Ethyl Formate as a Methyl Bromide Alternative for Fumigation of Citrus: Efficacy, Fruit Quality, and Workplace Safety
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Journal of Economic Entomology, XX(XX), 2021, 1–7
https://doi.org/10.1093/jee/toab175
Research
Commodity Treatment and Quarantine Entomology
Ethyl Formate as a Methyl Bromide Alternative for
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Fumigation of Citrus: Efficacy, Fruit Quality, and
Workplace Safety
Min-Goo Park,1,2 Byung-Ho Lee,2, Jeong-Oh Yang,1 Bong-Soo Kim,1 Gwang Hyun Roh,3,4
Paul E. Kendra,5 and Dong H. Cha3,6,
1
Animal and Plant Quarantine Agency (APQA), Gimcheon, Republic of Korea, 2Institute of Life Science, Gyeongsang National
University, Jinju, Republic of Korea, 3USDA-ARS, Pacific Basin Agricultural Research Center, Hilo, HI, USA, 4Oak Ridge Institute
of Science and Education, Oak Ridge, TN, USA, 5USDA-ARS, Subtropical Horticulture Research Station, Miami, FL, USA, and
6
Corresponding author, e-mail: dong.cha@usda.gov
Subject Editor: Lisa Gail Neven
Received 15 May 2021; Editorial decision 24 August 2021
Abstract
Ethyl formate (EF) was evaluated as a potential alternative to methyl bromide (MB) for phytosanitary treat-
ment of imported citrus fruit in the Republic of Korea. Planococcus citri (Risso) (Hemiptera: Pseudococcidae),
a mealybug with known tolerance against EF and MB, was used as a representative pest to test efficacy of the
two fumigants against eggs. In nine commercial-scale refrigerated container (67.5 m3) trials using imported
orange, lemon and grapefruit, EF applied at the currently approved dose for citrus (70 g·m-3 at 5°C for 4 h, de-
veloped for Aspidiotus excisus Green (Hemiptera: Diaspididae), a species less EF tolerant than P. citri) resulted
in 76.9–98.3% mortality of P. citri eggs. The EF treatment did not affect the sugar content or the color of peel and
pulp of the treated fruit. When oranges were treated according to the current MB (64 g·m-3 at >5°C for 2 h) or
EF treatment guidelines, the concentration of fumigant around the fruit fluctuated between 9.4 and 185.1 ppm
for EF and 9.5–203.0 ppm for MB during the 72-h post-fumigation processes (venting [0–2 h], transportation
to storage [2–24 h], and storage periods [24–72 h]) with both EF and MB maintained between 10 and 100 ppm
during the storage period. Considering the efficacy of EF, its apparent lack of phytotoxicity, and its more man-
ageable threshold limit value for humans (100 ppm EF compared to 1 ppm MB for an 8-h time weighted
average exposure), our results suggest that EF may be a promising alternative to MB for the phytosanitary
treatment of imported citrus in Korea.
Key words: Fumigant, citrus, Planococcus citri, mortality, permissible exposure limit, threshold limit value
Citrus is one of the world’s major tropical fruit crops with global effective MB alternative treatments to address concerns regarding
availability and the distinction of being the most traded horticul- ozone depletion (Yang et al. 2016) and human health risks associ-
tural commodity in the world (Liu et al. 2012, Matheyambath et al. ated with MB. In particular, there have been frequent reports of MB
2016). For example, the Republic of Korea (Korea) annually im- related acute chronic inhalation toxicity in humans (Jo et al. 2003)
ports about $250M worth of citrus fruits including orange (Citrus × and incidents of worker toxication or functional degradation of ner-
sinensis (L.) Osbeck), lemon (C. × limon (L.) Brum.), and grapefruit vous systems due to workplace MB exposure exceeding the 1 ppm
(C. × paradisi Macfad.), (Sapindales: Rutaceae), from different coun- acceptable limit (CDC 2011, Baur et al. 2015, Shin et al. 2016, Choi
tries including Australia, Chile, Spain, South Africa, and the United et al. 2021, Park et al. 2020a, 2021).
