ARTICLE IN PRESS

Journal of Stored Products Research 43 (2007) 468–473

www.elsevier.com/locate/jspr

Evaluation of two new diatomaceous earth formulations,

enhanced with abamectin and bitterbarkomycin,
against four stored-grain beetle species
C.G. Athanassioua,, Z. Korunicb
a
Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 11855 Athens, Greece
b
Diatom Research and Consulting Inc., 14 Greenwich Dr., Guelph, ON, Canada, N1H 8B8

Accepted 22 December 2006

Abstract

Two new natural diatomaceous earth (DE) formulations, enhanced with abamectin (DEA-P/WP), or bitterbarkomycin (DEBBM-
P/WP), were tested under laboratory conditions against adults of the rice weevil, Sitophilus oryzae, the lesser grain borer, Rhyzopertha
dominica, the red flour beetle, Tribolium castaneum and the rusty grain beetle, Cryptolestes ferrugineus. The bioassays were carried out on
wheat, at 3071 1C and 7075% r.h. The two enhanced DEs were applied either as dusts (P) or as wettable powders (WP) at the dose rates
of 75, 100 125 and 150 ppm. Adult mortality was assessed after 14 and 21 d of exposure on the treated substrate. After this interval, the
treated wheat was retained for an additional period of 49 d in the case of S. oryzae and 63 d in the case of the other three species, in order
to evaluate progeny production. Generally, for all species tested, mortality was higher on wheat treated with powders in comparison with
wettable solutions. After 14 d of exposure, all adult rice weevils were dead even at the lowest dose rate of DEA-P, while 100% mortality
was noted at doses X125 ppm of DEBBM-P. For the other species, mortality was 100% on wheat treated with 75 ppm of DEBBM-P,
with the exception of T. castaneum for which all adults were dead at doses X100 ppm. Progeny production was low, and no progeny were
produced in the cases of R. dominica and C. ferrugineus, for both DEs. However, for S. oryzae, progeny production was high on wheat
treated with WP formulations, at dose rates p100 ppm. Similar trends were noted for T. castaneum, at p100 ppm of DEBBM-WP. The
results of the present study indicate that both DEA-P/WP and DEBBM-P/WP are more effective against the four beetle species examined
in comparison with the currently commercially available DE formulations.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Enhanced diatomaceous earth; Sitophilus oryzae; Rhyzopertha dominica; Tribolium castaneum; Cryptolestes ferrugineus; Stored wheat

1. Introduction main drawback in the use of DEs is that they need to be

Diatomaceous earths (DEs) are very promising alter-
natives to traditional residual grain protectants. They are
of natural origin, have negligible toxicity to mammals and
can be applied with similar technology to that needed for
residual pesticides (Korunic, 1998; Subramanyam and
Roesli, 2000). Several DE formulations are now commer-
cially available (Subramanyam and Roesli, 2000), and
many studies document that they are very effective against
a wide range of stored-product insect species. However, the
Corresponding author. Tel.: +30 2105294582; fax: +30 2105294572.
E-mail address: athanas@aua.gr (C.G. Athanassiou).
applied at high-dose rates, and these affect the physical
properties, chiefly bulk density, of the stored grains
(Korunic et al., 1998). Also, the presence in the atmosphere
of high DE concentrations containing crystalline silica may
cause respiration problems (silicosis) to workers after
long exposure. Many DE formulations are only effective
at dose rates of 1000 ppm or more (Fields and Korunic,
2000; Fields et al., 2003; Arthur, 2003; Athanassiou
et al., 2003, 2004, 2005a), while most traditional grain
protectants should be applied at dose rates that usually
do not exceed 10 ppm. Hence, it is essential to evaluate the
use of novel DEs that are effective against insects at lower
dose rates.

