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300
Research Article
Received: 8 April 2008 Revised: 5 September 2008 Accepted: 12 September 2008 Published online in Wiley Interscience: 18 December 2008
(www.interscience.wiley.com) DOI 10.1002/ps.1688
Toxicity of δ-phenothrin and resmethrin to
non-target insects
Frank B Antwi and Robert KD Peterson∗
Abstract
BACKGROUND: The susceptibility of adult house cricket, Acheta domesticus (L.), adult convergent lady beetle, Hippodamia
convergens (Gu´
erin-M´
eneville), and larval fall armyworm, Spodoptera frugiperda (JE Smith), to resmethrin and δ-phenothrin
synergized with piperonyl butoxide (PBO) was evaluated in a laboratory bioassay procedure.
RESULTS: The 1 day LC50 values for resmethrin +PBO were 23.2, 32.08 and 307.18 ng cm−2for A. domesticus, H. convergens and
S. frugiperda respectively. The 1 day LC50 values for δ-phenothrin +PBO were 26.9, 74.91 and 228.57 ng cm−2for A. domesticus,
H. convergens and S. frugiperda respectively. The regression relationship between species mortality and concentration explained
51–81% of the variation for resmethrin +PBO and 72–97% of the variation for δ-phenothrin +PBO. The LC50 values decreased
with time for these insecticides for all surrogate species. In terms of sensitivities among the insects to resmethrin +PBO and
δ-phenothrin +PBO, A. domesticus was most sensitive, followed by H. convergens and then S. frugiperda.
CONCLUSION: The results indicate that resmethrin +PBO was generally more toxic than δ-phenothrin +PBO. Based on the
results, A. domesticus seems to be a good surrogate species for estimating potential non-target terrestrial insect impacts from
exposure to pyrethroids used in public health applications.
c
2008 Society of Chemical Industry
Keywords: risk assessment; ecotoxicology; house cricket; convergent lady beetle; fall armyworm; Acheta domesticus;Hippodamia
convergens;Spodoptera frugiperda; insecticide toxicity
1INTRODUCTION
Pyrethroids are broad-spectrum insecticides with rapid
knockdown activity and are among the most effective
groups of insecticides.1In public health, δ-phenothrin [3-
phenoxybenzyl (1RS,3RS;1RS,3SR)-2,2-dimethyl-3-(2-methylprop-
1-enyl)cyclopropanecarboxylate] and resmethrin [5-benzyl-3-
furylmethyl (1RS)-cis-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)-
cyclopropanecarboxylate] are synthetic pyrethroids used against
mosquitoes, human lice and flying and crawling insects in homes
and recreational areas.2–6 With the increased prevalence of
mosquito-borne pathogens such as West Nile virus in recent years,
pyrethroids such as permethrin, resmethrin and δ-phenothrin are
being used more widely to manage adult mosquitoes.7,8
Associated with the increased use of permethrin, resmethrin
and δ-phenothrin are public concerns about the environmental
effects of their usage. This is especially true for adult mosquito
management using these insecticides because, although the
application rates are very low, the application method, ultralow-
volume (ULV) spray, essentially causes the insecticides to drift over
relatively large areas. Most pyrethroids are toxic to both target
and non-target insects.1However, most studies of the impact
of pyrethroids have been limited to aquatic invertebrates. Data
on resmethrin and δ-phenothrin toxicity for non-target terrestrial
invertebrates are limited to honey bees.3,4
Risk assessment has been used to quantify risks from
mosquito management tactics.9–12 In pesticide risk assessment,
dose–response relationships for surrogate non-target species are
determined from bioassay studies and are used to compare toxic
doses to estimated or actual environmental exposures.13,14 This is
often accomplished using the risk quotient (RQ) method, whereby
the estimated environmental concentration (EEC) is compared
with a toxic endpoint (e.g. LC50 or no-effect concentration).14
Ecological risk assessments of pyrethroids such as resmethrin
and δ-phenothrin are limited because of a lack of toxicity data for
non-target terrestrial invertebrates. Therefore, the objective of this
study was to estimate LC50 values for resmethrin and δ-phenothrin
against three non-target, surrogate insect species.