States of America (USDA 2018). Upon arrival at Korean ports, these Ethyl formate (EF) is one of the potential MB alternatives for
fruits are normally treated with methyl bromide (MB) under the cur- fumigation of fruits and vegetables (Simpson et al. 2007, Yang et al.
rent phytosanitary disinfestation guidelines available for imported 2016). The U.S. Food and Drug Administration (FDA 1984) has
citrus (APQA 2018). However, there is an urgent need to develop reviewed the use of EF as a flavoring agent and has characterized
Published by Oxford University Press on behalf of Entomological Society of America 2021. This work is written by (a) US Government employee(s) and is in the 1
public domain in the US.2 Journal of Economic Entomology, 2021, Vol. XX, No. XX
this compound as generally regarded as safe (GRAS). Other advan- Insects
tages of EF include efficacy comparable to MB, short fumigation Stock colonies of P. citri were reared on potato sprouts following
period, low toxicity to mammals and the environment, and rapid methods described by Yang et al. (2016) at 25 ± 1°C with 60%
breakdown into formic acid and ethanol with no deleterious residues RH and 12:12 h (L:D) and maintained at the Plant Quarantine
(Haritos et al. 2003, Lee et al. 2007, Lee and Kim 2017, Park et al. Technology Center of Animal and Plant Quarantine Agency
2020b, Walse et al. 2021). Moreover, in terms of workplace safety, (Gimcheon, Korea).
the exposure limit (e.g., threshold limit value for 8-h time-weighted
average, TLV-TWA) of EF in the workplace is 100 ppm, which is Measurement of EF Concentration and
much lower than the 1 ppm TLV-TWA of MB (American Conference
Determination of Ct (concentration × time) Products
of Government Industrial Hygienists 2019), suggesting that EF is
For laboratory trials, the concentration of EF was measured timely
safer and more manageable to maintain below the limit value than
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using GC-FID after separation on a HP-5 column (J&W Sci. 19091J-
MB (Moon et al. 2015, Yang et al. 2017).
413). The oven temperature was maintained at 150°C and injector
Results from inspections during 2007–2011 revealed that the pri-
and detector temperatures at 240°C. The concentration of EF was
mary quarantine pests on citrus imported into Korea are external
calculated on the basis of peak area against external EF gas standard.
fruit feeders such as mealybugs and scale insects, 73% of which were
The peak areas were calibrated periodically using four different
invasive species (Suh et al. 2013). EF has been shown to be effective
levels of EF standards (a known volume of liquid EF injected into a
in disinfestation of mealybugs and scale insects (Simpson et al. 2007,
1L Tedlar gas sampling bag). The concentrations of fumigant in fu-
Misumi et al. 2013, Pupin et al. 2013, Jamieson et al. 2014, Yang
migation chambers and refrigerated shipping containers were moni-
et al. 2016) and has similar efficacy as MB. For example, Park et al.
tored at time intervals of 0.5, 1.0, 2.0, and 4.0 h exposure periods.
(2020b) recently reported similar efficacies of EF and MB (treated
The concentration × time (Ct) products were calculated as described
at approved doses of 35.0 g m−3 for 4 h for EF and 48.0 g m−3 for
by Ren et al. (2011).
2 h for MB both at 13°C; APQA 2018) for the disinfestation of
imported banana. In that study, the egg stage of citrus mealybug,
Planococcus citri (Risso) (Hemiptera: Pseudococcidae), was used as Small Scale Trials of Liquid EF on P. citri Eggs
a fumigant tolerant surrogate, as it was the most EF tolerant species/ at Current Recommended Exposure of EF for
life stage identified among several pest species considered, including Imported Citrus
Cyanotis scale, Aspidiotus excisus Green (Hemiptera: Diaspididae). The fumigation with EF on P. citri eggs was performed in glass des-
Aspidiotus excisus was the target species used to generate the cur- iccators (Duran, 6.9 L) with 30 eggs transferred to newly sprouted
rent recommended EF fumigation guidelines for banana and citrus potatoes placed in an insect breeding dish (10 × 4 cm, 0.053 µm
(APQA 2018). screen on top; n = 10). A small battery-operated fan was placed at
In this study, to evaluate the feasibility of using EF fumiga- the bottom of desiccators to improve uniform mixing of fumigants.