0022-474X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jspr.2006.12.008
 ARTICLE IN PRESS
C.G. Athanassiou, Z. Korunic / Journal of Stored Products Research 43 (2007) 468–473
One of the possible solutions to the problems caused by
the use of DEs in high doses is the combined use of other,
reduced-risk, methods, such as extreme temperatures
(Dowdy, 1999; Dowdy and Fields, 2002), entomopatho-
genic fungi (Lord, 2001; Akbar et al., 2004; Michalaki
et al., 2006) or low doses of insecticides (Korunic, 2001;
Stathers, 2003; Athanassiou et al., 2004). In the present
work, we evaluated the insecticidal effect of two new
natural but modified and enhanced DE formulations. Four
of the most important stored-grain species worldwide, two
internal feeders, the rice weevil, Sitophilus oryzae (L.)
(Coleoptera: Curculionidae), and the lesser grain borer,
Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), and
two secondary colonizers, the red flour beetle, Tribolium
castaneum (Herbst) (Coleoptera: Tenebrionidae) and the
rusty grain beetle, Cryptolestes ferrugineus (Stephens)
(Coleoptera: Cucujidae) were chosen for the study.
Parental mortality and the capability for progeny produc-
tion were assessed.
2. Materials and methods
2.1. Formulations
Two enhanced DE formulations were used in the tests:
(a) DEA-P/WP which was an abamectin-enhanced DE and
(b) DEBBM-P/WP which was a bitterbarkomycin-
enhanced DE. DEA-P/WP is a mixture of two natural
active ingredients: diatomaceous earth and abamectin
(0.25% of active ingredient) dissolved in a solvent and
emulsifier. DEBBM-P/WP is also a mixture of two natural
active ingredients: DE and the Chinese plant extract
bitterbarkomycin (BBM). This formulation contains 90%
of DE and 0.05% of BBM active ingredient dissolved in
solvent and emulsifier. For both formulations, a fresh
water DE containing 89% amorphous silicon dioxide,
4.0% Al2O3, 1.7% Fe2O3, 1.4% CaO, less than 1% of
MgO and K2O and 3% moisture was used. The median
particle size of this DE is 10 mm, specific gravity is 2.2,
surface area 35.7 m2/g, pH 8 and crystalline silica is 0.1%.
Both formulations were used either as powder (P) or as
wettable powder (WP). Finally, for both DEs, the oral
LD50 for rat is 44640 mg/kg.
2.2. Insects and commodity
The S. oryzae, C. ferrugineus and R. dominica adults used
in the tests were taken from cultures that were kept on
whole hard wheat in the laboratory at 3071 1C and
7075% r.h. The T. castaneum adults were taken from a
culture kept on wheat flour plus 5% brewers’ yeast (by
weight) under the same conditions. All adults used in the
tests were 7–21 d-old. Untreated, clean, with very little
dockage and infestation-free Canadian Western Hard
wheat with 13.6% moisture content acclimated for 7 days
at 3071 1C was used in the tests for all four species.
469
2.3. Bioassays
Exposure studies were carried out at 3071 1C and 7075%
r.h. One hundred grams of treated grain were placed in
200 ml jars sealed with waxed filter paper. The application
rates tested were 75, 100, 125 and 150 ppm, corresponding to
0.075, 0.1, 0.125 and 0.15 g of DE/kg of wheat, respectively.
For the dust application, the jars were shaken manually to
achieve distribution of the dust throughout the grain. For the
slurry application, DE at the required application rate was
mixed with water and the grain was sprayed with 0.2 ml
of the suspension per kg of grain (2L/1000 kg of grain). For
the application of 75, 100, 125 and 150 ppm, the quantity of
the formulation of 0.75, 1, 1.25 and 1.5 g, respectively, was
mixed with 20 ml of water and 100 grams was sprayed with
0.2 g of the suspension. There were six jars for each DE-
dose–species combination. An additional series of untreated
jars with wheat were used as controls. For each of the four
species, 50 adults were introduced into each jar. Mortality
was assessed after 14 d and 21 d, in two sets of three jars, for
each species. After the last mortality count, all jars were
retained under the same conditions as above. Then, 49 d later
for S. oryzae and 63 d later for the other species, the jars were
opened and the number of dead and live adults were counted.
These intervals were selected as the appropriate intervals for
the appearance of progeny, according to Aitken (1975).
2.4. Data analysis
Control mortality was corrected using Abbott’s (1925)
formula. Before the analysis, all data were arcsine trans-
formed to standardize means and normalize variances. For
each species, DE and exposure interval, the mortality and
progeny data were submitted to a one-way ANOVA, for DE
dose, by using SAS (SAS Institute, 1995). Progeny production
was measured as number of beetles/jar. Progeny production
in the control jars were not included in the analysis, since a
preliminary ANOVA indicated that significantly more
progeny were found in the control jars than in the treated
ones (Po0.0001). The numbers of offspring in the untreated
wheat (beetles/jar7SE) for S. oryzae, R. dominica, T.
castaneum and C. ferrugineus were 339.3749.4, 68.8713.1,
53.3717.7 and 39.679.4, respectively. For the comparison of
the means the Tukey–Kramer (HSD) test was used, at the 5%
level (Sokal and Rohlf, 1995).
3. Results
3.1. Mortality caused by DEA-P/WP
In the case of S. oryzae, at the 14-d exposure interval,
495% of the exposed individuals were dead (Table 1). All
adults were dead even at the lowest dose rate on wheat
treated with DEA-P. Some weevils were still alive in the
DEA-WP treatments, but significant differences were not
noted. After 21 days of exposure, all adults were dead, for
both DEA-P and DEA-P (Table 2). For R. dominica, after
 ARTICLE IN PRESS
470 C.G. Athanassiou, Z. Korunic / Journal of Stored Products Research 43 (2007) 468–473

