g·m⁻³·h⁻<fnref n="1" /> for CH₃Br).

2. CO₂ has a specific insecticidal effect only from a concentration in air of 35% (v/v) (Fleurat-Lessard 1990; Adler 1994) and the time of exposure required to kill insects (especially eggs and pupal stages) is much longer than in using a fumigant (even hydrogen phosphide PH₃), except if CO₂ concentration is close to 100%.

These issues which theoretically limit the interest of MA packaging of processed dry food-products to prevent infestation by insect pests in the commercial channel before the purchase by a consumer have not been addressed adequately in the context of high-tech food industry. On the first hand, it is commonly accepted that the food-product manufacturer must secure the product for any damage, contamination or infestation until the limit date allowed for shelf-life before consumption is over. In

<footnote n="1">¹INRA, Laboratoire des Insectes des Denrees Stockées, B P No 81, F 33883 Villenave d'Ornon cedex (France)
E-mail: francis fleurat-lessard @ bordeaux.inra fr-tel: +33 5 56843290</footnote>
order to satisfy this major constraint, the combination of plastic film packaging and injection of CO₂-enriched MA is under rapid development for dry food-products and this is recognised as a true alternative to post-fabrication fumigation. On the other hand, the long-term protection against insect infestation offered by the airtight enclosure is one of the advantages of this technique compared to the absence of persistent protection after a classical fumigation. Additionally, the recent progresses on the objective knowledge on selective permeability properties of plastic films available for industrial packaging of food products for the main natural gases such as nitrogen, oxygen, CO₂ or water vapour enable to keep constant the CO₂ concentration over a long period of time after the sealing operation. This technique of airtight packaging under a CO₂-enriched MA is becoming a current practice in the food-industry for high added-value dry food products such as cereal flours, rice, pasta, cereal flakes, breakfast mixes, dried fruits, pet-food, dehydrated plants, etc.

However, if many studies have been done on the effects of CO₂ at a high concentration on stored-product insects, there is little information about the effects of a relatively low rate of CO₂ during long-term conservation in the commercial channel, before customer will purchase it. The killing effect of moderately low concentrations of CO₂ during the long-term stay of food products in the commercial companies stores or on the shelves in the marketing shops has not been thoroughly studied, especially on the insect species which are major pests of these processed dry food products such as the Indian meal moth, *Plodna interpunctella* (Huebner), the hide beetle, *Dermestes maculatus* (de Geer) and the confused flour beetle, *Tribolium confusum* Jacquelin du Val. The interactions of CO₂ concentration level with temperature level are not clearly established for these species, which have been rarely taken as models for the prediction of the combined effects of temperature and CO₂.

Consequently, the objective of the present work was the determination of the tolerance of the different stages of the most noxious species potentially present in packaged dry food-products, to different levels of CO₂ concentration from the efficiency limit of 40% in air (v/v), in conditions of long-term exposure periods in a highly airtight enclosure and at different levels of temperature.

# Material and Method

## Insects and foodstuffs

All insects were taken from laboratory stock cultures maintained either at 25°C or 30°C and 70% relative humidity (rh). *P. interpunctella* was reared at 25°C in a climatic chamber with a photoperiod of 16 h on a mixture of crushed maize supplemented with 5% glycerol. The larvae were collected from the harbourage of corrugated cardboard when the last larval instar moved from the substrate to a pupation shelter. *D. maculatus* was reared at 30°C in the

