Role of CO2 and Ozone for stored pest management

1. Role of CO2 and Ozone for stored pest
management
Speaker
Sachin Kumar Jaiswal
Ph.D Scholar
IGKV, Raipur (C.G.)
Seminar In-charge
Dr. S. S. Shaw
Professor
IGKV, Raipur (C.G.)
CO2
DOCTORAL SEMINAR

2. INTRODUCTION
• Insects not only attack field crops during the growing season (22%), but also
damage grains stored in granaries (10%) (Weaver and Petro, 2005; Sadeghi et
al., 2011).
• More than 1200 species of 70 insect-pests have been identified which attacked
store grains and cereal products in store houses (Rajendra, 2002).
• Accor. to FAO estimate of world-wide annual losses in store has been given as
10% of all store grain i.e. 13 million tonnes of grain is lost due to insects.
• In India, food grain worth nearly Rs 50,000 crore is lost every year due to storage
pest infestation (10- 40%) (Upadhyay and Ahmad, 2011).

3. • Losses caused by insects include not only the direct consumption of kernels, but
also include accumulations of frass, exuviae, webbing, and insect cadavers.
• Stored grain insects in addition to obvious identifying characteristics, the feeding
habits of storage insect pests are used to separate them into two classes: Primary
pests and secondary invaders.
 Primary pests are those that are capable of penetrating and infesting intact
kernels of grain, and have immature stages that can readily develop within a
kernel of grain.
Secondary invaders cannot infest sound grain but feed on broken kernels,
debris, higher moisture weed seeds, and grain damaged by primary insect
pests.
(David K. Weaver and A. Reeves, 2005)

5. Table 1: Some examples of stored pests
Common
Name
Scientific Name Family Order Grains damaged
Pulse beetle Callosobruchus chinensis Bruchidae Coleoptera All pulses
Rice weevil Sitophilus oryzae Curculionidae Coleoptera Rice, wheat
Lesser grain
borer
Rhizopertha dominica Bostrychidae Coleoptera Wheat, Rice
Cigarette
beetle
Lasioderma serricorne Anobiiidae Coleoptera Tobacco, turmeric, ginger, chillies
Almond moth Cadra cautella Pyralidae Lepidoptera Dried fruits
Angoumois
grain moth
Sitotroga cerealella Gelichiidae Lepidoptera All cereals
Khapra beetle Trogoderma granarium Dermestidae Lepidoptera Wheat, oyher cereals
Red rust flour
beetle
Tribolium castaneum Tenebrionidae Coleoptera Processed foods like flour, grains, dry fruits
Indian meal
moth
Plodia interpunctella Phycitidae Lepidoptera Pistachio nuts
Rice moth Corcyra cephalonica Galleridae Lepidoptera -
Saw toothed
grain beetle
Oryzaephilus
surinamensis
Cucjidae Coleoptera Oat, wheat, barley, animal feed, flax,
sunflower
Flat grain
beetle
Cryptolestes minutus Cucjidae Coleoptera Mainly wheat, malt barley, filberts, nutmeg,
rice, sorghum, soybeans,cottonseed,
cowpea
Long headed
flour beetle
Latheticus oryzae Tenrebrionidae Coleoptera Pasta, dried cassava, oatmeal, provender,
tea, sorghum, maize, cereal grains
Pawar et al.,2015

6.  Presently, fumigation with chemical insecticides is the most effective and
widely used method to control all stages and kinds of pests in grain bins,
warehouses, and other mass grain-storage structures (Cheng et al., 2012).
 The two most commonly used chemicals approved for fumigation:
PHOSPHINE and METHYL BROMIDE.
 Methyl Bromide has been found to deplete the ozone, and its use is now
restricted.
 Grain handlers are therefore left with only one alternative: PHOSPHINE
• Single selection pressure can result in insects overcoming the effect.
• Future development of insect resistance to phosphine, could make it
difficult to maintain the “zero tolerance” requirement unless alternative
methods are found.
Present Situation of Stored Pest Management

7. Modified or controlled atmospheres (MAs or CAs)
 Modified Atmosphere (MA) – the atmospheric
composition within the treated enclosure may
change over time and is controlled in an indirect
fashion.
 Controlled Atmosphere (CA) – atmospheric
composition within the treatment is controlled or
maintained with addition of gas to sustain desired
gas levels.

