BioControl (2022) 67:213–224

https://doi.org/10.1007/s10526-021-10126-2 (0123456789().,-volV)
( 01234567
89().,-volV)

What would be representative temperatures for shelf-life

studies with biopesticides in tropical countries? Estimates
through long-term storage of biocontrol fungi
and calculation of mean kinetic temperatures
Marcos Faria . Luı́s Alberto Martins Palhares . Daniela Aguiar Souza .
Rogerio Biaggioni Lopes

Received: 3 August 2021 / Accepted: 8 December 2021 / Published online: 29 January 2022
 The Author(s), under exclusive licence to International Organization for Biological Control (IOBC) 2021

Abstract In many tropical countries, studies to
determine the shelf-life of biopesticides for commer-
cialization and storage without refrigeration have been
conducted under mild (B 25 C) temperature regimes.
Are these temperatures representative of non-refrig-
erated rural deposits? To answer this question, we set
up trials in five Brazilian geographical regions.
Representative temperatures were estimated using
two distinct approaches: (1) calculation of the mean
kinetic temperature (MKT) inside packages stored in
rural deposits for up to 19 months, and (2) comparison
of germination counts of fungi (Beauveria bassiana
and Trichoderma spp.) kept under these conditions
with those of conidia kept at constant temperatures.
MKTs calculated for deposits located in the Northern,
Northeastern, Central-Western, Southeastern, and
Southern regions were 28.0, 26.7, 23.6, 22.2, and
21.7 C, respectively. For all regions, MKTs calcu-
lated for the long-term trials were very close
(B 0.5 C) to the values calculated for the year
2013. However, in certain periods, MKTs in the South
and Southeast deposits were near 25 C, whereas
variations in other regions were minimal (B 0.6 C).
The less precise estimate of representative tempera-
tures in rural deposits, based on germination counts of
B. bassiana and T. harzianum, were in the 23.8–35.4
and 24.0–27.5 C ranges, respectively, depending on
the geographical region. Our results indicate that
guidelines that propose 30 C to be adopted for
stability tests for pharmaceutical and food products
in tropical countries must also be applied to biopes-
ticides intended for country-wide commercialization
and storage under non-refrigerated conditions in
Brazil.
Keywords Mycopesticides  Beauveria bassiana 
Trichoderma asperellum  Trichoderma harzianum 
Active packaging  Shelf-life

Handling Editor: Nicolai Meyling.

Introduction
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/
s10526-021-10126-2.
Numerous biological products based on living
microbes are marketed worldwide for control of pests,
M. Faria (&)  L. A. M. Palhares  including insects, mites, ticks, plant pathogens, and
D. A. Souza  R. B. Lopes weeds (Chandler et al. 2000; Menaria 2007; Harman
Embrapa Genetic Resources and Biotechnology, PqEB
W5 North Avenue, Federal District, Brasilia 70770-917,
2011; Lacey et al. 2015). Over recent years, these
Brazil biopesticides, including fungus-based products, have
e-mail: marcos.faria@embrapa.br