2 MATERIALS AND METHODS
2.1 Insecticides
Technical-grade resmethrin (94.3% purity), δ-phenothrin (94.9%
purity), and a synergist, piperonyl butoxide (PBO) (98.2% purity),
were obtained from Sigma-Aldrich (St Louis, MO). Stock solutions
for δ-phenothrin, resmethrin and PBO were prepared for each
pesticide before each experiment by dissolving the insecticides
and PBO in acetone, and serial dilutions were made by volumetric
pipetting. Concentrations were expressed on a weight/volume
basis.
∗Correspondence to: Robert KD Peterson, Department of Land Resources and
Environmental Sciences, Montana State University, Bozeman, MT 59717-
3120, USA. E-mail: bpeterson@montana.edu
Department of Land Resources and Environmental Sciences, Montana State
University, Bozeman, MT 59717-3120, USA
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Table 1. Insecticide treatments and concentrations used
Concentration (ng cm−2)a
Species Treatment 0X0.25X0.30X0.35X0.50X0.75X1X2X3X5X10X
Acheta domesticus Resmethrinb0.00 19.75 23.7 27.65 39.5 59.24 78.99
δ-Phenothrinc0.00 10.11 12.13 14.15 20.21 30.32 40.43
Hippodamia convergens Resmethrin 0.00 19.75 23.7 27.65 39.5 59.24 78.99
δ-Phenothrin 0.00 10.11 12.13 14.15 20.21 30.32 40.43 80.85
Spodoptera frugiperda Resmethrin 0.00 19.75 23.7 27.65 39.5 59.24 78.99 157.97 236.96 394.93 789.87
δ-Phenothrin 0.00 10.11 12.13 14.15 20.21 30.32 40.43 80.85 121.28 202.13 404.26
aConcentration: 0X, control (acetone); 0.25X,0.30X,0.35X,0.50X,0.75X,1X,2X,3X,5Xand 10Xthe maximum field application rate.
bPBO concentrations for resmethrin were 0X, control (acetone);59.24 ng cm−2(0.25X), 70.79 ng cm−2(0.30X), 82.59 ng cm−2(0.35X), 117.98 ng cm−2
(0.50X), 176.98 cm−2(0.75X), 235.97 ng cm−2(1X), 471.93 ng cm−2(2X), 707.90 ng cm−2(3X), 1179.83 ng cm−2(5X), and 2359.66 ng cm−2(10X).
cPBO concentrations were the same as those of δ-phenothrin.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Mortality (%)
0
20
40
60
80
100
resmethrin
0
20
40
60
80
100
δ-phenothrin
Concentration (log10)
Figure 1. Percentage mortality of Acheta domesticus (•), Spodoptera frugiperda larvae (o) and Hippodamia convergens () versus log concentration of
resmethrin and δ-phenothrin at day 1.
Using serial dilution preparations from the stock solutions, a
preliminary experiment was conducted to determine the range
of insecticide concentrations to use. This was done by exposing
the insects to resmethrin +PBO and δ-phenothrin +PBO at
decreasing concentrations above and below the field application
rate until mortality rates greater than 0 and lower than 100% were
observed.
2.2 Insects
Three taxa of terrestrial insects were chosen for bioassay:
house cricket, Acheta domesticus (L.), convergent lady beetle,
Hippodamia convergens (Gu ´
erin-M´
eneville) and fall armyworm,
Spodoptera frugiperda (JE Smith). Acheta domesticus adults (mean
dry weight =147.7mg; SD =35.5 mg) were chosen because
of ease of handling and rearing and known susceptibility to
many insecticides.15 Hippodamia convergens adults (mean dry
weight =10.4mg; SD =9.6 mg) were chosen because of ease
of handling and predator trophic level. Spodoptera frugiperda
larvae were chosen because of ease of handling and rearing and
immature life stage. Acheta domesticus adults were purchased
from Premium Crickets (Thomson, GA), H. convergens adults were
provided by Planet Natural (Bozeman, MT) and S. frugiperda
larvae were purchased in the third stage from Benzon Research
(Carlisle, PA).
2.3 Bioassays
The LC50 was determined under laboratory conditions. For
the establishment of the concentration– mortality relationships,
insects were exposed to 7, 8 and 11 concentrations, depending
on experiment and species (0, 0.25, 0.30, 0.35, 0.50, 0.75, 1, 2,
3, 5 and 10 times the maximum field application rates for each
insecticide) (Table 1). ‘Field application rate’ means the maximum
rate in g ha−1. The field rates for resmethrin and δ-phenothrin are
7.85 g ha−1and 4.03 g ha−1respectively.
