A series of tests was conducted to determine the efficacy of ozone and chlorine dioxide gases in controlling phosphine resistant field strains of the lesser grain borer, red flour beetle, sawtoothed grain beetle, rice weevil, and maize weevil. Laboratory strains of these five species served as phosphine susceptible strains. Both gases were effective against all strains of the five species, and suppressed progeny production. There were delayed toxicity effects with both gases against all species. However, the dosages required for complete kill varied with the gas and the species. The paper will provide an overview of tests conducted and highlights of significant research findings.

1. biosecurity built on science
PB CRC 3038: Evaluating chlorine dioxide and ozone
as alternative methods for controlling phosphine-
resistant insects in on-farm and commercial storages
Bhadriraju Subramanyam, Xinyi E
Department of Grain Science and Industry Kansas State
University
Plant Biosecurity Cooperative Research Centre

2. biosecurity built on science
What is the main message of this slide?
 April, 2013: Signing of agreement.
 April-December, 2014: Preliminary laboratory experiments to
determine baseline data on chlorine dioxide and ozone dosages to
control phosphine resistant insects.
 February-June, 2015: Establishing baseline dosages of chlorine dioxide
and ozone needed for controlling adults of phosphine resistant insects.
 July, 2015-March, 2016: Full scale studies to establish realistic chlorine
dioxide and ozone dosages.
 April-August, 2016: Final report was prepared and submitted to PBCRC.
Milestones

3. biosecurity built on science
What is the main message of this slide?
 Fumigant
A fumigant is a chemical that can exist in the gaseous state in sufficient
concentration to be lethal to a given target organism at a required
temperature and pressure (Bond, 1989). It diffuses as individual molecules
and can penetrate materials easily. Mists, fogs, and smokes are
suspensions of liquids or solids in the air, and unable to penetrate the
materials.
Responsive tactic.
Little or no residues.
Uniform distribution (a challenge) in the treated commodities/spaces.
Rationale

4. biosecurity built on science
What is the main message of this slide?
 Search for the new fumigants
• Environmentally benign
• High efficacy against all life stages of most species
• No cross-resistance to existing fumigants
• Effective suppression of progeny
• Minimal impacts on grain quality
• Cost-effective
Chlorine dioxide (ClO2)
Water treatment: legionella,
beverage and food industry, odor
control, oil industry, municipal and
waste water treatment.
Surface and air treatment: food
processing plants, biomedical
devices, hospitality, residential.
Ozone (O3)
Water treatment: cooling tower,
bottling, hydroponics,
aquaculture, drinking water.
Soil remediation, odor control,
industry laundries.
Rationale

5. biosecurity built on science
What is the main message of this slide?
 Chlorine dioxide
Project summary

6. biosecurity built on science
What is the main message of this slide?Species Strain County, state Collection
year
Resistance
status
Tribolium castaneum LAB Laboratory 1999 Susceptible
AB1 Dickinson, Kansas 2011 Medium
PD Russell, Kansas 2011 Medium
CF Washington, Kansas 2011 Weak
Rhyzopertha dominica LAB Laboratory 1999 Susceptible
CS Chase, Kansas 2011 Weak
RL Riley, Kansas 2007 Strong
FL Florida - Strong
Oryzaepilus
surinamensis
LAB Laboratory 1999 Susceptible
AB2 Abilene, Kansas 2011 Weak
Sitophilus zeamais LAB Laboratory 1999 Susceptible
TX Texas 2011 Weak
Sitophilus oryzae LAB Laboratory 1999 Susceptible
TX Texas 2011 Weak

7. biosecurity built on science
What is the main message of this slide?
Post-exposure time (day)
1 2 3 4 5
0
20
40
60
80
100
2.02 g/m3
, 6 h
Mean+SEmortality(%)
0
20
40
60
80
100
0.54 g/m3
, 8 h
RFB-lab
RFB-MN
RFB-CF
RFB-AB1
RFB-AB2
1.35 g/m3
, 6 h
2.70 g/m3
, 3 h
1 2 3 4 5 Mean+SEmortality(%)
0
20
40
60
80
100
0.54 g/m3
, 8 h
Post-exposure time (day)
1 2 3 4 5
0
20
40
60
80
100
2.02 g/m3
, 6 h
1 2 3 4 5
2.70 g/m3
, 3 h
LGB-lab
LGB-CF
LGB-RL
1.35 g/m3
, 6 h