8. Ambient atmosphere consists of approximately 79% N2, 20–
21% O2, and 0.04% CO2.
Modified or controlled atmospheres (MA or CA) with higher
or lower concentrations of atmospheric gases, mainly oxygen
(O2), carbon dioxide (CO2), ozone (O3), and nitric oxide (NO),
provide a cost-effective method to kill target pests and protect
stored products.
MAs with specific mechanisms by which insects are affected
by and adapt to low O2 (hypoxia/ anoxia) and high carbon CO2
(hypercapnia/ hypercarbia in airtight storage, with O2
maintained at a level, have been used for preventing insect
damage in stored grains.
(Rasool et al., 2017 )

9. • Insect tolerance to hypoxia/anoxia and hypercapnia / hypercarbia
involves:
 decrease in aerobic metabolism,
 decreased ( Nicotinamide Adenine Dinucleotide Phosphate)
NADPH enzyme activity, hence decreases carbohydrate (trehalose)
synthesis, nucleotide synthesis, cholesterol synthesis, and fatty-acid
synthesis (Feron, 2009).
 decreases in glutathione production and catalase, superoxide
dismutase, glutathione-S-transferase, and glutathione peroxidase
activities, which are involved in the protection against the toxic
effects of reactive oxygen species (Boardman et al., 2011).
 increase in carboxyl esterase and phosphatase activities.
 hypoxia induces energy and nutrient production, and
 Downregulation of glycolysis and pyruvate carboxylase fluxes in
adapted insects, accompanied with O2 consumption and acetate
production.

10. • The knowledge about the changes in insect energy and nutrient
sources, metabolic enzymes, and molecular pathways in response to
modified O2, CO2, NO, and O3 concentrations, as well as the role of
MAs in pest control will be useful for applying MAs in combination
with temperature control for pest control in stored food products.
For example-
MA with 8% O2, 60% CO2, and 32% N2 at 30°C killed 100% of 4th
instar larvae of E. cautella within 72 h, and resulted in 95% mortality
in Amyelois transitella Walker after 60-h exposure at 27°C (Brandle
et al., 1983).
Under the same MA, the mortality of E. cautella significantly
increased when the temperature was increased from 25 to 35°C
(Husain et al., 2015). These results indicate that an MA combined
with higher temperature is an effective method for pest control in
stored products in future.

11. Advantages of the Modified Atmospheres Technique:
• No influence on Quality, Color and Taste of the exposed
products
• 100% non-toxic treatment
• No residual, after opening the products can be used
immediately.
• Reduced health and safety compared to chemical fumigations
• Independent of ambient atmospheres
• Can be retrofitted in existing production processes
• Eradicates insects in all development stages: egg,
larvae/nymph, pupa, adult.
• No resistance has been ever observed

12. Role of CO2 for stored pest
management

13. Role of CO2 for stored pest management
• Carbon dioxide (CO2) is a fumigant that can be used as
an alternative to Phosphine as,
• produces no harmful residues,
• less hazardous to handle and relatively safer to use,
• effective in killing insects in all stages of their life
cycles
• could be used for long-term storage of products.
• as insects cannot develop resistance.
• can be used to control insect pests in organic product
storage.

14. • CO2 fumigation should be practiced under completely sealed storage,
and concentration must be maintained at 35% or higher during the first
15 days.
• For example,
Exposure for 17 days to a mixture of 40% CO2 and 2% O2 resulted
in 100% mortality of grain weevils, Calandra granaria Linnaeus
(Bailey, 1955).
Egg laying in insects decreases with increasing CO2 concentration
(Azzam et al., 2010).
• Factors Affecting Required Exposure Time for CO2 Fumigation
Depends On:
- Concentration of the atmosphere
- Grain temperature
- Moisture content of the grain
- Species and life stage