Acetone solutions of resmethrin +PBO and δ-phenothrin +PBO
were applied in glass vials (length 4.5 cm, diameter 2.5 cm, volume
20 cm3; Thermo Fisher Scientific Inc., Waltham, MA). Pure acetone
was used as the control. A quantity of 1 mL of the insecticide
dilution (resmethrin or δ-phenothrin) as well as PBO was dispensed
with a micropipette into the vials. The vials were then placed on
hot dog rollers (model HDR-565; The Helman Group, Ltd, Oxnard,
CA) and rotated mechanically to coat the vials uniformly until they
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0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Mortality (%)
0
20
40
60
80
100
resmethrin
0
20
40
60
80
100
δ-phenothrin
Concentration (log10)
Figure 2. Percentage mortality of Acheta domesticus (•), Spodoptera frugiperda larvae (◦)andHippodamia convergens () versus log concentration of
resmethrin and δ-phenothrin at day 2.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Mortality (%)
0
20
40
60
80
100
resmethrin
0
20
40
60
80
100
δ-phenothrin
Concentration (log10)
Figure 3. Percentage mortality of Acheta domesticus (•), Spodoptera frugiperda larvae (◦)and Hippodamia convergens () versus log concentration of
resmethrin and δ-phenothrin at day 3.
were dried. One insect was placed in each vial and covered with
a perforated cap. Four experimental replicates were used for each
concentration of each insecticide. For S. frugiperda, the caps were
lined with a fine-mesh nylon fabric to prevent escape and to allow
air circulation in the vials.
Treated vials were placed on large plastic trays and left on the
laboratory bench at 24 ±0.5◦C with a photoperiod of 16 : 8 h
light : dark. Insect mortalities were assessed and recorded daily
for 3 days. The insects were observed for 3 days to account for
delayed mortality and exposures over time to potential residues
after a single application scenario. Because 3 days represents a
considerable time for the insects in the vials, the 1 day mortality
data are most likely the most informative. Insects that did not
move when prodded with forceps were considered dead. Abbott’s
formula,16 as defined by Perry et al.17 and Antwi et al.,18 was used
to correct for control mortality, in which insects were exposed
to vials treated with acetone only. Control mortalities were less
than 5%.
2.4 Data analysis
The data were analyzed with SAS 9.1.19 The LC50 values were
determined with PROC PROBIT, mortality was regressed on
concentration using PROC REG19 and graphs of percentage
mortality against log10 concentration were plotted with Sigma
Plot 8.0 (SPSS Inc., Chicago, IL). However, for S. frugiperda larvae,
the poor fit of the models was accounted for by multiplying the
variances by a heterogeneity factor (χ2/k−2), where kis the
number of concentrations, to account for extrabinomial variations
due to genetic and environmental influences that caused poor
fit.20 – 23 Differences in LC50 values among the insects and between
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Table 2. Relationship between mortality and concentrations of resmethrin and δ-phenothrin with piperonyl butoxide
Species Treatment Day Regression modelaFR
2P
Acheta domesticus Resmethrin 1 Y=18.52 +1.09X21.76 0.8131 0.0055
Acheta domesticus Resmethrin 2 Y=32.73 +1X10.21 0.6712 0.0241
Acheta domesticus Resmethrin 3 Y=38.03 +0.83X5.28 0.5135 0.07
Hippodamia convergens Resmethrin 1 Y=17.94 +0.78X11.36 0.6943 0.0199
Hippodamia convergens Resmethrin 2 Y=26.37 +0.9X10.4 0.6752 0.0234
Hippodamia convergens Resmethrin 3 Y=30.44 +0.73X6.2 0.5534 0.0552
Spodoptera frugiperda larvae Resmethrin 1 Y=16.58 +0.11X17.83 0.6646 0.0022
Spodoptera frugiperda larvae Resmethrin 2 Y=22.73 +0.1X12.98 0.5906 0.0057
Spodoptera frugiperda larvae Resmethrin 3 Y=17.35 +0.09X14.09 0.6102 0.0045
Acheta domesticus δ-Phenothrin 1 Y=−10.94 +2.12X71.08 0.9343 0.0004
Acheta domesticus δ-Phenothrin 2 Y=−3.70 +2.02X181.03 0.9731 <0.0001
Acheta domesticus δ-Phenothrin 3 Y=1.31 +1.62X103.55 0.9539 0.0002
Hippodamia convergens δ-Phenothrin 1 Y=−5.69 +0.7X52.96 0.8982 0.0003
Hippodamia convergens δ-Phenothrin 2 Y=3.61 +0.72X23.64 0.7976 0.0028
Hippodamia convergens δ-Phenothrin 3 Y=12.76 +0.91X25.57 0.81 0.0023
Spodoptera frugiperda larvae δ-Phenothrin 1 Y=6.43 +0.19X40.4 0.8178 0.0001
Spodoptera frugiperda larvae δ-Phenothrin 2 Y=9.28 +0.23X31.81 0.7795 0.0003
Spodoptera frugiperda larvae δ-Phenothrin 3 Y=14.79 +0.22X23.7 0.7247 0.0009
aY=mortality (%); X=concentration (ng cm−2).