123
214
experienced two-digit annual growth rates (Glare et al.
2012; Shoram 2020). For the adoption of mycopesti-
cides and other biopesticides to continue at an
increased rate, many obstacles need to be overcome,
such as factors related to mass production, release
strategies aimed at better control, and longer environ-
mental persistence (Lacey et al. 2015; Glare et al.
2017; Grzywacz 2017). Another relevant factor,
especially under tropical conditions, is a satisfactory
shelf life, which allows the concentration of active
propagules to be kept within acceptable limits during
storage (Jones and Burges 1998; Grzywacz 2017). The
stability of mycopesticides is highly influenced by the
range of temperatures during transportation and stor-
age before their use by farmers. It is not unusual that
the storage of this sort of product under tropical
conditions results in loss of quality in a matter of
weeks or even days, with a high proportion of killed or
debilitated fungal cells (Sandhu et al. 1993; Marques
and Alves 1996; Silva 2006; Kassa 2003).
The international conference on harmonization
(ICH) proposed the establishment of different climatic
zones and the conditions to be employed in long-term
stability tests. Stability testing was initially proposed
for pharmaceutical products to ensure that their full
efficacy would be retained up to the end of their
expiration date (Grimm 1993). Regions of the globe
like the USA, Europe, and Japan were designated as
climatic zone II, where the tests of thermal stability
with drugs should be conducted at 25 C and 60% RH
parameters that were later adopted for tests with
foodstuffs. Hot and humid countries were classified as
climatic zone IVb, and tests there should be conducted
at 30 ± 2 C, and when packaging material are
permeable to water vapor the RH must be 75 ± 5%
(WHO 2004). In 2005, the Brazilian National Health
Surveillance Agency (ANVISA 2005) established the
same condition for pharmaceutical products recom-
mended to be stored in the 15–30 C range. The
temperature of 30 ± 2 C is the calculated mean
kinetic temperature (MKT) for hot countries, which
can be understood as a single calculated temperature at
which the total amount of degradation over a particular
period is equal to the sum of the individual degrada-
tions that would occur at various cycles of higher and
lower temperatures (Grimm 1993; Bott and Oliveira
2007) or, put in another way, the temperature corre-
sponding to the effects of a given temperature–time
123
M. Faria et al.
distribution on the chemical reaction kinetics (Huynh-
Ba and Zahn 2009).
In Brazil, shelf-life studies with microbial pesti-
cides have not been conducted under these conditions,
but under milder ones. For fungus-based biopesticides,
also known as mycopesticides, the highest tempera-
ture adopted in such studies has been 27 C, although
for most commercial mycopesticides it is usually
determined at B 25 C, while in other cases the
subjective and undesirable expression ‘‘room temper-
ature’’ is used on package inserts or labels (Agrofit
2021). This is worrisome, since incorrect conclusions
could be drawn, such as the validity period of
biopesticides advertised by manufactures. Changes
in storage temperature may result in different shelf
lives, as shown by Faria et al. (2012). In their study, the
time for the initial germination (93–96%) of actively
packaged Beauveria bassiana conidia to fall to the
level of 80% was approx. 100 and [ 480 d when
subjected to storage at 40 and 25 C, respectively.
These temperatures can be reached in the summer of
some temperate regions and, even more frequently, in
tropical countries.
We believe that, if regulators and biopesticide
manufacturers become aware of representative storage
temperatures in rural deposits, more realistic shelf-life
experiments would be conducted during biopesticide
development. Equally important, farmers would have
more accurate information about the stability of these
products during storage and, consequently, it could
lead to greater field efficacy in the control of pests.
Therefore, the main objective of the present study was
to estimate representative temperature(s) in rural
deposits located in different geographic regions of
Brazil, using two different approaches.
Material and methods
Preliminary trial: short-term storage on a rural
deposit in the Central-Western region
A three-month trial, from 08/30/2011 to 11/28/2011,
was conducted in a pesticide deposit in the Central-
Western region (from now on, referred to as CW
deposit), located at the Embrapa Cerrados experimen-
tal station, in Planaltina, Federal District. This location
was chosen due to its proximity (\ 30 km) to the
Invertebrate Mycology Laboratory at Embrapa
What would be representative temperatures for shelf-life studies with biopesticides in tropical…
Genetic Resources and Biotechnology (Brası́lia, Fed-
eral District), as well as the availability of a weather
station close to the deposit. The test helped to
determine the relationship between the ambient tem-
perature inside the deposit and the temperature
readings inside airtight packages, as well as estimate
the representative temperature (RT) in this deposit
during the trial. To each 1 l aluminized package
(Lamipack Embalagens e Laminados Plásticos Ltda.,
Brazil), one RP-20A O2/H2O-absorbing sachet (Mit-
subishi Gas Chemical Company Inc., Japan) and 10 g
of Trichoderma asperellum URM 5911, formulated as
a water dispersible granule formulation (Quality WG,
Laboratório de BioControle Farroupilha Ltda, Patos
de Minas, MG Brazil) were added, in addition to
dataloggers in some packages to monitor the temper-
ature and RH every 30–60 min. Previously calibrated
dataloggers, models U10-003, U12-012, and U14-001
(Hobo, Onset Computer Corp., Bourne, MA, USA),
were used, the former being adopted only in experi-
ments in which the RH of the environment or inside
packages was measured. Due to the slower loss of
conidial germination after packaging with O2/H2O-
absorbing sachets, and the consequent risk of exces-
sive prolongation of the experimental period, a
product batch with low conidial germination (38.3%)
was used. Then, packages were kept for eight days
inside an incubator set at 25 C to guarantee reduction
of the internal levels of O2 and UR (equilibrium
period). After the equilibrium period, four packages
were destructively sampled to determine the water
activity (aw) and conidial germination. Determina-
tions of aw in this study were performed using a water
activity meter (LabMaster-aw, Novasina, Pfäffikon,
Switzerland) calibrated at 25 C. Germination was
estimated by plating the conidial suspension with
approx. 1 9 106 con/ml-1 in 1/5 potato dextrose agar
(PDA) medium followed by incubation at 25 C for
16 h. The germination assessment methodology fol-
lowed guidelines recommended by Lopes et al.
(2013). The remaining pouches (four per treatment)
were transferred to the CW pesticide deposit or
incubators regulated to constant temperatures (25,