8. biosecurity built on science
What is the main message of this slide?Mean+SEmortality(%)
0
20
40
60
80
100
0.54 g/m3
, 8 h
Post-exposure time (day)
1 2 3 4 5
0
20
40
60
80
100
2.02 g/m3
, 2 h
1 2 3 4 5
1.35 g/m3
, 6 h
2.70 g/m3
, 3 h
STGB-lab
STGB-PD
STGB-AB2
STGB-CF
Mean+SEmortality(%)
0
20
40
60
80
100
RW-lab
RW-TX
MW-lab
MW-TX
1.35 g/m3
, 6 h 2.02 g/m3
, 6 h
Post-exposure time (day)
1 2 3 4 5
0
20
40
60
80
100
1 2 3 4 5
2.70 g/m3
, 3 h 2.70 g/m3
, 9 h

9. biosecurity built on science
What is the main message of this slide?
Species Strain
Exposure time (h) 24.8 °C
with wheat without wheat
T. castaneum LAB 26 15
AB1 26 15
MN 26 15
O. surinamensis LAB 16 3
AB2 16 3
R. dominica LAB 24 20
CS 34 18
RL 34 20
S. zeamais LAB 24 15
TX 18 7
S. oryzae LAB 15 5
TX 18 7
Exposure times required for complete mortality of adults of five
stored-product insect species at 0.54 g/m3 (200 ppm) of chlorine
dioxide based on mortality assessments made 5 d after the exposure.
Exposure times required for complete mortality of adults of five
stored-product insect species at 1.40 g/m3 (520 ppm) of chlorine
dioxide based on mortality assessments made 5 d after the exposure.
Species Strain
Exposure time (h) with wheat
24.8 °C 32.8 °C
T. castaneum LAB 12 8
AB1 12 8
CF 12 8
O. surinamensis LAB 4 4
AB2 4 4
R. dominica LAB 12 8
CS 12 8
RL 12 8
S. zeamais LAB 12 8
TX 12 8
S. oryzae LAB 12 8
TX 12 8

10. biosecurity built on science
What is the main message of this slide?Progeny production (mean±SE) and percentage reduction (in the parenthesis) of laboratory and field strains of five insect
species after exposure to 1.40 g/m3 of chlorine dioxide for various time periods in vials with wheat during October, 2015.
Exposure
Time (h)
R. dominica S. zeamais S. oryzae
LAB CS RL LAB TX LAB TX
0 111.7±11.9 41.7±10.7 41±13.3 230±28.4 209±23.9 286.3±12.4 263.7±39.6
1 109.7±22.7
(1.8%)
32.3±7.0
(22.5%)
39±18.5
(4.9%)
119.7±28
(48.0%)
98.3±22.8
(53.0%)
169.7±21.8
(40.7%)
185.7±23.5
(29.6%)
2 41.3±8.7
(63.0%)
21.0±5.2
(49.6%)
21.0±6.4
(48.8%)
89.0±8.5
(61.3%)
11.0±1.0
(94.7%)
138.7±15.8
(51.6%)
65.7±23.4
(75.1%)
3 40.7±23.4
(63.6%)
20.3±1.3
(51.3%)
7.3±3.7
(82.2%)
112.3±19.4
(51.2%)
16.7±9.3
(92%)
73.7±3.0
(74.3%)
0.7±0.3
(99.7%)
4 37.7±14.9
(66.2%)
13.0±6.1
(68.8%)
48.3±11.2
(-17.8%)
4.0±3.5
(98.3%)
8.3±5.8
(96%)
25.0±16.8
(91.3%)
2.0±0.6
(99.2%)
5 11±2.9
(90.2%)
7.7±1.9
(81.5%)
15.0±14.5
(63.4%)
27.3±12.5
(88.1%)
1.0±0.6
(99.5%)
_ _
6 19±6.4
(83.0%)
4.7±2.3
(88.7%)
12.7±4.5
(69.0%)
12.3±10.3
(94.7%)
0.7±0.7
(99.7%)
_ _
7 13±6.4
(88.4%)
5.0±2.1
(88.0%)
8.0±4.6
(80.5%)
2.7±1.8
(98.8%)
24.3±24.3
(88.4%)
_ _
8 3.3±2.0
(97.0%)
2.0±2.0
(95.2%)
11.7±9.7
(71.5%)
1.3±0.9
(99.4%)
0±0
(100%)
_ _