Table 1
Mean mortality (%7SE) of S. oryzae, R. dominica, T. castaneum and C. ferrugineus adults, exposed for 14 d on wheat treated with each of four DE
formulations at four doses. (Within each column and species, means followed by the same letter are not significantly different; where no letters exist, no
significant differences were noted; HSD test at 5%.)

Species Dose DE formulation

DEA-WP DEA-P DEBBM-WP DEBBM-P

S. oryzae 75 ppm 95.774.0 10070.0 74.573.5a 98.471.3

100 ppm 10070.0 10070.0 78.673.5a 96.372.3
125 ppm 96.473.3 10070.0 10070.0b 10070.0
150 ppm 10070.0 10070.0 10070.0b 10070.0
F 0.55 — 29.59 0.80
P 0.59 — o0.01 0.52
R. dominica 75 ppm 88.072.3 88.372.5a 10070.0 10070.0
100 ppm 86.772.5 84.372.6a 97.371.3 10070.0
125 ppm 87.372.6 85.472.3a 97.371.3 10070.0
150 ppm 94.771.9 96.571.6b 10070.0 10070.0
F 1.22 6.50 2.66 —
P 0.36 0.02 0.11 —
T. castaneum 75 ppm 8.172.3a 85.373.5a 56.374.9a 96.072.3
100 ppm 45.272.5b 92.173.2a 76.473.2b 10070.0
125 ppm 64.072.9c 10070.0b 92.072.5c 10070.0
150 ppm 74.273.2d 10070.0b 10070.0d 10070.0
F 137.90 22.00 47.16 3.00
P o0.01 o0.01 o0.01 0.09
C. ferrugineus 75 ppm 65.473.9a 10070.0 95.172.1a 10070.0
100 ppm 97.371.3b 10070.0 91.173.6a 10070.0
125 ppm 10070.0b 10070.0 10070.0b 10070.0
150 ppm 10070.0b 10070.0 10070.0b 10070.0
F 6.05 — 5.79 —
P 0.01 — 0.02 —