30, 35, or 40 C). For transportation from our lab to
the CW deposit, the packages were kept in a styrofoam
box. Both in the deposit and in each incubator,
dataloggers were placed close to packages to record
the environmental temperature. Although hermetic
packaging was adopted in our study and, therefore, the
215
internal content was insensitive to the RH of the
surrounding external space, aw levels of the mycopes-
ticide were monitored. At the end of the storage
period, the packages were taken to the laboratory to
determine aw and conidial germination as already
explained.
Half-lives for mycopesticides at controlled
temperatures
This test had a dual purpose—to estimate the time
required for initial germination of stored conidia to
drop 50% (half-life) in the laboratory under different
controlled temperatures and to indicate completion
time for the long-term tests in pesticide deposits (see
Long-term storage in rural deposits in different
geographic regions). The same active packaging
technique previously described was used, but this
time pure conidia (1.6 g per package) of either
Beauveria bassiana PL63 (active ingredient of the
product Boveril WP, Koppert Biological Systems,
Piracicaba, SP Brazil) or Trichoderma harzianum
ESALQ 1306 (active ingredient of the product
Trichodermil, from the same company), were used.
After a 12–13 day equilibrium period at 25 C, four
packages were used to determine conidial germination
and aw. The remaining packages were transferred to
incubators set at 26, 29, 32, 35, or 38 C. Between 16
and 20 foil pouches were randomly incubated in each
temperature. Germination rates were assessed on
different dates over a storage period of 24 and
30 months for T. harzianum and B. bassiana, respec-
tively. In each assessment, between three and four foil
pouches per treatment were destructively sampled.
The initial conidial germination rates for T. harzianum
and B. bassiana were 43.9 and 83.0%, respectively.
The choice for a T. harzianum batch with low quality
was to ensure that at the end of the study there would
be significant loss of germination, since the active
packaging of high-quality conidia could substantially
extend the duration of this test. In addition to half-life,
the parameter known as shelf-life, here considered as
the time for the initial concentration of active ingre-
dients to fall by 20% at a given temperature, was
calculated whenever possible. Ambient RH was not
considered for calculation of either half-lives or shelf-
lives, because hermetic pouches were always used.
123
216
Long-term storage in rural deposits in different
geographic regions
The experiments that aimed to determine representa-
tive temperatures (RTs) in pesticide deposits located
in the five geographic regions of Brazil were carried
out for almost 19 months. The packages with
mycopesticides were prepared on the same day and
in exactly the same way as described in Half-lives for
mycopesticides at controlled temperatures. Experi-
ments described in this section and in the previous one
were set up with the same conidial batches and on the
same days and, therefore, the initial conidia germina-
tion rates for T. harzianum and B. bassiana were also
43.9 and 83.0%, respectively. The equilibrium period
at 25 C was 12 (B. bassiana) or 13 days (T.
harzianum) and, at the end of this, all packages were
placed in styrofoam boxes and delivered personally
(CW deposit) or sent via courier to the other
geographic regions on Nov 26, 2012. With the
exception of the CW deposit, in which the packages
were stored on the same day, packages arrived
between two (deposit in Northern region) and nine -
days after shipment (deposit in Southeastern region).
Packages were placed in deposits in the five geo-
graphic regions: Central-West (same as before), North
(N deposit – Associação Rural de Pimenta Bueno;
municipality of Pimenta Bueno, Rondonia state),
Northeast (NE deposit—Nossa Senhora Aparecida
Farm; municipality of Formosa do Rio Preto, Bahia
state), Southeast (SE deposit—ABC Foundation;
municipality of Itaberá; Sao Paulo state), and South
(S deposit—Caracol Farm; municipality of Ponta
Grossa; Parana state). The geographical coordinates
and characteristics of the deposits are described in
Table 1. Between four and five packages of each
mycopesticide were kept in each deposit, always
between 1.2 and 2.0 m above the floor. The same
number of packages were kept in the laboratory in
incubators regulated at fixed temperatures (26, 29, 33,
35, or 38 C). In each treatment with T. harzianum,
one package contained a datalogger for recording,
every hour, the temperature during the transport
(laboratory-deposit-laboratory) and the long-term
conditions in the deposits. As the preliminary test
showed that the temperatures inside the packages are
very close to those in the deposits (outside the
packages), the ambient temperature was not moni-
tored, except in the CW deposit. The storage periods in
123
M. Faria et al.
deposits in the N, NE, CW, SE, and S regions were
11/28/2012 to 6/7/2014, 12/4/2012 to 6/5/2014, 11/26/
2012 to 06/11/2014, 12/05/2012 to 6/3/2014, and
11/30/2012 to 6/5/2014, respectively. At the end of the
storage periods, the packages were removed from the
deposits and sent back to the Invertebrate Mycology
Laboratory taking the same precautions as before,
where they arrived 0–6 days later. The packages were
kept in a refrigerator (4–5 C) for up to 11 days, and
then they were all processed at the same time. The
germination percentages were determined by plating
the conidial suspension of T. harzianum and B.
bassiana on 1/5 PDA for 16 h or PDA for 22 h,
respectively.
Calculation of mean kinetic temperatures (MKTs)
in rural deposits
Temperatures in rural deposits were recorded every
hour with dataloggers kept inside some biopesticide
pouches, as explained in Long-term storage in rural
deposits in different geographic regions. The follow-
ing equation was used to calculate the MKT in each
deposit:
DH
Tk ¼   R 
DH DH
þþeð RTn Þ
RT1 RT2 DH
þe
 ln e n
where, Tk = MKT in kelvin; DH = activation energy
(83.14472 kJ/mole-1); R = gas constant
(0.008314472 kJ/mole-1/K-1); T1 to Tn are the tem-
peratures (in K) at different sample points and
n = number of temperature sample points.
Statistical analysis
Experiments were arranged in completely randomized
designs and several analyses were performed using the
the R program (R Development Core Team 2012).
Conidia germination data were fitted to a generalized
linear model (GLM) with binomial distribution and
logit link function (logistic regression) to estimate the
representative temperatures and half-lives for conidia
stored at controlled temperatures and rural deposits.
Akaike information criterion (AIC), Bayesian infor-
mation criterion (BIC) and likelihood ratio tests (LRT)
were used to choose the best model to fit proportional
data. Then, the data adequacy test (goodness-of-fit) to
What would be representative temperatures for shelf-life studies with biopesticides in tropical… 217