11. biosecurity built on science
What is the main message of this slide?
• 800 ppm (2.16 g/m3) for 4 hours
• 6 kg of wheat for each mini silo
• Gas entered from the bottom and vented
from the top.
• Insects were placed at the bottom, the
center and the top of the mini silo.
• 25°C, 20 % r.h.
Species Strains
% Mortality (Mean ± SE) at day 5
Bottom Center Top
T. castaneum
LAB 100 ± 0 100 ± 0 92.9 ± 3.6
CF 100 ± 0a 77.5 ± 1.9b 88.5 ± 4.1ab
MN 96.6 ± 1.7 91.2 ± 6.3 87.8 ± 6.1
R. dominica
LAB 100 ± 0 98.1 ± 1.9 94.3 ± 5.7
CS 100 ± 0 95.6 ± 4.4 97.8 ± 2.2
FL 96.7 ± 1.6 86.0 ± 6.3 95.2 ± 2.9
O. surinamensis
LAB 100 ± 0 100 ± 0 100 ± 0
AB2 100 ± 0 100 ± 0 100 ± 0
S. zeamais
LAB 47.6 ± 6.5 36.2 ± 1.7 35.0 ± 1.5
TX 29.6 ± 3.7a 9.7 ± 2.3b 5.6 ± 5.6b
S. oryzae
LAB 91.7 ± 4.5 89.0 ± 1.6 78.2 ± 6.3
TX 37.9 ± 6.0 37.9 ± 6.0 30.2 ± 4.8
Means by species and strains followed by different letters was significantly
different (P < 0.05) within a species and strain (by Bonferroni t-tests)

12. biosecurity built on science
What is the main message of this slide?
Strains
Column (% mortality at day 5) Box (% mortality at day 5)
50 g 250 g 500 g 250 g 500 g
RFB-LAB 100 ± 0a 66.7 ± 6.7b 13.7 ± 3.2c 85.0 ± 2.9ab 68.8 ± 8.5b
RFB-CF 100 ± 0a 67.4 ± 11.3b 26.6 ± 6.6c 100 ± 0a 100 ± 0a
LGB-LAB 100 ± 0a 23.6 ± 1.6b 0 ± 0b 98.1 ± 1.9a 73.2 ± 15.5a
LGB-FL 100 ± 0a 9.8 ± 5.8c 6.7 ± 4.4c 81.2 ± 4.7a 42.0 ± 0.5b
STGB-LAB 100 ± 0a 100 ± 0a 70.0 ± 0b 100 ± 0a 100 ± 0a
STGB-AB2 100 ± 0a 100 ± 0a 72.3 ± 11.3b 100 ± 0a 100 ± 0a
RW-LAB 100 ± 0a 98.3 ± 1.7a 38.5 ± 11.3b 100 ± 0a 100 ± 0a
RW-TX 89.1 ± 7.5ab 60.0 ± 9.2b 59.9 ± 9.8b 96.6 ± 3.4a 96.7 ± 3.3a
Means by species and strains followed by different letters was significantly different
(Bonferroni, P=0.05).

13. biosecurity built on science
What is the main message of this slide?Ozone:
Ozone exposure time (h)
0 5 10 15 20 25
Mean±SEmortality(%)
0
20
40
60
80
100
0 5 10 15 20 25
A
C
B
D
0
20
40
60
80
100
LAB
CS
FL
A
Mortality assessed on
day 5 for
R. dominica exposed
with and without wheat
to 0.21 or 0.42 g/m3 of
ozone.
(A) Without wheat and
0.21 g/m3 of ozone;
(B) Without wheat and
0.42 g/m3 of ozone;
(C) With wheat and 0.21
g/m3 of ozone;
(D) With wheat and 0.42
g/m3 of ozone.