tion as a phytosanitary treatment for imported citrus, commercial After tightly sealing the desiccator using vacuum seal (Dow Corning,
scale trials using nine 12 m refrigerated shipping containers were Midland, MI), liquid EF was injected into the fumigation chamber
conducted with the egg stage of P. citri as a tolerant surrogate for (after removing the volume of air equivalent to the volume of fu-
EF treatment. Specifically, using three species of imported citrus migant injected) using a gas-tight syringe (SGE Analytical Science,
(orange, grapefruit, and lemon), we 1) evaluated the efficacy of www.sge.com, Australia) to achieve EF Ct product around 110–
EF on P. citri eggs at the current recommended exposure for im- 120 g h m-3 at 5°C with 4-h exposure, similar to the current recom-
ported citrus (70 g m-3 for 4 h at 5°C; (APQA 2018) in small mended level of EF exposure guidelines available for phytosanitary
scale laboratory and larger commercial scale trials, 2) assessed disinfestation of imported oranges in Korea (APQA 2018). At the
the effect of EF fumigation on sugar content and color of citrus completion of fumigation, the desiccators were opened and aerated
fruit, and 3) compared the concentrations of MB and EF desorbed for 1-h in a fume hood. Then, the treated mealybug samples were
from treated orange fruit into refrigerated shipping containers removed from desiccators and moved to a growth chamber (25 ±
and cold storage units during the simulated 72-h post-fumigation 2°C, 75 ± 5% RH, 16:8 L:D). The assessment of treatment efficacy
processes, which generally take place before citrus delivery to on P. citri eggs was based on nymphal emergence from eggs over the
consumers. For EF fumigation treatments in this study, liquid EF 14 d post-fumigation period and reported as corrected % egg hatch
was vaporized and delivered by nitrogen gas into fumigation con- (see below) calculated with egg hatch rate of control eggs at 93.4%.
tainers as a nonflammable EF + nitrogen formulation (Park et al.
2020b).
Commercial Scale Trials of Liquid EF Plus Nitrogen
on P. citri Eggs and Citrus at Current Recommended
Exposure of EF for Imported Citrus
Materials and Methods
Commercial scale trials using nine 12 m refrigerated shipping con-
Fumigants tainers (67.3–67.9 m3, average size 67.5 m3) were performed at the
Methyl bromide (MB) was supplied by registered fumigators in port of Pusan, South Korea. Detailed information of the trials con-
Korea (Dong-A Limited, Busan, Korea). Liquid ethyl formate ducted is summarized in Table 1. Each container was fitted with a
(Fumate, 99%) was supplied from Safefume Inc. (Hoengseong, circulation fan inside the top of the container to promote circula-
Korea). For both scale-up experiments conducted using 12 m re- tion of fumigant during the trial. Planococcus citri eggs were col-
frigerated shipping containers (average 67.5 m3) and phytotoxicity lected from the colony and placed into breeding cages (400–500
assessment conducted using 10 m3 refrigerated shipping containers, eggs/breeding cage) containing newly sprouted potatoes. Generally,
liquid EF was vaporized using a commercial EF vaporizer (SFM- for each trial, three breeding cages with eggs were placed inside
1, Safefume Inc., Hoengseong, Korea) and discharged into the con- three different citrus boxes each located at front top, middle center,
tainers with nitrogen gas as a propellent. and rear bottom parts of the container (one egg cage/citrus box),Journal of Economic Entomology, 2021, Vol. XX, No. XX 3
respectively, with the front section close to the door. For some of
CTP indicates concentration × time products. Corrected % egg hatch was calculated based on % egg hatch of untreated control eggs at: No 1 (88%), No 2, 3 (64.4%), No 4 (93.1%), No 5 (82%), No 6 (84.6%), and No 7,
Table 1. Summary of conditions used during nine commercial scale fumigation trials conducted in nine 12 m refrigerated shipping containers (‘Reefer’) using liquid ethyl formate with N2 at 70 g
2.7 ± 2.7
3.3 ± 4.5
2.4 ± 3.1
2.4 ± 2.1
3.9 ± 5.5
4.2 ± 3.3
9.2 ± 9.9
23.8 ± 1.3
Corrected
egg hatch
the lemon trials, it was difficult to access fruit boxes in the rear and/
(%)
14.8
or center parts of the container, so only 2 (one each in the front
and center parts of the container), 1 (front part), and 2 (one each in
the front and center parts) egg breeding cage(s) were treated in #5,
7, and 9 trials, respectively (Table 1). Since these trials were con-
# nymph emerged
28.0 ± 10.0 ducted using imported commercial citrus fruit, the loading ratio (%,
32.0 ± 14.4
23.0 ± 10.0
32.0 ± 22.6
53.0 ± 13.9
106.0 ± 38.2
30.0 ± 8.7
183.0 ± 4.9
w/v) of citrus fruits varied in each trial between 27.4 and 37.0%
(Table 1), which was calculated by metric tons of citrus × 100/cubic
57
meters of fumigation chamber. After sealing the containers, EF was
injected through an injection port located above the container door
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(front part). A dose of 70.0 g m-3 EF was applied at 5–8°C for 4 h
using liquid EF vaporized by the SFM-1 EF vaporizer (SafeFume,
Hoengseong, Korea) and delivered by nitrogen gas from cylinders.