14 d of exposure, increasing dose increased adult mortality,
but significant differences were noted only for DEA-P
(Table 1). Mortality reached 100% in all cases after 21 d of
exposure (Table 2).
In contrast with the preceding species, significant
differences were noted among mortality levels after 14 d
of exposure for both DEA-P and DEA-WP in the case of
T. castaneum (Table 1). Mortality was extremely low
(o10%) on wheat treated with 75 ppm of DEA-WP, but
reached 74% at 150 ppm. Mortality levels were substan-
tially improved in the case of DEA-P, reaching 100% on
wheat treated with X125 ppm. Mortality was low on wheat
treated with 75 ppm of DEA-WP after 21 d of exposure,
but reached 100% at 150 ppm (Table 2). In addition, all
adults were dead after 21 d exposure to DEA-P, even at
75 ppm. Finally, for C. ferrugineus, almost one-third of the
exposed adults were still alive in the treatments with DEA-
WP after 14 d of exposure, but mortality was 100% at
application rates X125 ppm (Table 1). All adults died
during the treatments with DEA-P. At the 21 d exposure,
all adults were dead with the exception of the lowest dose
rate of DEA-WP (Table 2).
3.2. Mortality caused by DEBBM-P/WP
For S. oryzae, after 14 d of exposure, mortality was
100% on wheat treated with X125 ppm of either DEBBM-
WP or DEBBM-P (Table 1). After 21 d of exposure,
mortality levels were 100% on 75 ppm of DEBBM-P,
but only 80% at the same dose of DEBBM-WP (Table 2).
In the case of R. dominica, most adults were dead after 14 d
of exposure (Table 1), and all were dead after 21 d
(Table 2).
For T. castaneum, DEBBM-WP caused 100% mortality
after 14 d of exposure only at 150 ppm (Table 1). In
contrast, with DEBBM-P mortality was 100% after 14 d of
exposure except at 75 ppm (Table 1). After 21 d of
exposure, all adults were dead, with the exception of 75
and 100 ppm of DEBBM-WP (Table 2). Finally, for C.
ferrugineus, after 14 d of exposure, all adults were dead
even with the lowest dose of DEBBM-P (Table 1).
Moreover, mortality was 498% for the lowest dose of
DEBBM-WP after the 21-d exposure (Table 2).
3.3. Progeny production on wheat treated with DEA-P/WP
Progeny production for S. oryzae was high at dose rates
of DEA-WP o125 ppm, while very few progeny were
noted on wheat treated with the two higher dose rates
(Table 3). In contrast, p0.3 weevils/jar was noted in the
case of DEA-P. In comparison with S. oryzae, less progeny
were found for R. dominica (p1 adults/jar), especially on
wheat treated with DEA-P where no emergence occurred at
doses X100 ppm (Table 2).
 ARTICLE IN PRESS
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Table 2
Mean mortality (%7SE) of S. oryzae, R. dominica, T. castaneum and C. ferrugineus adults, exposed for 21 d on wheat treated with each of four DE
formulations at four doses. (Within each column and species, means followed by the same letter are not significantly different; where no letters exist, no
significant differences were noted; HSD test at 5%.)

Species Dose DE formulation

DEA-WP DEA-P DEBBM-WP DEBBM-P

S. oryzae 75 ppm 10070.0 10070.0 80.474.1a 10070.0

100 ppm 10070.0 10070.0 90.573.2b 10070.0
125 ppm 10070.0 10070.0 10070.0c 10070.0
150 ppm 10070.0 10070.0 10070.0c 10070.0
F — — 12.75 —
P — — o0.01 —
R. dominica 75 ppm 10070.0 10070.0 10070.0 10070.0
100 ppm 10070.0 10070.0 10070.0 10070.0
125 ppm 10070.0 10070.0 10070.0 10070.0
150 ppm 10070.0 10070.0 10070.0 10070.0
F — — — —
P — — — —
T. castaneum 75 ppm 26.974.1a 10070.0 58.673.8a 10070.0
100 ppm 73.573.6b 10070.0 80.374.8b 10070.0
125 ppm 89.573.8c 10070.0 10070.0c 10070.0
150 ppm 10070.0d 10070.0 10070.0c 10070.0
F 112.17 — 53.46 —
P o0.0001 — o0.01 —
C. ferrugineus 75 ppm 88.974.4a 10070.0 98.871.1 10070.0
100 ppm 10070.0b 10070.0 10070.0 10070.0
125 ppm 10070.0b 10070.0 10070.0 10070.0
150 ppm 10070.0b 10070.0 10070.0 10070.0
F 100.00 — 1.00 —
P o0.0001 — 0.44 —