Table 1 Characteristics of rural deposits located in Brazil used for storage of mycopesticides under non-refrigerated conditions
Deposit Municipality Deposit characteristics
Geographic coordinates/ Aprox. area Wall Roof and ceiling types Ceiling
altitude (m) (m2) type heighta (m)

N Pimenta Bueno, 11380 21.3600 S 61110 47.0200 12 Masonry Clay roof/PVC lining n.i.b
Rondonia W/185 m
NE Formosa do Rio 10 340 4700 S 45 410 6.400 100 Masonry Zinc roof/no internal 4.0
Preto, Bahia W/81 m lining
CW Planaltina, Federal 15360 12.0900 S 47420 58.8200 40 Masonry Asbestos roof/no 2.8
District W/1.017 m internal lining
SE Itaberá, Sao Paulo 24040 2000 S 49090 1900 17 Masonry Asbestos roof/concrete 2.7
W/738 m slab
S Ponta Grossa, Parana 24570 39.4500 S 50130 53.2600 40 Masonry Zinc roof/no internal n.i.b
W/921 m lining
a
Heights refer to the lowest point of the ceiling
b
Ceiling height was not informed

each model was performed, and the pearson dispersion
statistic was used to verify the presence of overdis-
persion in the models (Hilbe 2015). Additionally,
deviance residuals, Anscombe residual plots and
residual envelope graphs (half normal plots) were
constructed to identify outliers and adjust the model.
Simultaneous confidence intervals at 95% were con-
structed and pairwise comparisons were done via odds
ratios. As in the previous experiment, the actual
controlled temperatures in incubators (and not those
for which the incubators had been set to) were
considered in the analyses.
Results
Short-term storage in a rural deposit in the Central-
western region
The aw and germination of T. asperellum conidia after
the 8-day equilibrium period were 0.054 ± 0.004 and
38.300% ± 4.150, respectively. The three-month test
carried out in incubators regulated to fixed tempera-
tures and in the CW deposit with a variable temper-
ature regime confirmed, as expected, that the
temperature of the air inside the packages is coincident
with that of the surrounding environment (data not
shown). The temperatures recorded by dataloggers
kept inside incubators set to 25, 30, 35, or 40 C were
25.00 ± 0.18, 30.40 ± 0.11, 35.00 ± 0.22, and
40.90 ± 0.11 C, respectively. In turn, the
temperatures recorded inside packages kept in these
incubators were 24.90 ± 0.11, 30.30 ± 0.13,
34.70 ± 0.20, and 39.90 ± 0.35 C, respectively.
During the study period, the average daily temperature
recorded at the meteorological station near the CW
deposit was 21.8 C, ranging from 10.0 to 34.6 C.
Inside the deposit the variation was narrower, with
average daily temperature of 23.9 C, ranging from
18.5 (lowest temperature recorded was 17.6 C) to
27.9 C (highest temperature recorded was 32.3 C).
Inside packages, the average daily temperature was
23.8 C, varying from 18.1 (lowest temperature
recorded was 16.6 C) to 27.9 C (the highest tem-
perature recorded was 32.5 C), i.e., values very close
to those registered inside the deposit. The daily
average RH recorded at the weather station was
64.1%, varying from 10.0 to 98.0%. While inside the
deposit, the daily average RH was 58.2%, ranging
from 26.8 to 84.9%. As expected, the RH readings
inside the hermetic packages with O2/H2O-absorbing
sachets were considerably lower than those of the
surrounding air, reaching and maintaining 0.0% in less
than one week after packaging (data not shown).
At the end of the three-month storage period, the aw
determined for conidia kept in the CW deposit and
incubators at 25, 30, 35, or 40 C were, respectively,
0.031 ± 0.000, 0.030 ± 0.001, 0.029 ± 0.000,
0.030 ± 0.000, and 0.029 ± 0.001. The germination
rates for conidia kept in the deposit and incubators
were influenced by the storage conditions according to
the best fit model (v2 = 127.1; df = 5; p \ 0.0001).

123
218
Conidial germinations were 30.00 ± 1.91,
33.60 ± 3.34, 27.90 ± 1.55, 24.00 ± 2.85, and
16.60 ± 2.27% in the CW deposit and incubators at
25, 30, 35, or 40 C, respectively. According to the
general linear model applied to our data, germination
percentage for conidia kept in the deposit indicates a
representative temperature of 27.5 [23.1–31.5] C
(Fig. 1). Throughout the experimental period, esti-
mate of half-life was only possible for conidia kept at
constant 40 C.
Half-lives for mycopesticides at controlled
temperatures
The average temperatures recorded every hour by
dataloggers kept inside incubators set at 26, 29, 32, 35,
and 38 C were 26.00 ± 0.34, 29.10 ± 0.66,
32.90 ± 0.83, 34.30 ± 0.46, and 37.10 ± 2.40 C,
respectively. In turn, the average temperatures
recorded every hour by dataloggers inside packages
kept in incubators set at 26, 29, 32, 35, and 38 C were
25.90 ± 0.30, 29.20 ± 0.32, 32.70 ± 0.34,
34.40 ± 0.37, and 37.10 ± 2.40 C, respectively.
The greatest variation, which were observed in the
readings recorded by dataloggers kept in the incubator
set to 38 C, could be due to periodic opening of this
incubator during mild ambient temperatures and,
50
Conidial germination (%)
0.054
40
0.030
0.031
a 0.029
30
b bc
20 bc
10
0 period of equilibrium. According to the best fit
Pre- 25 °C CW 30 °C
storage
Treatments
Fig. 1 Germination (means and 95% confidence intervals) of
Trichoderma asperellum conidia in a commercial water
dispersible granule formulation (WG) stored in incubators set
at constant temperatures (25, 30, 35, or 40 C) or at a fluctuating
temperature regime in deposit located in the Central-Western
(CW) region (airtight pouches with O2/H2O-absorbing
sachets ? 10 g of conidia) for 90 days. Numbers on the top of
bars refer to the average water activity of the samples after the
equilibrium period. Bars with same letters are not significantly
different by pairwise odds ratio comparisons (P \ 0.05). The
horizontal stippled line corresponds to 50% of the initial
germination
123
M. Faria et al.
above all, from a failure in the incubator, which
operated in approx. 28.50 ± 1.78 C (ranging from
24.40 to 36.80 C) during 5.5% of the experimental
period.
After the equilibrium period at 25 C (immediately
before transfer to fixed temperatures), B. bassiana
conidia showed an average initial germination of
82.30 ± 4.30% at 22 h after plating on PDA, and aw
of 0.066 ± 0.007. According to the best-fit logistic
regression models, storage conditions inside incuba-
tors influenced the germination rates of B. bassiana
conidia. Pouches transferred to incubators operating at
different temperatures (26–35 C) had conidial ger-
mination evaluated at 371, 582, 744, and 899 days
after storage. Evaluations for pouches stored at 38 C
were performed only at 371 and 582 days after
storage. In an evaluation carried out at 582 days that
included all the treatments, the germination of conidia
kept at 38 C was statistically lower than those of
conidia kept at 26, 29, and 32 C (v2 = 32.1; df = 4;
p \ 0.0001). In the evaluation conducted at 899 days
(30 months), when there were no more pouches at
38 C, the germination readings at fixed temperatures
(26 to 35 C) did not differ (v2 = 4.5; df = 3;
p = 0.2087), ranging from 67.80 ± 6.91 to
71.10 ± 3.50%, as shown in Supplementary
Table S1. It was not possible to calculate the actual
half-life for B. bassiana conidia stored at each
temperature, although it was possible to estimate the
half-lives, which varied from 2368 days at 26 C to
1049 days at 38 C (Fig. 2). As expected, the shelf-
0.030
lives were quite shorter, being 1507 and 582 days at
26 8C and 38 8C, respectively.
0.029
Conidia of T. harzianum had an average germina-
c
tion of 43.90 ± 1.22% after 16 h of plating in 1/5
d
PDA and aw of 0.029 ± 0.001 immediately after the
35 °C 40 °C
models, the germination rates for T. harzianum conidia
kept in incubators were influenced by the storage
conditions. Contrary to what was observed for B.
bassiana, we were able to calculate the actual half-
lives of stored conidia at all temperatures evaluated, as
shown in Fig. 3. For temperatures in the 26–35 C
range, half-lives (22% of conidia germination) were in
the range of 350–700 days. For the highest storage
temperature (38 C), germination was 22.7%
(20.0–25.8) at 134 days and differed (v2 = 29.2;
df = 2; p \ 0.0001) from data calculated at 35 C
(27.2%) and 32 C (32.6%) (Supplementary
What would be representative temperatures for shelf-life studies with biopesticides in tropical…
Fig. 2 Germination probabilities (means and 95% confidence
intervals) for Beauveria bassiana conidia in airtight pouches
with O2/H2O-absorbing sachets stored in incubators set to
constant temperatures: a 26 C, b 29 C, c 32 C, d 35 C, and
e 38 C. Initial conidial viability (after equilibrium period) was
82.3%. The curves are the best fit of the data to logistic
regression models, whereas the vertical dotted lines indicate the
half-life estimate for each temperature
Table S2). Shelf-lives in the 26–35 C range varied
from 87 to 131 days.
Long-term storage in rural deposits at different
geographic regions
The temperatures recorded during transport (labora-
tory to deposits) and until storage of pouches in the
deposits ranged from 22.10 ± 2.49 to
27.00 ± 2.83 C, depending on the region. The aver-
age daily temperatures as well as the maximum and
219
Fig. 3 Germination probabilities (means and 95% confidence
intervals) for Trichoderma harzianum conidia in airtight
pouches with O2/H2O-absorbing sachets stored in incubators
set to constant temperatures: a 26 C, b 29 C, c 32 C, d 35 C,
and e 38 C. Initial conidial viability (after equilibrium period)
was 44.6%. The curves are the best fit of the data to logistic
regression models, whereas the vertical dotted lines indicate the
half-life estimate for each temperature
minimum temperatures during storage are provided in
Table 2. In a few cases, temperature readings inside
the packages did not cover the entire storage period,
mainly because the battery life of dataloggers was too
short. For both B. bassiana and T. harzianum,
germination rates of conidia kept in rural deposits
and incubators were influenced by the storage condi-
tions according to the best fit models.
The germination of B. bassiana conidia kept in
incubators and deposits for 19 months can be seen in
Fig. 4a, with significant differences among treatments
123
220 M. Faria et al.