#egg treated
EF levels inside the citrus boxes with P. citri eggs were determined at
1,000b
a
1,500a
1,500a
1,350a
1,500a
1,350a
1,200
900b
450c
timed interval of 0.1, 2.0, and 4.0 h. The gas was withdrawn into 1
liter gas bags (SKC Tedlar bag, Eighty Four, PA) using an air pump
through gas sampling hoses connecting the fruit boxes and gas sam-
pling ports located at the front, center, and rear parts of the con-
tainer. The concentration of EF was measured one time from each
(g h m-3)
122.4
133.6
125.4
111.5
112.8
90.4
112.2
103.2
98.0
CTP
gas sampling bag using a portable EF analyzer (iBRD MX, Industrial
Scientific Corp., Pittsburgh, PA). The total number of P. citri eggs
tested was 14,950 (10,750 eggs treated in EF trials and 4,200 eggs
in untreated control trials). Untreated control trials were conducted
without EF injection but otherwise using exactly same methods as
Temp.(°C)
the EF trials to determine the baseline % hatch rate of P. citri eggs.
5±1
6±1
6±1
6±1
6±1
6±1
7±1
8±2
8±2
The % egg hatch was determined by evaluating the nymphal emer-
gence over the 14 d post-fumigation. Corrected % egg hatch was cal-
culated as described below with the control egg hatch rate of 81.5%
determined from six scale-up untreated control trials.
Fruit loading ratio (w/v)
Effect of EF Fumigation on Citrus Fruit Quality
27.5
27.4
27.4
31.9
34.6
36.7
34.9
37.0
34.6
The effects of EF treatment on soluble sugar content and color of
orange, grapefruit, and lemon were evaluated. Imported citrus fruits
from a local retailer were treated with the scheduled dose of 70.0 g
m-3 of EF for 4 h at 5 ± 1°C in the 10 m3 refrigerated fumigation con-
tainer at the Animal and Plant Quarantine Agency (Gimchon, Korea)
with 25 % loading ratio (w/v) of orange (3 trials, 18.5 ton/trial),
grapefruit (1 trial, 21.6 ton/trial), and lemon (5 trials, 24.2 ton/trial).
Fruit weight(Ton)
After completion of the 4-h fumigation treatment, the fumigation
container was ventilated for 2 h at 5°C with the door opened (0–2 h
18.5
18.5
18.5
21.6
23.5
24.8
23.5
24.9
24.1
post-fumigation) and transported under 5°C to a storage facility
with the door closed (2–24 h post-fumigation). Once in storage,
grapefruit and lemon are generally released to the retail chain after
a minimal storage period. Thus, we evaluated quality of fumigated
grapefruit and lemon fruits at 15 ± 1°C storage condition over 7 d
Citrus × paradisi
to simulate conditions at retail chains. For orange, due to a greater
Citrus × sinensis
Citrus × sinensis
Citrus × sinensis
m-3 for 4 h at 5–8°C on imported citrus fruits
Citrus variety
market demand and greater amount of fruit imported, fumigated
Citrus limon
Citrus limon
Citrus limon
Citrus limon
Citrus limon
oranges delivered to storage are distributed to consumers either
within 1–2 d of the delivery without a cold storage or after a 3–14
d in cold storage. To simulate these conditions, EF fumigated orange
fruits were divided into three groups of post-fumigation processes:
1) stored at 15 ± 1°C over 14 d simulating the condition of direct dis-
tribution to retail, 2) stored at 5 ± 1°C over 14 d simulating the con-
Three eggs cages treated;
Actual size
two egg cages treated;
dition of 2-wk cold storage before distributed to retail, and 3) stored
one egg cage treated.