15. CO2 FUMIGATION TECHNIQUE
• Stacking - The stack should be built
based on good storage practice
condition (Fig. 1). The floor sheet
should be at least 0.5 mm thickness
and should be longer and wider than
the base of the proposed stack. The
floor should be inspected for holes,
tears, weak spots, and manufacturing
faults before the stack is built.
• Sealing - Seal the cover sheet and floor
sheet together with solvent-based PVC
glue (Fig. 2). Silicone mastics can be
used to completely seal gaps or small
holes.
Fig. 1. Stacking
Fig. 2. Sealing

16. Inlet Port - Set the inlet port for filling the gas
near the floor at the bottom of the stack (Fig.
3).
Outlet Port - Set the outlet port on top of the
stack (Fig. 4).
Pressure Standard Test -The tightness of the
gas enclosure is measured by the time required
for negative pressure of 500 Pascals (5 cm of
water gauge) to fall to 250 Pascals (2 cm of
water gauge) (Fig. 5).
Fig. 4. Outlet port
Fig. 3. Inlet port
Fig. 5. Pressure test standard

17. Gas Introduction - Filling gas into the sealed stack must be done
quickly. Invert the cylinder and connect it to the inlet pipe (Figs. 6
and 7). As the gas is released, the pressure inside the cylinder falls
rapidly, causing the temperature to fall substantially and the carbon
dioxide to freeze.
Fig. 6. Gas introduction Fig. 7. Gas-filled stack

18. • Dosage Required
The required dosage is determined by monitoring the concentration
of carbon dioxide at the top of the stack (outlet). Gas filling should
stop when the concentration exceeds 75%. The concentration of
carbon dioxide must be monitored during the first 15 days of
exposure period. It must remain at or above 35%.
• Precaution:
 Before Using CO2: It is essential that the storage bins and any
inter-connecting ductwork and fans be sealed to ensure to
maintain the concentration within the desired treatment time.
 Pressure testing to ensure gas tightness is recommended

19. The first problem with using CO2 to fumigate is that to be effective,
the CO2 level must be maintained above 40% over 7-15 days in order to kill
every type of insect.
For example:- Carbon dioxide as a potential fumigant for termite control,
researchers at the University of Hawaii found that exposure to 50% carbon
dioxide for 60 hours resulted in approximately 70% termite mortality, while
complete mortality was recorded after 120 hours.
Grain Temp.
C°
Concentration
CO2
Exposure
Days
40 60 % 1
25 60 % 5
20 60% 14
10 – 20 60% 14 – 56
* At 25Cº, 60% CO2, all life stages succumb – Navarro et al. 2012

20. Typical Installation
Grain Silo
Bulk CO2
Tan
k
CO2
injection
Flow control &
analyzer
panel

21. Control Panel
Pressure
Regulater
LCO2 Valve
Solenoid
Valve
Probe (CO2 Concentration
60-100% )
Vaporizer
Flow meter
%CO2
70
22
° C
Silo
Bulk CO2
Tank
Typical
Installation

22. | Praxair Business Confiential | 7/3/2014
Volume
needed(ft3
)
Void Space
CO2 Usage
(lbs)
12,260 0.4 560
24,530 0.4 1,120
36,800 0.4 1,680
CO2 is required to treat a typical grain silo

23. Role of ozone for stored pest
management

24. Ozone (O3)
 Ozone was first discovered by the European researcher C. F.
Schonbein in 1839.
 It was first used commercially in 1907 in municipal water
supply treatment in Nice and in 1910 in St. Petersburg
(Kogelschatz, 1988).
 At room temperature-
ozone is a nearly colorless gas,
has a pungent, characteristic odor described as similar to ‘‘fresh air
after a thunderstorm’’ (Coke, 1993).
 readily detectable at 0.01–0.05 ppm level (Miller et al., 1978;
Mustafa, 1990; Mehlman and Borek, 1987).
 found in low concentration in nature.
decomposes rapidly and, thus, does not accumulate substantially
without continual ozone generation (Peleg, 1976; Miller et al., 1978).