Table 2 Mean kinetic temperatures (MKTs) calculated for rural deposits located in different geographic regions of Brazil
Deposit Whole storage period Year 2013
b
Storage period Average, maximum and Calculated Storage Average, maximum and Calculated
(duration) minimum temperatures MKT (C) period minimum temperatures MKT (C)
(C) (C)

N 11/29/12 to 05/27/ 27.9, 36.7, 23.2 28.0 –c – –

2013 (6.0 months)
NE 12/05/2012 to 26.0, 37.4, 17.3 26.7 01/01/2013 26.0, 37.4, 17.3 26.8
05/15/2014 to 12/31/
(17.5 months) 2013
CW 11/27/2012 to 23.3, 31.7, 14.9 23.6 01/01/2013 23.3, 31.7, 14.9 23.7
06/11/2014 to 12/31/
(18.7 months) 2013
CWa 08/30/2011 to 23.8, 32.5, 16.6 24.2 –d – –
11/28/2011
(3.0 months)
SE 12/06/2012 to 21.7, 29.7, 10.0 22.2 01/01/2013 21.2, 29.7, 10.0 21.8
01/14/2014 to 12/31/
(13.5 months) 2013
S 12/01/2012 to 20.1, 35.9, - 0.1 21.7 01/01/2013 19.6, 35.9, - 0.1 21.2
12/18/2013 to 18/12/
(12.7 months) 2013
a
Same rural deposit as before, but this three-month long study was conducted before the long-term trials initiated at Nov/Dec 2012
b
For the long-term trials, biopesticide packages, some of them with dataloggers inside, were prepared on the same day and sent to
rural deposits in styrofoam boxes on Nov 26, 2012. With the exception of the CW deposit, in which the packages were stored on the
same day (and readings started in the following day), the arrival in the other deposits occurred between two (deposit in the Northern
region) and nine days after shipment (deposit in the Southeastern region)
c
The battery of the datalogger was not enough to record the temperature readings inside the biopesticide pouche after 05/27/2013
d
Study conducted before 2013

(v2 = 199.50; df = 10; p \ 0.0001). At 26 and 29 C,
levels between 73.9 and 74.7% were recorded, which
were statistically higher than those of conidia stored at
35 and 38 C, which ranged from 63.9 to 64.9%. The
germination rates for conidia maintained in the
deposits in the Southern, Southeastern, Central-west-
ern, and Northeastern regions did not differ signifi-
cantly. The estimated representative temperatures in
the N, NE, CW, SE, and S deposits, based on B.
bassiana germination, were 35.4 [30.8–40.0], 29.7
[21.6–37.8], 24.4 [22.0–26.9], 25.0 [23.0–27.1], and
23.8 [22.7–24.9] C, respectively.
Germination readings for T. harzianum conidia
kept in incubators and deposits for 19 months are in
Fig. 4b, and significant differences were detected
among treatments (v2 = 626.60; df = 10;
p \ 0.0001). At 26 and 29 C, germination levels
between 24.9 and 26.8% were recorded, respectively,
which were statistically higher than those of conidia
kept at higher temperatures. The germination levels of
conidia kept at 32–35 C (between 16.6 and 17.5%)
were higher than for those kept at 38 C (only 9.6%).
The estimated representative temperatures in the N,
NE, CW, SE, and S deposits, based on T. harzianum
germination, were 26.4 [25.8–27.0], 27.1 [26.4–27.7],
24.0 [23.4–24.5], 25.1 [24.0–26.2], and 27.5
[26.9–28.2] C, respectively. Statistical parameters
for all models used in our study are shown in the
Supplementary Table S3.
Calculation of mean kinetic temperatures (MKTs)
in rural deposits
For all rural deposits, differences of 5 C were
recorded. Therefore, the mean kinetic temperature
was calculated applying the equation derived by