67.3
67.5
67.3
67.6
67.9
67.5
67.4
67.5
67.9
(m3)
initially at 5 ± 1°C over 3 d then stored at 15 ± 1°C over 11 additional
days. After the 7- or 14-d storage periods, sugar content and color
of fruit were compared between EF treated and untreated citrus. The
8, 9 (85.6%).
soluble sugar content (% Bx) was determined using a portable re-
Reefer
fractometer (Hand refractometer ATC-1E, Atago Co., Kyoto, Japan)
b
a
c
(Tolman and Smith 1906). Pulp from whole fruit was ground with
ID
1
2
3
4
5
6
7
8
94 Journal of Economic Entomology, 2021, Vol. XX, No. XX
a tissue grinder (Kontes, Vineland, NJ) and filtered through a funnel ranging from 6.7 to 25.0%, was achieved with a mean Ct product of
covered with filter paper (No. 1, model # N0111.0CM, Advantech, 114. 3 ± 5.0 g h m-3 (Table 2).
Taipei, Taiwan). The color of citrus peel and pulp was measured as
Hunter L, a, b values using colorimeter and expressed as hue values Commercial Scale Trials of Liquid EF Plus Nitrogen
(Techkon GmbH, Wiesbaden, Germany). Peel color was measured on P. citri Eggs and Citrus at Current Recommended
inside the three 10 mm circles marked over the equatorial zone of Exposure of EF for Imported Citrus
each fruit using white paper tape with 10 mm circle perforated as the
Cumulative Ct products of EF, P. citri egg mortality, and specific
background. The color of pulp was measured from the cut surface
conditions from the nine scale-up trials are summarized in Table
after cutting the fruit in half horizontally.
1. In three orange trials with 27.4–27.5% loading ratio (w/v), one
grapefruit trial with 31.9% loading ratio, and five lemon trials with
Evaluation of EF and MB Desorption from
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34.6–37.0% loading ratio, Ct products were 122.4–133.6 g h m-3,
Fumigated Orange 111.5 g h m-3, and 90.4–112.8 g h m-3 for orange, grapefruit, and
EF and MB desorbed from treated orange fruits into surrounding lemon trials, respectively. Similar to glass chamber trials, complete
area was evaluated using fruits treated under current MB and EF control of P. citri eggs was not achieved with % egg hatches of 2.4–
fumigation guidelines for imported citrus in Korea (64 g m-3 MB for 3.3, 2.4, and 3.9–23.8% from orange, grapefruit, and lemon trials,
2 h at > 5°C or 70 g m-3 EF for 4 h at >5°C). Fumigation treatment respectively (Table 1). By the end of the fumigation treatment, the
was conducted in a mini-shipping container (0.65 m3) that simulated concentration of EF in grapefruit and lemon trials appeared much
the 12 m refrigerated shipping container with 25% (w/v) of fruit lower than that of orange trials (Fig. 1a and b).
loading ratio. Simulating the commercial venting and delivery condi-
tions used for imported orange, at the end of each fumigation treat- Effect of EF Fumigation on Citrus Fruit Quality
ment, the fumigation container was opened for ventilation for 2 h There were no significant differences in soluble sugar content (% Bx)
using a circulation fan inside the container (ventilation: 0–2 h post- between the untreated and EF treated orange, grapefruit, and lemon
fumigation). After the 2-h ventilation cycle, the container was closed fruits after 7-d (lemon and grapefruit) at 15°C or 14-d (orange)
and transported to storage (transport: 2–24 h post-fumigation) storages at 5, 5–15, and 15°C (Table 3). EF treatment did not induce
with passive ventilation through a ventilation hole (50 mm diam- significant changes in chromatic values of fruit with no differences
eter) located at the rear end of the container (during 18–24 h post- in hue values from peel and pulp of orange, grapefruit, and lemon
fumigation prior to arrival at storage). During the transportation, fruit (Table 3).