25.  Ozone has the potential to kill storage pests hence used as
alternative to other fumigants (phosphine)
 Can cause insect, bacteria and fungal mortality – disrupts
cell walls
 excess ozone auto decomposes rapidly to produce oxygen
and thus leaves no residues in food.
 efficacy against a wide range of micro-organisms
including bacteria, fungi, viruses, protozoa, and bacterial
fungal spores has been reported (Cullen et al., 2009;
Khadre et al., 2001; Restaino et al., 1995).
 attractive to the food industry and consequently it has
been affirmed as Generally Recognized as Safe (GRAS)
for use in food processing (Graham, 1997).
Advantages Of Ozone Fumigation

26. Table 2: Effect of ozone treatment storage insect
Feed
grain
Insects Conditions Mortality rate Reference
Maize
RFB (Tribolium castaneum)
MW (Sitophilus zeamais)
IMM (Plodia interpunctella)
50 ppm for 3 days
94.5% IMM
100% MW
92.2% RFB
Kells et al.
(2001)
Maize
RFB (Tribolium castaneum)
MW (Sitophilus zeamais)
IMM (Plodia interpunctella)
25 ppm for 5 days
77.0% IMM
99.9% MW
91.4% RFB
Kells et al.
(2001)
Wheat
Ephestia kuehniella and
Tribolium confusum,
Ozone
concentration
of 13.9 mg/L
T. confusum,
72.6% (Larvae)
1.3–22.7% (Adult)
90–100% (larve, adult)
Isikber and
Oztekin
(2009)
Stored
products
Tribolium castaneum
Rhyzopertha dominica
Oryzaephilus surinamensis
50 ppm 30 _C and
70% RH
50% mortality 11.39– 20.10 h (TC)
9.22–12.19 h (RD)
6.1–9.66 h (OS)
95% mortality 22.17– 37.9 h (TC)
21.85–35.17 h (RD)
11.03–18.72 h (OS)
Sousa et al.
(2008)
Maize T. castaneum 50 ppm
50% mortality (71.4 h) at 20 _C
95% mortality (151.8 h) at 20 _C
Pereira et
al. (2008)

27. Case study 01
Ozone: A New Controlled Strategy for Stored Grain Structures
Pawar et al., 2015
• The concentration of 50 ppm ozone for three days resulted in 92–
100% mortality of adult red flour beetle, Tribolium castaneum
(Herbst), adult maize weevil, Sitophilus zeamais (Motsch.), and
larval moth, Plodia interpunctella and reduced by 63% the
contamination level of the fungus Aspergillus parasiticus Speare
on the kernel surface.
• The attractive aspect of ozone is that it decomposes rapidly (half-
life of 20–50 min) to molecular oxygen without leaving a residue
and proved as an effective technology for grain protection without
affecting its end-use quality.

28. Case study 02
Combined effect of ozone mixed with carbon dioxide on the mortality
of five stored product insects
Golam Reza et al., 2011• Adults of Sitophilus oryzae (L.), Tribolium castaneum (Herbst), Rhyzopertha
dominica(F.), Oryzephilus surinamensis (L.) and 3rd
instar larvae of Plodia
interpunctella (Hubner) were exposed to the mixture of ozone and carbon dioxide.
• After exposure periods of 24 h, the insects were transferred to clean jars containing
food and held at 27±2ºC and 65 ±5% R.H. Experiments were performed in different
heights (30, 40, 50 and 100 cm) and nutrition materials (date, wheat and rice), in
penetration tests and empty-space tests.
• In empty-space trials, the highest mortality was for P. interpunctella. In penetration
tests, treatment with high-pressure ozone and carbon dioxide under different height
and foodstuff may result in different rates of mortality. The mixture of ozone and
carbon dioxide in the interaction between height and diet (heigh×diet) are not
significant for the S. oryzae, T. castaneum, R. dominicaand P. interpunctellabut for
O. surinamensis is significant. The influence of ozone gas and carbon dioxide in the
date is more than rice and wheat. The mixture of ozone with carbon dioxide can be
as suitable fumigant for decreasing phosphine and methyl bromide under ambient
storage conditions in penetration and empty-space fumigations