123
What would be representative temperatures for shelf-life studies with biopesticides in tropical… 221

a 100
0.061 constant temperatures (o C) deposits agricultural settings, in which biopesticides and other
90
< 0.010 < 0.010
products are stored before field applications. Signif-
Conidial germination (%)

80 icant differences among MKT were seen for rural

a
70 b b b deposits in different geographic regions of Brazil,
60 bc bc bcd
50
cde
de de
ranging from 21.7 (Southern region) to 28.0 C
e
40 (Northern region). Therefore, the adoption of
30 30 ± 2 C for stability tests with biopesticides should
20 be applied in Brazil. This must be done to guarantee
10
the effectiveness of biopesticides following field
0
Pre- 26°C
storage
29°C 32°C 35°C 38°C N NE SE S CW applications throughout the entire Brazilian territory.
b 50 Exceptions would be commercial products for which
0.029 constant temperatures (o C) deposits
storage is recommended in refrigerated conditions or
Conidial germination (%)

40 < 0.010 < 0.010

a products sold only in particular regions of the country,
30
20 bc bc bc
c
d d
10
e
0
Pre- 26°C
storage
29°C 32°C 35°C 38°C N NE
Treatments
Fig. 4 Germination (means and 95% confidence intervals) of
unformulated Beauveria bassiana (a) and Trichoderma
harzianum (b) stored in airtight pouches with O2/H2O-absorbing
sachets in incubators set to constant temperatures (26, 29, 32, 35,
or 38 C) or at fluctuating temperature regimes in rural deposits
located in the five Brazilian geographic regions [Central-West
(CW), North (N), Northeast (NE), Southeast (SE), and South
(S)] for 19 months. Numbers on the top of bars refer to the
average water activity of samples after the equilibrium period
(except for the treatments ‘‘Pre-storage’’, the water activity (aw)
values were always B 0.010). Bars with same letters are not
significantly different by pairwise odds ratio comparisons
(P \ 0.05). For each fungus, the horizontal stippled line
corresponds to 50% of the initial germination
Haynes (1971) based on the Arrhenius equation.
Calculated MKTs for each rural deposit in two
different periods are provided in Table 2. In certain
periods (not shown in Table 2), such as from the
beginning of the experiment (December 2012) until
the end of summer 2013, MKTs in the South and
Southeast deposits were 24.4 and 24.8 C, respec-
tively, whereas variations in other regions were very
small (up to 0.6 C).
Discussion
Most studies with MKT estimates refer to populated
sites, and there has been a lack of studies in
in which the MKT is considerably lower than 30 C.
Temperature excursions may occur when biopesti-
b
bc bc
cides are exposed to temperatures outside the
range(s) prescribed for transport and/or storage, thus
the safety margin of 2.0 C calculated in our study is
quite reasonable. This margin is close to the safety
SE S CW margin of 2.6 C calculated by Grimm (1998) for
tropical countries. Additionally, some municipalities
in the N/NE regions, for example, have average air
temperatures of approximately 2 C higher than the
municipalities considered in our study (Alvares et al.
2013). This is important since temperatures [ 30 C
were recorded in all rural deposits, except the one
located in the Southeastern region, in which the
maximum recorded temperature was 29.7 C. The
average number of days with temperatures over 30 C,
determined for Brazilian capital cities by Bott and
Oliveira (2007), ranged from 3.6 (Brasilia, Federal
District) to 94.3 days (Boa Vista, Roraima state), but
even for the latter city the calculated MKT was
28.9 C. The MKT values calculated for these capi-
tals, varying from 21.7 to 30.1 C, were in accordance
with values calculated in our work. Special attention
must be paid to very stressful temperatures (C 30 C)
during shipment of biopesticides, since additional
stability testing would have to be considered for such
conditions. For example, temperatures during the non-
refrigerated transport can exceed 50 C (International
Safe Transit Association 2002). Therefore, the impact
of this thermal stress must be considered in the
determination of shelf-life, unless the product manu-
facturer clearly states on the package inserts or labels
the need to transport biopesticides under less severe
conditions, preferably at temperatures no higher than
those adopted in thermal stability tests. The Haynes
version of the Arrhenius equation, adopted in our