14. biosecurity built on science
What is the main message of this slide?
Lethal times (LT99) of phosphine susceptible and resistant strains of four insect species
after exposed to 0.42 g/m3 of ozone in the presence or absence of wheat
Species Strains Lethal time (95% CL) (h) Ratio (95% CL)
LT99 with wheat LT99 without wheat
T. castaneum LAB 13.64 (8.64-92.79) 10.65 (8.31-19.90) 1.26 (0.58-2.73)
CF 15.91 (11.79-24.91) 14.35 (11.37-20.30) 1.11 (0.71-1.73)
PD 25.81 (16.19-64.99) 29.89 (21.74-47.59) 1.16 (0.57-2.36)
O. surinamensis LAB 7.07 (4.24-25.46) 4.33 (3.49-5.98) 1.63 (0.82-3.25)
AB2 11.18 (9.33-14.16) 9.05 (7.64-11.25) 1.24 (0.94-1.63)
S. zeamais LAB 5.56 (4.65-7.20) 4.20 (3.56-5.29) 1.32 (1.00-1.75)
TX 3.80 (3.37-4.50) 3.14 (2.52-4.59) 1.21 (0.89-1.64)
S. oryzae LAB 3.11 (2.76-3.65) 2.32 (2.02-2.87) 1.34 (1.08-1.65)
TX 2.00 (1.90-2.13) 2.91 (2.07-8.17) 1.45 (0.96-2.20)

15. biosecurity built on science
What is the main message of this slide?
Progeny production (% reduction) of laboratory and field strains of four insect species after
exposure to 0.42 g/m3 of ozone for different time periods in the presence of wheat
Time (h) T. castaneum O. surinamensis S. zeamais S. oryzae
LAB CF PD LAB AB2 LAB TX LAB TX
0 115.8 14.2 41.0 260.6 355.8 278.4 160.4 223.0 190.8
1 70.8
(38.9%)
16.4
(-15.5%)
57.0
(-39.0%)
242.4
(7.0%)
229.4
(35.5%)
215.4
(22.6%)
93.2
(41.9%)
102.4
(54.1%)
98.2
(48.5%)
2 58.0
(49.9%)
13.8
(2.8%)
41.4
(-1.0%)
49.4
(81.0%)
149.8
(57.9%)
125.6
(54.9%)
56.6
(64.7%)
4.4
(98.0%)
1.8
(99.1%)
3 45.0
(61.1%)
46.4
(-226.8%)
35.8
(12.7%)
69.4
(73.4%)
92.0
(74.1%)
103.0
(63.0%)
4.0
(97.5%)
1.4
(99.4%)
0.6
(99.7%)
4(5) 65.2
(43.7%)
54.4
(-283.1%)
66.0
(-61.0%)
2.4
(99.1%)
3.8
(98.9%)
4.8
(98.3%)
3.8
(97.6%)
2.0
(99.1%)
1.2
(99.4%)
6 5.6
(95.2%)
10.6
(25.4%)
60.6
(-47.8%)
2.4
(99.1%)
1.8
(99.5%)
0
(100%)
1.2
(99.3%)
1.4
(99.4%)
4.2
(97.8%)
8 8.4
(92.7%)
29.2
(-105.6%)
46.4
(-13.2%)
2.6
(99.0%)
0.4
(99.9%)
1.0
(99.6%)
0.2
(99.9%)
3.8
(98.3%)
0.4
(99.8%)
10 3.6
(96.9%)
19.8
(-39.4%)
40.6
(1.0%)
1.2
(99.5%)
0
(100%)
- - - -

16. biosecurity built on science
What is the main message of this slide?
Progeny production (% reduction) of laboratory and field strains of four insect species
after exposure to 0.42 g/m3 of ozone for different time periods in the absence of wheat
Time (h) T. castaneum O. surinamensis S. zeamais S. oryzae
LAB CF PD LAB AB2 LAB TX LAB TX
0 47.6 11.4 39.8 339.6 194.8 285.8 179.6 162.8 114.6
1 73.6
(-54.6%)
16.2
(-42.1%)
65.0
(-63.3%)
282.2
(16.9%)
271.6
(-39.4%)
47.0
(83.6%)
7.8
(95.7%)
65.4
(59.8%)
11.8
(89.7%)
2 46.6
(2.1%)
24.4
(-114%)
89.2
(-124.1%)
138.2
(59.3%)
129.6
(33.5%)
49.6
(82.6%)
0.8
(99.6%)
0
(100%)
0
(100%)
3 46.2
(2.9%)
69.6
(-510.5%)
92.4
(-132.2%)
29.0
(91.5%)
42.6
(78.1%)
15.4
(94.6%)
0
(100%)
0
(100%)
0
(100%)
4(5) 45.8
(3.8%)
55.6
(-387.7%)
95.6
(-140.2%)
1.0
(99.7%)
0.6
(99.7%)
0
(100%)
0.4
(99.8%)
0
(100%)
0
(100%)
6 11.2
(76.5%)
46.8
(-310.5%)
90.8
(-128.1%)
0.6
(99.8%)
0.8
(99.6%)
0.2
(99.9%)
0.4
(99.8%)
0
(100%)
0
(100%)
8 6.0
(87.4%)
12.6
(-10.5%)
86.0
(-116.1%)
1.6
(99.5%)
0.4
(99.8%)
0
(100%)
0.4
(99.8%)
0
(100%)
0
(100%)
10 2.6
(94.5%)
0.6
(94.7%)
49.4
(-24.1%)
0.4
(99.9%)
0.4
(99.8%)
- - 0
(100%)
0
(100%)