29. Case study 03
Utilization of ozone to control potato tuber moth, Phthorimaea
operculella (Lepidoptera: Gelechiidae), in storage
Ibrahim R.A. and Al-Ahmadi S.S., 2014
The susceptibility of different stages of the potato tuber moth,
Phthorimaea perculella, to different modified atmospheres containing
various concentrations of ozone was studied as an alternative to methyl
bromide fumigation. The ozone concentrations used against larvae and adults
were 5, 10, 20 40, 60 and 80 ppm at different exposure times. For the eggs
and pupae, a range of 1 to 5 ppm of ozone was used. Results showed that 5
ppm was adequate to kill all eggs and pupae of the moth. The exposure time
needed to achieve a 100%mortality of ggs and pupae was 1 h, whereas
exposure at 80 ppm for 1 h caused 80 and 84%mortality of larvae and adults
of P. operculella, respectively. The order of sensitivity of P. operculella to
ozone was: eggs > pupa > adults and larva. In conclusion, the eggs and
pupae of potato tuber moth were more sensitive to modified atmosphere than
larvae and adults. The results showthat ozone could be an effective alternative
to control potato tuber moth in the storage.

30. Case study 04
Influence of grain mass temperature on ozone toxicity to Sitophilus zeamais
(Coleoptera: Curculionidae)
De Sousa et al., 2015
Considering that grain mass temperature can influence the
exposition time to fumigants, the objective of this work was to evaluate the
influence of grain mass temperature during the ozonization on the ozone
toxicity to Sitophilus zeamais Mots. (Coleoptera: Curculionidae). Corn
grains were placed into PVC cylindrical containers with an ozone gas
injectionexhaustion system. The insects were restrained in cages placed in
the medium layer of the grain mass and subjected to an atmosphere
modified with 50 ppm ozone, 8 L min-1 flow, at grain mass temperature of
20, 30, 35 and 40 °C. Insects were exposed to ozone for 24 and 48-h at
each temperature. Insect mortality was calculated at the end of each assay.
During the period of 24-h exposition the ozone toxicity increased with the
increase in the grain mass temperature, in which insect mortality was
higher at 35 and 40 °C. In the-48 h exposition period there was 100%
insect mortality.

31. Case study 05
Can Ozone be a New Control Strategy for Pests of Stored Grain?
Jian et al., 2015
Ozone (O3) is a strong oxidant with a long history of safe use in
many fields. Researches have been recently focused on the application of
O3 as a fumigant to control stored-grain insects and microorganisms and
to reduce mycotoxins. This review found the following facts: (1) O3
significantly suppressed insect populations at B50 ppm with 4 days
treatment; (2) to eradicate insect infestation,[135 ppm with more than 8
days treatment would be required; (3) O3 at 50 ppm with 3 days treatment
reduced 63 % of stored fungi; (4) O3 at 5–30 ppm could reduce
mycotoxin contamination; however, high concentration and long
treatment time were required to eliminate mycotoxins; (5) application of
O3 at doses that were sufficient for the effective disinfestation of grain
might affect qualities of grain; and (6) O3 at 47–106 ppm could
noticeably damage equipment in 2 months by corrosion. Based on these
facts, we recommended that B50 ppm O3 should be used in the stored-
grain industry and its potential method of application was also analyzed.

32. Conclusion
Modified or controlled atmospheres (MAs or CAs) with higher or lower
concentrations of atmospheric gases, carbon dioxide (CO2) and ozone (O3),
provide a cost-effective method to kill target pests and protect stored products.
And it is having no residual effect, after opening the products can be used
immediately. It overcome the health hazard and is safe as compared to chemical
fumigations.
Eradicates insects in all development stages: egg, larvae/nymph, pupa, adult.
No resistance has been ever observed

33. Thank you

34. • Trehalose is the primary carbohydrate in insects, and plays an important role in
insect development and all physiological activities by serving as an instant energy
source as well as by mitigating abiotic stressors (Shukla et al., 2015). Trehalose
protects cells against various environmental stresses, such as heat, cold,
desiccation, dehydration, and oxidation.
• Chen and Haddad (2004) reported that trehalose can protect Drosophila and
mammalian cells from hypoxic and anoxic injury. The mechanism underlying this
protective action might be related to the decrease in protein denaturation through
protein-trehalose interactions (Chen et al., 2003). In the presence of trehalose,
cells can be maintained in the dry state for up to 5 days.