For T. castaneum, no significant differences were noted
in progeny production among doses for DEA-WP, but
progeny decreased with the increase in dose (Table 3). No
progeny were obtained in the DEA-P treatments. Similar
results were also recorded for C. ferrugineus, with no beetle
emergence on wheat treated with DEA-P (Table 3).
3.4. Progeny production on wheat treated with DEBBM-P/WP
The number of F1 individuals of S. oryzae was extremely
high (4120 weevils/jar) at 75 ppm of DEBBM-WP, but it
decreased with the increase of dose to o1 weevil/jar. On
the other hand, very few progeny were noted on wheat
treated with DEBBM-P. For R. dominica, no progeny
emerged from treatments with either formulation (Table 3).
For T. castaneum, progeny production was high after
treatment with DEBBM-WP at 75 ppm, while no progeny
were found at dose rates X125 ppm. For DEBBM-P,
progeny production was recorded only in the case of
75 ppm. Finally, for C. ferrugineus, on wheat treated with
DEBBM-WP progeny production was generally low, and
ranged from 0.3 to 1 adults/jar (Table 3). No progeny
emerged on wheat treated with DEBBM-P.
4. Discussion
Based on the results of the present study, by using the
two enhanced DEs tested here, high mortality can be
obtained against adults of S. oryzae, R. dominica, T.
castaneum and C. ferrugineus with dose rates of 150 ppm or
less. In contrast, Athanassiou et al. (2004, 2005a) by using
the DEs SilicoSec, PyriSec and Insecto, noted that
1000 ppm were required for a satisfactory level of mortality
for S. oryzae. Similar results have been reported in the case
of T. castaneum by Fields and Korunic (2000). These dose
rates question the viability of using DEs, as alternatives to
traditional grain protectants, which are normally effective
at dose rates of 10 ppm or less.
DEs vary remarkably in their insecticidal effect among
different stored-grain insect species (Fields and Korunic,
2000). Among the species tested here, C. ferrugineus is
considered as the most DE-susceptible, followed by S.
oryzae, R. dominica and T. castaneum (Korunic, 1998;
Arthur, 2000; Fields and Korunic, 2000; Athanassiou et al.,
2005a; Kavallieratos et al., 2005). Based on our results,
with the exception of T. castaneum, for the other three
species high mortality can be obtained by using 75 ppm of
DEA-P/WP and DEBBM-P/WP, either in dust or in slurry
applications. For complete mortality of T. castaneum
adults, 100 ppm or more is required, which is still an
extremely low dose rate, as compared with the DEs known
so far. Although both enhanced DEs tested here were very
effective against the four species examined, for S. oryzae
DEA-P/WP was more effective than DEBBM-P/WP. In
contrast, for the other three beetle species, DEBBM-P/WP
was more effective than DEA-P/WP, especially at the 14 d
 ARTICLE IN PRESS
472 C.G. Athanassiou, Z. Korunic / Journal of Stored Products Research 43 (2007) 468–473

Table 3
Progeny production (number of adults/jar7SE) of S. oryzae, R. dominica, T. castaneum and C. ferrugineus adults, on wheat treated with each of four DE
formulations at four doses. (Within each column and species, means followed by the same letter are not significantly different; where no letters exist, no
significant differences were noted; HSD test at 5%. Progeny in the control samples: 418.5718.6, 20.172.8, 21.873.3, 45.872.5 for S. oryzae, R. dominica,
T. castaneum and C. ferrugineus, respectively.)