123
222
study, is mainly concerned with the chemical stability
for which the laws of reaction kinetics are applicable
(Grimm 1998), but sometimes the shelf-life of a
product can be limited by its physical stability
(Waterman 2009). For example, biopesticides with
hygroscopic components in their formulation and sold
in packages that are permeable to water vapor may
result in loss of physical consistency and biological
activity.
Following a 19 month storage period in rural
pesticide deposits, germination of actively packaged
B. bassiana conidia was not severely affected, and
estimated RTs ranged from 35.4 C in the Northern
down to 23.8 C in the Southern region. For T.
harzianum, the RTs estimated for the rural deposits
ranged from 27.5 C in the Southern down to 24.0 C
in the Central-western region. For a given region, the
difference between estimates was usually small
(B 3.7 C). However, this difference was 9.0 C for
the rural deposit located in the Northern region. Such
differences are expected for biological systems. Nev-
ertheless, these values were far from the more reliable
MKTs values (21.7–28.0 C) calculated for rural
deposits in the five geographic regions.
Under laboratory conditions, B. bassiana conidia
with initial germination of approx. 82% achieved
germination in 67–71% range after 30 months at fixed
temperatures ranging from 26 to 35 C. The actual
half-life for the B. bassiana batch with satisfactory
quality could not be calculated, but it is expected to be
longer than 900 days at 35 C. These high values for
B. bassiana were only possible because the small
volume of fungal material (1.6 g) were sealed in small
airtight packages with the inclusion of a sachet that
absorbed oxygen and water vapor from the headspace.
Since the adoption of active packaging (O2/H2O-
absorbing sachets ? barrier pouches) with small
amounts of conidia can considerably extend their
longevity as previously shown by Faria et al. (2012),
for T. harzianum a deliberate choice was made to use
an inferior quality batch (initial germination of
approx. 44%). The half-lives estimated for stored T.
harzianum conidia were 428 days at 26 C,
227–270 days at temperatures between 29 and
35 C, and only 94 days at 38 C.
In the pharmaceutical and food industries, the
temperature of 30 ± 2 C is usually adopted in shelf-
life tests in tropical countries, and the adoption of 75%
RH is also required when solid formulations are kept
123
M. Faria et al.
in packages permeable to water vapor (Zahn 2009;
ANVISA 2018, 2019). Additional harmonization
refers to the concept of shelf life itself and what
would be the most suitable parameter to assess the
concentration of active ingredients in mycopesticides.
Different manufacturers and even scientists have
adopted definitions that conflict with the usual concept
of shelf life. For example, some commercial mycopes-
ticides have been registered after stability tests carried
out at \ 30 C and until the initial number of colony
forming units drops by 50%. Close to the end of their
expiration date these products are unlikely to have
retained their efficacy, and this exemplifies how
mistaken methodologies for determination of shelf
life could cause inestimable financial losses to end
users. We expect the shelf lives for most mycopesti-
cides currently registered in Brazil to be \ one–
two months for shelf-life determinations conducted
at 30 C when parameters such as Colony Forming
Units or germination of fungal propagules are used to
estimate the time for initial concentration of active
ingredients to drop by 20%. Even shorter shelf lives
would be expected if parameters more closely related
to fungal virulence were considered, such as conidial
vigor for conidia-based mycoinsecticides (Faria et al.
2015). The production of high-quality batches and
development of advanced packaging systems has been
previously shown as ways to substantially improve the
shelf life of mycopesticides (Faria et al. 2015, 2017).
Our study has demonstrated the need to harmonize
the stability testing of biopesticides in Brazil, which is
also likely true in other countries located in hot
climatic zones. Given the current lack of specific
guidelines applicable to biopesticides, finding ways of
harmonizing regulatory requirements is a key step to
make effective products available to farmers. Our
results indicate that the storage temperature of 30 C,
which is already adopted by the food and pharmaceu-
tical industries and regulatory bodies, is highly
recommended for stability studies with biopesticides.
This information may guide companies in the devel-
opment of formulations and packaging systems that
guarantee acceptable shelf lives under realistic non-
refrigerated storage conditions.
Acknowledgements Sincere thanks to the owners of farms
and to the partners that collaborated in the delivery/collection of
mycopesticide packages in pesticide deposits in different
regions of Brazil, including Eduardo José da Silva (EMATER/
RO), Marco Tamai (Universidade do Estado da Bahia); Todi
What would be representative temperatures for shelf-life studies with biopesticides in tropical…
Sujuki (Fazenda Nossa Senhora Aparecida); Antônio Celso
Pinheiro, Renato Ledesma Aleixo, and Ariclenes Anibal
Ballarotti (Koppert Biological Systems; formerly Itaforte
Bioprodutos); Gilmar Robert de Jesus (Fundação ABC); Pedro
Luiz Aiçar de Suss (Fazenda Caracol); and Roberto Teixeira
Alves and Anselmo de Castro (Embrapa Cerrados).
Author contributions MF: Planning; collected the data, wrote
the paper. LAMP: Conceived and designed the analysis;
performed the analysis. DAS: Collected the data. RBL:
Planning, wrote the paper.
Funding Not applicable.
Data availability Data available within the article or its
supplementary materials.
Code availability Not applicable.
Declarations
Conflict of interest The authors declare that they have no
conflict of interest.
References
AGROFIT (2021) Sistema de agrotóxicos fitossanitários, do
Ministério da Agricultura, Pecuária e Abastecimento.
http://agrofit.agricultura.gov.br/agrofit_cons/principal_
agrofit_cons. Accessed 01 July 2021
Alvares CA, Stape JL, Sentelhas PC, Gonçalves JLM, Sparovek
G (2013) Köppen’s climate classification map for Brazil.
Meteorol Z 22:711–728
ANVISA (2005) Resolução—RE n8 1 de 29 de julho de 2005.