the container door remained closed with circulation fans automat-
ically on and off to maintain the target temperature (5°C) inside the
Evaluation of EF and MB Desorption from
container. After arrival at the storage facility, the fumigated fruits
inside the container were transferred to cold storage at 2–5°C (cold
Fumigated Orange
storage: 24–72 h post-fumigation). During the first 2-h ventilation process, the level of fumigants inside
Concentrations (ppm) of MB and EF released from MB or the 0.65 m3 mini-shipping container decreased rapidly from 518 to
EF treated fruits were determined during 0–2 and 2–24 h post- 18 ppm for EF and from 236 to 51 ppm for MB (Fig. 2). After the 2-h
fumigation inside the mini-shipping container and during 24–72 h ventilation, the door of the container was closed for transportation
inside the cold storage unit. The concentrations of EF were measured to storage. During the transportation period, the levels of EF and
using GC-FID as described above. The concentrations of MB were MB inside the mini shipping container increased due to the EF and
determined using a MB detector (MiniRAE 3000, RAE systems, San MB desorbed from treated orange. During the 2–18 h period, the
Jose, CA) calibrated to read MB using 10.6 eV lamp with a correc- concentrations of EF and MB inside the container were maintained
tion factor of 1.7–1.8. Headspace samples (~500 ml) of EF and MB between 94 and 203 ppm for EF and between 145 and 185 ppm for
were taken from 3 gas sample ports installed in the mini-shipping MB. During the 18–24 h period with passive ventilation, levels of
container and in cold storage.
Table 2. Efficacy of ethyl formate fumigation on eggs of
Planococcus citri with concentration × time products (CTP) of ethyl
Statistical Analysis
formate ranging between 106.5 and 123.3 g h m-3 with an average
Corrected % egg hatch was calculated by 100 – [(% Egg hatch of CTP of 114.3 at 5 ± 1.5°C
control – % Egg hatch of treatment)/Egg hatch of control] × 100,
modified from Abbott’s formula for corrected mortality (Abbott CTP (g # egg # nymph % Egg Corrected
1925). Mean % egg hatch and standard deviation was calculated h m-3) treated emerged hatch % egg hatch
using Proc Univariate in SAS ((SAS Institute 2009). Differences in 0.0 30 28 93.4 -
sugar content and fruit color between EF treated and untreated 123.2 31 3 9.7 10.4
citrus fruits were analyzed with Fisher’s least significant difference 112.1 30 6 20.0 21.4
using SAS (SAS Institute 2009). 117.1 30 4 13.3 14.3
108.6 30 5 16.7 17.9
106.5 32 8 25.0 26.8
Results 113.7 30 3 10.0 10.7
115.2 30 2 6.7 7.1
Small Scale EF Fumigation on P. citri Eggs at
110.9 30 4 13.3 14.3
Recommended EF Exposure for Imported Citrus 118.0 30 3 10.0 10.7
When P. citri eggs, surrogate for EF tolerant citrus pest, were treated 117.6 29 2 6.9 7.4
in 6.8 liter glass chambers with EF at the target Ct products under
the current fumigation guideline for imported citrus (110–120 g h Corrected % egg hatch was calculated based on control egg hatch rate of
m-3 with 4-h exposure), mean % egg hatch rate of 13. 2 ± 5.9%, 93.4%.Journal of Economic Entomology, 2021, Vol. XX, No. XX 5
EF and MB in the shipping container decreased from 112 to 46 ppm
for EF and from 185 to 15 ppm for MB (Fig. 2). After the treated
orange were transferred to cold storage, the levels of EF and MB in-
side the cold storage were maintained between 12.6 and 36.6 ppm
for EF and between 9.4 and 32.5 ppm for MB, which was below the
100 ppm TLV-TWA of EF and greater than the 1 ppm TLV-TWA
of MB.