darkness on dog food and larvae or adults of known age-range were directly collected in stock cultures. *T. confusum* was reared in the same conditions than previous species on whole wheat flour with a black cardboard sheet on the substrate where adults were regularly collected. Larvae were collected inside the substrate after separation by gentle sieving. *T. confusum* eggs were collected in white wheat flour substrate from oviposition of a limited number of females isolated from the stock culture at the pupal stage and then paired with an equal number of males for a week after adult emergence. After this period of sexual maturation, the couples were put in 'oviposition boxes' with a side replaced by a metallic sieve. This side of the oviposition boxes was placed directly on white wheat flour in a larger box. The females could lay eggs through the aperture of the within the white flour. This system limited the egg cannibalism which is a natural behaviour of adults of this species. For *P. interpunctella* two virgin moth couples were placed in an aerated box containing crushed maize kernels. For *D. maculatus*, mature young adults of the two sexes were put for an oviposition period of one week directly on finely ground dog food. After one week oviposition, the adults were removed and the substrate containing the eggs was dispatched in several fractions which were used indifferently as experimental parcels or as untreated control. In these conditions, all the different age instars were present in the egg collect.

For two of the three species, the three important stages (1 e eggs, larvae and adults) were exposed separately to the MAs. For the Indian meal moth, the adult (moth) was not exposed to the MAs.

## Exposure protocol and control of mortality in exposed insects

Insects were exposed in 1.5 L capacity glass containers equipped with inlet and outlet valves fitted to the system of gas flushing (Fig 1). The different concentrations of CO₂ were accurately fixed at 20, 40 and 60% with an air balance by the means of an electronic regulation of three massic flowmeters, each of them being connected to a pure gas supply of either N₂, O₂ or CO₂ gas flush was maintained at a flow-rate of 1 ml. min⁻¹ after the initial purge at a higher rate just after the insects were placed inside the containers. There was one run of the exposure chamber for each temperature of exposure, 1. e 20, 27.5 and 35°C (Table 1). Every day after initial purge, some insect boxes were withdrawn from the exposure containers and after a 24-h period allowed for vital function recovery in exposed insects, mortality was assessed by direct visual observation. For the eggs, the boxes with exposed insects and those with unexposed control batches were put in an incubation chamber in usual conditions of rearing and the control was delayed until the first larval instar has emerged. In each condition of the bioassay, there was a parallel untreated control where the
average number of emergent larvae was considered as the number of viable eggs present in all the corresponding treated batches. Each experimental condition was achieved with four replicates, and the control included eight replicates for one date of control for each developmental stage of each species for each temperature level.

## Method of presentation of results

The mortality rate of larvae or adults was plotted against time of exposure for all experimental conditions. After the translation of the percentages into probit units, the lethal time 50% (LT50) and 99% (LT99) were estimated from the linear regression. The survival rate in eggs was calculated by comparison of the counts observed in exposed batches and in untreated control batches. The LT50 and LT99 were then grouped for each developmental stage and for each species in a three-dimension graphical representation with the three explanatory variables on the same figure: CO₂ concentration, temperature level and exposure time by the means of EXCEL© 5 software.

## Results

The trends of the influence of temperature and CO₂

concentration level are similar with the three species (Table 2) when developmental stages are considered separately.

For *P. interpunctella*, the larval stage was the most tolerant one, compared to eggs. At 20°C, LT99 was observed respectively after 9, 4 and 3 days of exposure in CO₂ concentrations of 40, 60 and 80% respectively (Fig. 2d). For eggs, the LT99 was reduced between 3 to 2.5 days exposure in the same range of CO₂ concentrations (Fig. 2b). The effects of temperature enhancement of the mortality rate were particularly visible with the LT50 (Fig. 2a,c) while the effect of CO₂ concentration changes was not perceptible for the egg stage (Fig. 2a,b).

With *D. maculatus*, the different stage had a similar tolerance and adults were more tolerant than other stages only under the lower CO₂ concentration of 20% (Fig. 3e, f). The increase of CO₂ concentration had a significant effect both on larvae and on adults but not on eggs (Fig. 3). This situation is comparable with the one observed previously with the Lepidopteran pest *P. interpunctella*. The impact of temperature is reduced compared to the one observed in *P. interpunctella*. At the two lowest experimented temperatures of 20 and 27.5°C, the adults are killed only after a 5-day exposure time at the lowest level of CO₂ concentration of 40%.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Experimental system for insect exposure to hypercarbic Atmosphere.</figcaption></figure>