17. Percent reduction (mean ± SE) in adult emergence from four young stages of
Rhyzopertha dominica after exposure to 0.42 g/m3 of ozone for various time periods
Stages Exposure time
(h)
Percent reduction (mean ± SE)
in adult emergence after ozone exposure
LAB CS FL
Eggs 24 73.3 ± 3.9 64.4 ± 3.6 47.7 ± 12.4
48 85.4 ± 3.9 85.3 ± 4.5 92.4 ± 4.8
72 99.7 ± 0.3 96.3 ± 2.0 100.0 ± 0
Young larvae 10 100.0 ± 0 100.0 ± 0 99.0 ± 1.0
24 100.0 ± 0 99.2 ± 0.5 97.1 ± 2.0
34 99.7 ± 0.3 98.4 ± 0.7 99.0 ± 1.0
Old larvae 10 53.3 ± 3.9 b 64.6 ± 6.7 b 93.3 ± 4.3 a
24 99.1 ± 0.4 98.8 ± 1.2 98.3 ± 1.0
34 99.6 ± 0.3 98.8 ± 0.5 99.2 ± 0.8
Pupae 2 12.4 ± 8.9 12.1 ± 11.6 19.6 ± 17.7
6 16.4 ± 13.8 b 35.2 ± 6.0 b 76.5 ± 7.0 a
10 32.8 ± 8.6 b 54.4 ± 6.9 b 96.6 ± 3.4 a
Means by species and strains followed by different letters was significantly different (Bonferroni, P=0.05).

18. biosecurity built on science
What is the main message of this slide?Significant findings
 Both ozone and chlorine dioxide can effectively kill adults of phosphine susceptible and resistant strains
of five common insect pests after exposed to 200-500 ppm of gas for less than 24 hours. They can also
effectively suppress the progeny production of Rhyzopertha dominica, and Sitophilus spp.
 Young stages of Rhyzopertha dominica are susceptible to ozone exposure (200 ppm), especially for
larvae.
 Mortality of adults of all strains and species increased as the post-exposure time increased, indicating
delayed toxicity effect.
 Both gases have the ability to react with the active sites on the surface of the grain, and longer
equilibrium time is expected to reach to the targeted lethal concentration.
 There is no cross-resistance between ozone and phosphine or chlorine dioxide and phosphine.
 High temperatures promote the efficacy of chlorine dioxide against adults.
 Wheat restricts the penetration of chlorine dioxide and ozone by surface binding reaction and porosity,
resulting lower mortality.

19. biosecurity built on science
What is the main message of this slide?Research Outcomes
• Subramanyam, Bh., E, X.Y., Savoldelli, S., Sehgal, B. , Maier, D. E., Ren, YL (2014) Efficacy of ozone against
stored grain insect species in wheat: Laboratory and field observations. 11th International Working
Conference on Stored Product Protection.
• Subramanyam, Bh., E, X.Y. (2015) Efficacy of chlorine dioxide gas against five stored-product insect
species. 10th Conference of International Organization for Biological and Integrated Control (IOBC)-
Integrated Protection of Stored Products.
• Subramanyam, Bh., E, X.Y., Li, B. (2016) Efficacy of a high concentration of chlorine dioxide gas against
laboratory and field strains of five stored product insect species. XXV International Congress of
Entomology.
• E, X.Y., Li, B., Subramanyam, Bh. (2016) Efficacy of ozone fumigation against phosphine susceptible and
resistant strains of Rhyzopertha dominica. XXV International Congress of Entomology.

20. biosecurity built on science
What is the main message of this slide?Industry Impacts
 PureLine is planning to register chlorine dioxide as a fumigant for stored products in U.S.
A broader spectrum will be investigated with PureLine’s financial support including the deactivation of
microbes and denaturation of mycotoxins in stored grains.
 Popcorn county

21. biosecurity built on science
What is the main message of this slide?Future Plans