Discussion
Treated under the currently approved doses of EF and MB, our re-
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sults show no adverse effects of EF fumigation on citrus fruit quality
and a more manageable level of EF than MB in terms of the work-
place exposure limit of EF and MB. In fruit quality evaluation trials,
there were no significant differences in fruit color and sugar content
between EF treated and untreated citrus fruits during the 7–14 d
post-fumigation period. During the 24–72 h post-fumigation period,
when workers need to handle treated fruit (i.e., potentially exposed
to fumigants desorbed from treated fruit) to transfer it to storage (at
24-h post-fumigation) or retrieve it for distribution (any time after
being stored in storage), the levels of EF and MB were 46 ppm for EF
and 15 ppm for MB inside the shipping container at the 24-h mark
and fluctuated between 12.6–36.6 ppm for EF and 9.4–32.5 ppm for
MB inside the storage during the 24–72 h post-fumigation period.
These results indicate that the lowest level of MB during the post-
harvest period with potential worker exposure was 9.4 ppm, which
was > 9 times greater than the exposure limit (1 ppm TLV-TWA
of MB; American Conference of Government Industrial Hygienists
2019). In contrast, during the same post-treatment period, the levels
of EF around treated orange fruit were maintained between 12.6 and
46 ppm, well below the 100 ppm TLV of EF (American Conference
of Government Industrial Hygienists 2019), suggesting that EF is a
safer treatment option than MB.
In terms of EF efficacy, when treated under the current recom-
mended dose of EF for imported citrus in Korea, complete con-
trol of P. citri eggs was not achieved in either the small-scale glass
chamber experiments or the scale-up fumigation trials at a commer-
cial facility. This is somewhat expected and similar to a recent study
evaluating the recommended dose of EF on P. citri for imported ba-
Fig. 1. Concentration of ethyl formate (EF) monitored at 0.5, 2, and 4 h time nana. This is because the current EF treatment guidelines for disin-
intervals during commercial-scale fumigation trials conducted in nine 12 m festation of imported banana (35.0 g m−3 for 4 h at 13°C) and citrus
refrigerated shipping containers with 70 g m-3 for 4 h at 5°C on (a) imported fruit (70 g m-3 for 4 h at >5°C) were developed based on the control
orange (OR) and grapefruit (GF) and (b) lemon (LE) with % fruit loading ratio of Cyanotis scale that is known to be less EF tolerant than P. citri
(LR) for each trial listed next to fruit variety treated in each trial. (Park et al. 2020b). Although P. citri is already established in Korea
Table 3. Effect of ethyl formate (EF) fumigation (70 g m-3 for 4 h at 5 ± 1℃) on sugar contents and inside/surface hue values of fruit after
post-fumigation storage at different temperature conditions.
Hue value
Citrus type EF treatment Storage temperature (°C) Sugar content (% Brix) Pulp Peel
Citrus × sinensis No 5 13.0 ± 0.4 ns* 28.76 ± 2.63 ns 139.16 ± 4.17 ns
Yes 5 11.4 ± 0.3 ns 31.56 ± 1.83 ns 139.37 ± 0.62 ns
No 5→15 13.3 ± 0.6 ns 29.37 ± 2.58 ns 139.30 ± 3.60 ns
Yes 5→15 13.8 ± 0.2 ns 30.71 ± 1.75 ns 137.11 ± 1.01 ns
No 15 12.8 ± 0.2 ns 30.16 ± 2.03 ns 140.22 ± 2.46 ns
Yes 15 14.3 ± 0.6 ns 31.35 ± 1.21 ns 138.87 ± 1.56 ns
Citrus limon No 15 9.7 ± 0.4 ns 40.92 ± 3.61 ns 98.77 ± 2.02 ns
Yes 15 9.9 ± 0.5 ns 36.15 ± 2.08 ns 101.08 ± 1.36 ns
Citrus × paradise No 15 11.3 ± 0.6 ns 11.72 ± 0.94 ns 101.30 ± 3.06 ns
Yes 15 11.7 ± 0.4 ns 10.28 ± 2.51 ns 96.09 ± 1.99 ns
*For each of citrus species and storage temperature combinations, ‘ns’ next to means of sugar contents or hue values indicates no significant differences between
EF treated and untreated control at P < 0.05.6 Journal of Economic Entomology, 2021, Vol. XX, No. XX
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Fig. 2. Concentrations of methyl bromide (MB) and ethyl formate (EF) desorbed from MB and EF treated imported orange into fruit surrounding areas:
concentrations in mini refrigerated shipping container during 0–2 h ventilation cycle (container door opened), during 2–24 h transport period (container door
closed), and in storage during 24–72 h cold storage at 5°C. Threshold limit value for 8-h time weighted average exposure (TLV-TWA) of EF = 100 ppm, MB = 1 ppm.