Table 3. Lethal concentrations and risk quotients for Acheta domesti-
cus treated with resmethrin and δ-phenothrin with piperonyl butoxide
Treatment Day
LC50
(ng cm−2) CI (95%) P>χ
2
Risk quotient
(EEC/LC50)a
Resmethrinb1 23.2 17.34 – 25.41 0.3429 3.41
Resmethrin 2 13.39 5.03– 18.98 0.9104 5.9
Resmethrin 3 4.7 NDc0.8362 16.81
δ-Phenothrind1 26.9 23.51 –32.05 0.2008 1.5
δ-Phenothrin 2 24.37 20.85 –29.97 0.2957 1.66
δ-Phenothrin 3 28.14 22.97 –39.3 0.8045 1.44
aEEC =maximum field rate.
bResmethrin commercial maximum field rate =78.99 ng cm−2(0.007
lb acre−1).
cND, no data as confidence interval could not be determined by
statistical analysis.
dδ-Phenothrin commercial maximum field rate =40.43 ng cm−2
(0.0036 lb acre−1).
the treatments were determined by comparison of the 95%
confidence limits.
3RESULTS
Theresultsof residual or contact bioassays for the insects are shown
in Figs 1, 2 and 3. The lethal concentrations are also presented in
Tables 3, 4 and 5. There was generally a good fit to the model
assumptions. Table 2 shows the regression relationship between
mortality of the insects (Acheta domesticus, Hippodamia conver-
gens,andSpodoptera frugiperda larvae) and resmethrin +PBO and
δ-phenothrin +PBO concentrations. (Hereafter, ‘resmethrin +
PBO’ and ‘δ-phenothrin +PBO’ will be referred to as ‘resmethrin’
and ‘δ-phenothrin’ respectively.) The relationships were signifi-
cant. For resmethrin, the models explained 51.4 – 81.3% of the
Table 4. Lethal concentrations and risk quotients for Hippodamia
convergens treated with resmethrin and δ-phenothrin with piperonyl
butoxide
Treatment Day
LC50
(ng cm−2) CI (95%) P>χ
2
Risk quotient
(EEC/LC50)a
Resmethrinb1 32.08 20.76 –43.65 0.3542 2.46
Resmethrin 2 17.85 7.11– 24.73 0.5472 4.43
Resmethrin 3 10.95 NDc0.9206 7.21
δ-Phenothrind1 74.91 64.95–90.14 0.3498 0.54
δ-Phenothrin 2 56.02 45.71 –73.85 0.1227 0.72
δ-Phenothrin 3 28.29 15.45 –52.38 0.0114 1.43
aEEC =maximum field rate.
bResmethrin commercial maximum field rate =78.99 ng cm−2(0.007
lb acre−1).
cND, no data as confidence interval could not be determined by
statistical analysis.
dδ-Phenothrin commercial maximum field rate =40.43 ng cm−2
(0.0036 lb acre−2).
total response variation for A. domesticus, 55.3 –69.4% for H. con-
vergens and 59.1–66.5% for S. frugiperda larvae for days 1 to 3
(Table 2). For δ-phenothrin, the models explained 93.4 –97.3% for
A. domesticus, 79.8–89.8% for H. convergens and 72.5 –81.8% for S.
frugiperda larvae for days 1 to 3 (Table 2).