a = gas cylinders; b = massic flowmeter; c = electronic regulation box; d = gas partition module; e = flowmeter; f = hygrometric regulator; g = insect exposure chamber; h = bubling final control.
<table><caption>**Table 1.** Bioassay conditions in experimental study on the effects of temperature and CO₂ concentration levels on the survival of three insect species of dry food products to a CO₂-enriched modified atmosphere (MA)</caption><thead><tr><th>Insect species</th><th>Stages</th><th>Insect numbers</th><th>Feeding medium</th></tr></thead><tbody><tr><td rowspan="2">Plodia interpunctella</td><td>Eggs (1-4 days old)</td><td>100 to 150</td><td>crushed corn + glycerol 25 g</td></tr><tr><td>Larvae (15-18 d old)</td><td>50</td><td>crushed corn + glycerol 25 g</td></tr><tr><td rowspan="3">Dermestes maculatus</td><td>Eggs (1-3 d old)</td><td>20 to 30</td><td>dog-food: 6 damped pellets</td></tr><tr><td>Larvae (all instars)</td><td>30</td><td>dog-food: 6 damped pellets</td></tr><tr><td>Adults</td><td>30</td><td>dog-food: 6 damped pellets</td></tr><tr><td rowspan="3">Tribolium confusum</td><td>Eggs (1-5 d old)</td><td>80 to 120</td><td>whole wheat flour: 6 g</td></tr><tr><td>Larvae (15-22 d old)</td><td>100</td><td>whole wheat flour: 6 g</td></tr><tr><td>Adults</td><td>100</td><td>whole wheat flour: 6 g</td></tr></tbody></table><table><thead><tr><th colspan="2">studied variables</th></tr><tr><th>CO₂ concentration (air balance)</th><th>Insect species and stages</th></tr></thead><tbody><tr><td>CO₂/ Air 40/ 60 (v/v)</td><td>Plodia interpunctella (Phycitinae)</td></tr><tr><td>CO₂/ Air 60/ 40 (v/v)</td><td>eggs</td></tr><tr><td>CO₂/ Air 80/ 60 (v/v)</td><td>larvae</td></tr><tr><td>Temperature levels</td><td>Dermestes maculatus (Dermestidae)</td></tr><tr><td>20°C</td><td>eggs</td></tr><tr><td>27.5°C</td><td>larvae</td></tr><tr><td>35°C</td><td>adults</td></tr><tr><td>Exposure times</td><td>Tribolium confusum (Tenebrionidae)</td></tr><tr><td>one to 10 days</td><td>eggs</td></tr><tr><td></td><td>larvae</td></tr><tr><td></td><td>adults</td></tr></tbody></table>