Species Dose DE formulation

DEA-WP DEA-P DEBBM-WP DEBBM-P

S. oryzae 75 ppm 21.072.5a 0.070.0 127.179.9a 2.071.2

100 ppm 10.371.8b 0.370.3 12.371.8b 0.670.3
125 ppm 0.070.0c 0.370.3 4.371.2c 0.070.0
150 ppm 0.370.3c 0.070.0 0.770.7d 0.070.0
F 46.38 0.66 154.05 2.46
P o0.01 0.59 o0.01 0.14
R. dominica 75 ppm 0.370.3 0.770.3 0.070.0 0.070.0
100 ppm 1.070.5 0.070.0 0.070.0 0.070.0
125 ppm 1.070.3 0.070.0 0.070.0 0.070.0
150 ppm 0.070.0 0.070.0 0.070.0 0.070.0
F 2.25 4.00 — —
P 0.16 0.06 — —
T. castaneum 75 ppm 1.770.9 0.070.0 10.071.9a 0.770.3
100 ppm 1.370.9 0.070.0 8.772.2a 0.070.0
125 ppm 0.370.3 0.070.0 0.070.0b 0.070.0
150 ppm 0.070.0 0.070.0 0.070.0b 0.070.0
F 1.51 — 15.08 4.00
P 0.28 — o0.01 0.06
C. ferrugineus 75 ppm 1.370.8 0.070.0 1.070.5 0.070.0
100 ppm 0.370.3 0.070.0 0.770.3 0.070.0
125 ppm 0.070.0 0.070.0 0.370.3 0.070.0
150 ppm 0.070.0 0.070.0 0.370.3 0.070.0
F 2.86 — 1.22 —
P 0.10 — 0.36 —

exposure. However, after 21 d of exposure, these variations
were eliminated, and mortality in most cases reached
100%.
Apart from variations between the two enhanced DEs,
the most important variations were those recorded between
the slurry and the dust application of each DE. In all cases,
the slurry application of DEA-P/WP or DEBBM-P/WP
were less effective than the corresponding applications of
dust, especially at low dose rates. Generally, dusts are more
effective than aqueous solutions of DEs, as previously
reported by other researchers (Maceljski and Korunic,
1971; Subramanyam and Roesli, 2000; Arthur, 2004). This
could be attributed to the fact that the presence of water
partially decreases the effectiveness of DE particles, since
DEs are not very effective in humid conditions (Korunic,
1998; Vayias and Athanassiou, 2004). Consequently, high-
er doses are required in the case of slurries in comparison
with dust applications. However, the use of slurry avoids
the respiratory problems caused by DE dusts, and this is an
advantage for large-scale applications.
Progeny production in the treated substrate is perhaps
more important than parental mortality, because a grain
protectant should protect the grain for a long storage
period (Athanassiou et al., 2005b). In our work, progeny
production of S. oryzae was high on wheat treated with
p100 ppm of the slurries of DEA-P/WP or DEBBM-P/
WP, which suggest that, for long-term protection,
4100 ppm of the slurries is required. In contrast, progeny
production was low for all species tested in the dust
applications of both enhanced DEs.
In summary, our results indicate that the enhanced DEs
tested here were very effective against S. oryzae, R.
dominica, T. castaneum and C. ferrugineus, and, based on
the available literature, more effective than the currently
available commercial DE formulations. According to
Korunic (2001) and Arthur (2003) the combination of
DEs with other reduced-risk control techniques, such as
botanicals or low doses of insecticides, could lead to
increased use of DEs in stored-product IPM strategies.
Additional experimentation is required to clarify the
performance of these two enhanced DEs under a broader
range of conditions (target species, various commodities,
various temperature and moisture levels etc.).
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