Guia para a realização de estudos de estabilidade. https://
bvsms.saude.gov.br/bvs/saudelegis/anvisa/2005/res0001_
29_07_2005.html. Accessed 12 July 2021
ANVISA (2018) Guia para determinação de prazos de validade
de alimentos. Guia n8 16/2018. https://docero.com.br/doc/
8vxcc0. Accessed 12 July 2021.
ANVISA (2019) Resolução—RDC n8 318. https://www.in.gov.
br/web/dou/-/resolucao-rdc-n-318-de-6-de-novembro-de-
2019-226513805. Accessed 12 July 2021
Bott RF, Oliveira WP (2007) Storage conditions for stability
testing of pharmaceuticals in hot and humid regions. Drug
Dev Ind Pharm 33:393–401
Chandler D, Davidson G, Pell JK, Ball BV, Shaw K, Sunderland
KD (2000) Fungal biocontrol of Acari. Biocontrol Sci
Technol 10:357–384
Faria M, Hotchkiss JH, Wraight SP (2012) Application of
modified atmosphere packaging (gas flushing and active
packaging) for extending the shelf life of Beauveria
bassiana conidia at high temperatures. Biol Control
61:78–88
Faria M, Lopes RB, Souza DA, Wraight SP (2015) Conidial
vigor vs. viability as predictors of virulence of ento-
mopathogenic fungi. J Invertebr Pathol 125:68–72
223
Faria M, Martins I, Souza DA, Mascarin GM, Lopes RB (2017)
Susceptibility of the biocontrol fungi Metarhizium aniso-
pliae and Trichoderma asperellum (Ascomycota:
Hypocreales) to imbibitional damage is driven by conidial
vigor. Biol Control 107:87–94
Glare TR, Caradus J, Gelernter W, Jackson T, Keyhani N, Kohl
J, Marrone P, Morin L, Stewart A (2012) Have biopesti-
cides come of age? Trends Biotechnol 30:250–258
Glare TR, Jurat-Fuentes JL, O’Callaghan MO (2017) Basic and
applied research: entomopathogenic bacteria. In: Lacey LA
(ed) Microbial control of insect and mite pests. Elsevier,
Amsterdam, pp 47–67
Grimm W (1993) Storage conditions for stability testing in the
EC, Japan and USA; the most important market for drug
products. Ind Pharm 19:2795–2830
Grimm W (1998) Extension of the international conference on
harmonisation tripartite guidelines for stability testing of
new drug substances and products to countries of climatic
zones III and IV. Drug Dev Ind Pharm 24:313–325
Grzywacz D (2017) Basic and applied research: Baculovirus. In:
Lacey LA (ed) Microbial control of insect and mite pests.
Elsevier, Amsterdam, pp 27–46
Harman GE (2011) Trichoderma—not just for biocontrol any-
more. Phytoparasitica 39:103–108
Haynes JD (1971) Worldwide virtual temperature for product
stability testing. J Pharm Sci 60:927–929
Huynh-Ba K, Zahn M (2009) Understanding ICH guidelines
applicable to stability testing. In: Huynh-Ba K (ed)
Handbook of stability testing in pharmaceutical develop-
ment: regulations, methodologies, and best practices.
Springer, pp 9–41
Hilbe JM (2015) Practical guide for logistic regression. CRC,
New York
International Safe Transit Association (2002) ISTA temperature
project—data summary. Report. East Lansing, MI, USA,
p. 11. https://docplayer.net/5515399-Ista-temperature-
project-data-summary.html. Accessed 12 July 2021
Jones KA, Burges HD (1998) Technology of formulation and
application. In: Burges HD (ed) Formulation of microbial
biopesticides. Beneficial microorganisms, nematodes and
seed treatments. Kluwer Academic Publishers, Dordrecht,
pp 2–30
Kassa A (2003) Development and testing of mycoinsecticides
based on submerged spores and aerial conidia of the
entomopathogenic fungi Beauveria bassiana and Me-
tarhizium anisopliae (Deuteromycotina: Hyphomycetes)
for control of locusts, grasshoppers and storage pests. PhD,
Georg-August-Universität Göttingen (Germany)
Lacey LA, Grzywacz D, Shapiro-Ilan DI, Frutos R, Brown-
bridge M, Goettel MS (2015) Insect pathogens as biolog-
ical control agents: back to the future. J Invertebr Pathol
132:1–41
Lopes RB, Martin I, Souza DA, Faria M (2013) Influence of
some parameters on the germination assessment of
mycopesticides. J Invertebr Pathol 112:236–242
Marques EJ, Alves SB (1996) Otimização de formulações na
preservação de conı́dios de Beauveria bassiana (Bals.)
Vuill. e Metarhizium anisopliae (Metschn.) Sorok. em
diferentes condições de armazenamento. Arq Biol Tecnol
39:861–877
123
224
Menaria BL (2007) Bioherbicides: an eco-friendly approach to
weed management. Curr Sci India 92:10–11
R Development Core Team (2012) R: a language and environ-
ment for statistical computing. R Foundation for Statistical
Computing. http://www.r-project.org.
Sandhu SS, Rajak RC, Agarwal GP (1993) Studies on prolonged
storage of Beauveria bassiana conidia: effects of temper-
ature and relative humidity on conidial viability and viru-
lence against chikpea borer, Helicoverpa armigera.
Biocontrol Sci Technol 3:47–53
Shoham J (2020) The rise of biological products in the crop
protection and plant nutrition markets. Outlooks Pest
Manage 31:129–131
Silva RZ da (2006) Formulação e armazenamento de conı́dios
de Beauveria bassiana (Bals.) Vuill. PhD, Universidade
Estadual de Londrina (Brazil)
Waterman KC (2009) Understanding and predicting pharma-
ceutical product shelf-life. In: Huynh-Ba K (ed) Handbook
of stability testing in pharmaceutical development: regu-
lations, methodologies, and best practices. Springer, New
York, pp 115–135
WHO (2004) Stability studies in a global environment. Geneva
meeting. Working document QAS/05.146 with comments,
10 p. https://www.who.int/medicines/services/expertcom
123
M. Faria et al.
mittees/pharmprep/QAS05_146Stabilitywithcomments.pdf.
Accessed 12 July 2021
Zahn M (2009) Global stability practices. In: Huynh-Ba K (ed)
Handbook of stability testing in pharmaceutical develop-
ment: regulations, methodologies, and best practices.
Springer, New York, pp 43–91
Marcos Faria is an insect pathologist based at EMBRAPA,
Brazil. His main research focuses on microbial control of pests
with entomopathogenic fungi.
Luı́s Alberto Martins Palhares works as a system analyst at
EMBRAPA, developing softwares with R, PHP, Java and/or
Javascript. He also performs statistical analysis for various
kinds of experimental designs.
Daniela Aguiar Souza is a laboratory technician and works on
molecular biology of microorganisms and biological control of
agricultural pests using fungi.
Rogerio Biaggioni Lopes is a scientist based at EMBRAPA,
and his research focuses on invertebrate pathology, develop-
ment of biopesticides and microbial control of arthropods.