and not considered a quarantine pest, eggs of P. citri were tested in pitting, firmness, % Brix and fungal decay (Sung et al. 2008). In con-
this study as an EF tolerant surrogate pest and life stage. In light of trast, MB treatment has been shown to be phytotoxic for grapefruit
a recent increase in new species invasion from fruit trades in Korea in terms of peel injuries and mold injuries (Hatton and Cubbedge
(Suh et al. 2013), it is possible that new invasive pest species more 1979) and for lemon in terms of peel injury and browning (B.H.L.,
tolerant than Cyanotis scale will be intercepted in the future. Thus, unpublished data), suggesting EF as a feasible alternative of MB fu-
our results showing the incomplete control of P. citri eggs under the migation with consideration of fruit quality for imported citrus.
current treatment guideline suggests the potential need for the revi- In conclusion, the results from this study suggest that EF is a
sion of current EF guidelines to prepare for the interception of more promising alternative to MB for the phytosanitary disinfestation
tolerant invasive pests. of imported citrus. EF treatment showed expected efficacy on the
Although the same EF dose was applied throughout the nine P. citri eggs (e.g., Park et al. 2020b) and no adverse impact on citrus
commercial scale trials, Ct products of EF varied among different fruit quality. Moreover, the level of EF could be maintained well
trials with the greatest Ct products resulted from orange trials below the 100 ppm TLV limit during the post-fumigation periods
(122.4–133.6 g h m3) followed by grapefruit (111.5 g h m3) and when workers need direct access to treated fruit and are potentially
lemon (90.4–112.8 g h m3) trials. This may be due to differences in exposed to desorbed fumigant. In contrast, the level of MB during
EF sorption by different types of fruit and/or differences in loading the same period was always over the 1 ppm TLV limit. Although
ratio of fruit in each trial with 27.4–27.5% loading ratio (w/v) in promising, additional studies are required, including 1) additional
three orange trials, 31.9% loading ratio in one grapefruit trial, and confirmatory and efficacy trials using grapefruit, 2) evaluation of
34.6–37.0% loading ratio in five lemon trials. Regardless of the other quality parameters such as flavor (e.g., taste panel) and shelf
cause underlying the variation in Ct products, differences in Ct prod- life of treated citrus fruit, 3) estimation of MB and EF residues in
ucts appear to be related to differences in EF efficacy from different fruit over time to determine the acceptable limits in treated citrus,
trials. In particular, an average of 2.4% P. citri egg hatch rate was and 4) better characterization of EF sorption in different species of
observed from orange and grapefruit trials, while a greater egg hatch citrus fruit.
rate (10.9%) was observed from lemon trials. This suggests the im-
portance of achieving target Ct products by optimizing the loading
ratio and better understanding sorption characteristics of each citrus Acknowledgments
fruit species. In commercial applications prone to varying degrees of We thank the Animal and Plant Quarantine Agency for supporting
fruit loading ratio, increasing both the initial dose and duration of this project and assisting with safety assessment of MB fumigation,
fumigation treatment may be beneficial to ensure target Ct products Dong-A Limted in Busan, Korea for providing access to commercial
and effective phytosanitary disinfestation of citrus, especially when fumigation facilities, and SafeFume Inc for providing Fumate and its
loading ratio of citrus becomes > 30%. vaporizer. This research was supported in part by an appointment to
In the current study, there were no apparent negative impacts the ARS Research Participation Program, an agreement between the
of EF fumigation on either the color or the sugar content of citrus U.S. Department of Energy (DOE) and the USDA, managed under
fruit. This result is consistent with a previous report on phytotox- DOE contract number DE-SC0014664. Opinions, findings, conclu-
icity of EF fumigation on navel orange (Sung et al. 2008). When sions, or recommendations expressed in this publication are those
70 g m-3 of EF was applied for 4 h at 5°C, there were no significant of the authors and do not necessarily reflect the views of the USDA.
differences between the EF treated and untreated fruit in terms of USDA is an equal opportunity provider and employer.Journal of Economic Entomology, 2021, Vol. XX, No. XX 7
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