<table><caption>**Table 2a.** Lethal time 50 and 99 % for different stages of *T. confusum* exposed to increasing concentrations of CO₂ in air at three levels of temperature</caption><thead><tr><th rowspan="2">Temperature</th><th rowspan="2">[CO₂]</th><th colspan="2">egg</th><th colspan="2">larva</th><th colspan="2">adult</th></tr><tr><th>TL 50 (x)</th><th>TL 99 (x)</th><th>TL 50</th><th>TL 99</th><th>TL 50</th><th>TL 99</th></tr></thead><tbody><tr><td rowspan="4">20°C</td><td>40 %</td><td>1.3</td><td>6</td><td>6.5</td><td>&gt; 9</td><td>3.85</td><td>7</td></tr><tr><td>60 %</td><td>1.15</td><td>2.75</td><td>7.0</td><td>2.15</td><td>4.5</td><td></td></tr><tr><td>80 %</td><td>1.1</td><td>5</td><td>2</td><td>5.0</td><td>1.75</td><td>2.8</td></tr><tr><td>40 %</td><td>0.6</td><td>4.0</td><td>4.3</td><td>6.0</td><td>3.75</td><td>6</td></tr><tr><td rowspan="3">27.5°C</td><td>60 %</td><td>0.8</td><td>4.0</td><td>2.85</td><td>5.0</td><td>1.7</td><td>2.9</td></tr><tr><td>80 %</td><td>0.35</td><td>3.0</td><td>1.3</td><td>3.5</td><td>1.05</td><td>2.8</td></tr><tr><td>40 %</td><td>0.7</td><td>3.0</td><td>2.35</td><td>4.75</td><td>2.4</td><td>3.8</td></tr><tr><td rowspan="2">35°C</td><td>60 %</td><td>0.5</td><td>2.0</td><td>1.4</td><td>2.15</td><td>1.25</td><td>1.9</td></tr><tr><td>80 %</td><td>0.75</td><td>3.0</td><td>0.9</td><td>1.8</td><td>0.75</td><td>1.6</td></tr></tbody></table>
<table><caption>**Table 2b.** Lethal time 50 and 99 % for different stages of *D. maculatus* exposed to increasing concentrations of CO₂ in air at three levels of temperature.</caption><thead><tr><th rowspan="2">Temperature</th><th colspan="3">egg</th><th colspan="2">larva</th><th colspan="2">adult</th></tr><tr><th>[CO<sub>2</sub>]</th><th>TL 50 (x)</th><th>TL 99</th><th>TL 50</th><th>TL 99</th><th>TL 50</th><th>TL 99</th></tr></thead><tbody><tr><td rowspan="4">20°C</td><td>40 %</td><td>1.3</td><td>3.0</td><td>1.45</td><td>3.0</td><td>1.55</td><td>4.8</td></tr><tr><td>60 %</td><td>1.7</td><td>4.5</td><td>1.3</td><td>2.9</td><td>1.3</td><td>3.0</td></tr><tr><td>80 %</td><td>1.4</td><td>3.0</td><td>1.3</td><td>2.9</td><td>1.3</td><td>3.0</td></tr><tr><td>40 %</td><td>1.25</td><td>1.9</td><td>0.6</td><td>3.0</td><td>1.35</td><td>4.8</td></tr><tr><td rowspan="4">27.5°C</td><td>60 %</td><td>1.25</td><td>1.9</td><td>0.5</td><td>2.0</td><td>0.65</td><td>1.9</td></tr><tr><td>80 %</td><td>1.0</td><td>1.9</td><td>0.4</td><td>1.0</td><td>0.65</td><td>1.9</td></tr><tr><td>40 %</td><td>0.7</td><td>1.9</td><td>1.75</td><td>3.0</td><td>1.7</td><td>3.0</td></tr><tr><td>60 %</td><td>0.5</td><td>1.8</td><td>0.75</td><td>1.2</td><td>0.8</td><td>1.9</td></tr><tr><td rowspan="2">35°C</td><td>80 %</td><td>0.5</td><td>2.5</td><td>0.45</td><td>0.95</td><td>0.45</td><td>0.95</td></tr></tbody></table>

<table><caption>**Table 2c.** Lethal time 50 and 99 % for different stages of *P. interpunctella* exposed to increasing concentrations of CO₂ in air at three levels of temperature (time in days)</caption><thead><tr><th rowspan="2">Temperature</th><th colspan="3">egg</th><th colspan="2">larva (caterpillar)</th></tr><tr><th>[CO<sub>2</sub>]</th><th>TL 50 (x)</th><th>TL 99 (x)</th><th>TL 50</th><th>TL 99</th></tr></thead><tbody><tr><td rowspan="4">20°C</td><td>40 %</td><td>1.6</td><td>3.0</td><td>3.0</td><td>&gt; 8</td></tr><tr><td>60 %</td><td>1.75</td><td>3.0</td><td>1.35</td><td>5.0</td></tr><tr><td>80 %</td><td>2.1</td><td>2.9</td><td>1.15</td><td>3.0</td></tr><tr><td>40 %</td><td>0.9</td><td>1.5</td><td>2.75</td><td>6.0</td></tr><tr><td rowspan="4">27.5°C</td><td>60 %</td><td>1.2</td><td>2.0</td><td>1.15</td><td>5.0</td></tr><tr><td>80 %</td><td>1.25</td><td>2.0</td><td>0.75</td><td>3.0</td></tr><tr><td>40 %</td><td>0.3</td><td>3.0</td><td>1.6</td><td>4.0</td></tr><tr><td>60 %</td><td>0.3</td><td>2.0</td><td>0.85</td><td>2.0</td></tr><tr><td rowspan="2">35°C</td><td>80 %</td><td>0.3</td><td>1.0</td><td>0.55</td><td>1.0</td></tr></tbody></table>

In *T. confusum*, the larval instar is the most tolerant compared to eggs or adults. Under 40% CO₂ and at 20°C, LT99 was observed close to 10 days of exposure. In the same conditions, LT99 for eggs and adults was observed respectively at 6 and 7 days. Additionally with this particular species, it has been observed mainly in adult stage a great difference between LT50 or LT99 at 40% CO₂ and LT50 and LT99 at the two other higher concentration levels (Fig 4e, f). At the concentration level of 80% CO₂, the larval stage and the eggs of *T. confusum* were observed significantly more tolerant than the adult stage (Fig 4). As in the case of the two previous species, the egg stage of *T. confusum* has a similar tolerance whatever the concentration of CO₂ for a given temperature level (Fig. 4a,b).

## Discussion

### Effect of temperature on CO₂ efficiency and vice-versa

The increase of the efficiency of CO₂ exposure with temperature is particularly visible for the egg stage in all the

three species. Egg is generally the most frequent stage of noxious insect species infesting processed food-products because female moths or beetles can rapidly lay eggs onto unprotected finished product during the cooling phase after product extrusion or cooking for instance. This observation implies that a high concentration of carbon dioxide is not necessary to kill eggs of the insect species involved in this study. The efficiency of the MA will be improved only with higher temperatures of storage. For the larval and adult stage, whatever the species involved, the increase in the two variables, i.e. CO₂ concentration level and temperature had a complementary effect. When temperature is increasing from 20 to 35°C, the killing rate of CO₂ is multiplied at least two fold. In the same time, when CO₂ concentration increases from 40 to 80%, the efficiency is multiplied by a two fold factor. Consequently, the response surface has a two-directional pattern in most cases (Figs. 2-4).

## Conclusion

Enriched-CO₂ MA packaging can eliminate residual infestation of dry food-products provided the resting time after sealing before opening would be at least 10 days with a level of CO₂ concentration as low as 40%. These conditions are efficient to kill the most tolerant stage of the two important insect pests, the Indian meal moth and the confused flour beetle, even at a low temperature level of 20°C. Additional useful information has been produced enabling optimal scheduling the combination of conditions such as the storage temperature and CO₂ concentration levels for an application of this preventative disinfestation (also called disinfestation for sanitary and hygienic quality insurance) to a large range of situations and various processed food products sensitive to insect attack post-fabrication. In these conditions of low carbon dioxide concentration inside the sealed packages, the cost of this preventative control measure should be supportable for a large range of dry food products.
Thibolium Confusum

<figure><img src="image_3.png" /><figcaption>Eggs</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Larvae</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Adults</figcaption></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

Fig. 2. Time (in days) leading to 50 or 99% kill of different stages of the Indian meal moth when exposed to different CO₂ concentration levels at different temperatures.
Dermestes maculatus

<figure><img src="image_9.png" /><figcaption>Eggs</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Larvae</figcaption></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>Adults</figcaption></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

Fig. 3. Time (in days) leading to 50 or 99% kill at different stages of the hide beetle when exposed to different CO₂ concentration levels at different temperatures.
Dlodia interpumctella

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 4.** Time (in days) leading to 50 or 99% kill of different stages of the confused flour beetle when exposed to different CO₂ concentration levels at different temperatures.</figcaption></figure>

Aknowledgements

A part of the equipment required for this study was granted
by the 'Conseil Régional d'Aquitaine'.

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