For resmethrin, the slopes varied from 0.83 to 1.09 for A.
domesticus, from 0.73 to 0.9 for H. convergens and from 0.09 to
0.11 for S. frugiperda larvae (Table 2). For δ-phenothrin, the slopes
ranged from 1.62 to 2.12 for A. domesticus, from 0.7 to 0.91 for
H. convergens and from 0.19 to 0.23 for S. frugiperda larvae for days
1 to 3 (Table 2).
In terms of sensitivities among the insects to resmethrin and
δ-phenothrin, A. domesticus were most sensitive, followed by H.
convergens and then S. frugiperda larvae. Achetadomesticus was the
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Table 5. Lethal concentrations and risk quotients for Spodoptera frugiperda larvae treated with resmethrin and δ-phenothrin with piperonyl
butoxide
Treatment Day LC50 (ng cm−2) CI (95%) P>χ
2Risk quotient (EEC/LC50)a
Resmethrinb1 307.18 169.62– 664.63 <0.0001 0.26
Resmethrin 2 256.67 99.77– 684.57 <0.0001 0.31
Resmethrin 3 361.65 203.98 – 862.8 <0.0001 0.22
δ-Phenothrinc1 228.57 158.83 –394.3 <0.0001 0.18
δ-Phenothrin 2 165.02 107.55 –309.14 <0.0001 0.25
δ-Phenothrin 3 151.94 91.99– 294.3 <0.0001 0.27
aEEC =maximum field rate.
bResmethrin commercial maximum field rate =78.99 ng cm−2(0.007 lb acre−1).
cδ-Phenothrin commercial maximum field rate =40.43 ng cm−2(0.0036 lb acre−1).
most sensitive species for resmethrin (LC50 =4.7–23.2ngcm
−2)
and for δ-phenothrin (LC50 =24.37– 28.14 ng cm−2) (Table 3).
Hippodamia convergens LC50 values were 10.95 –32.08 ng cm−2for
resmethrin and 28.29 –74.91 ng cm−2for δ-phenothrin (Table 4).
Spodoptera frugiperda larvae were the least sensitive species
for resmethrin (LC50 =256.67– 361.65 ng cm−2)andforδ-
phenothrin (LC50 =151.94– 228.57 ng cm−2) (Table 5).
For A. domesticus, the lethal concentrations decreased with
time, except at day 3 for δ-phenothrin. The LC50 values were
3.41–16.81-fold less than the field rate for resmethrin and
1.44 –1.66-fold less than the field rate for δ-phenothrin for days 1 to
3 (Table 3). For H. convergens, lethal concentrations decreased with
time for resmethrin (32.08 –10.95 ng cm−2)andforδ-phenothrin
(74.91– 28.29 ng cm−2). The LC50 values were 2.46 – 7.21-fold less
than the field rate for resmethrin and 0.54–1.43-fold less than the
field rate for δ-phenothrin for days 1 to 3 (Table 4). Except at day 3
for resmethrin, the LC50 for S. frugiperda larvae decreased with time
(307.18– 256.67 ng cm−2for resmethrin and 228.57–151.94 ng
cm−2for δ-phenothrin) (Table 5). The LC50 values were 0.22 –0.31-
fold less than the field rate for resmethrin and 0.18–0.27-fold less
than the field rate for δ-phenothrin for days 1 to 3 (Table 5).
4 DISCUSSION AND CONCLUSIONS
The use of surrogate species is an established testing strategy to
assess the potential impact of a chemical on species residing in the
habitat of concern. The results of this study indicate that resmethrin
and δ-phenothrin are inherently more toxic to Acheta domesticus
than to Hippodamia convergens or Spodoptera frugiperda larvae.
For each active ingredient there was a good relationship be-
tween the observed mortality of the insects and the concentrations
used. The regression relationship explained 51–97% of the vari-
ation in the models for all treatments and insect species tested.
The slope of the regression relationship line indicates how fast
the insects responded with increasing concentration. The slopes
were generally higher for A. domesticus, varying between 0.83 and
2.12 for both resmethrin and δ-phenothrin. Spodoptera frugiperda
larvae had the lowest slopes of about 0.09–0.23 for resmethrin
and δ-phenothrin.
The results demonstrate that non-target surrogate insect
species representing three orders and families vary in their
sensitivity to resmethrin and δ-phenothrin. Comparison of the LC50
values revealed that S. frugiperda larvae were least susceptible
to resmethrin and δ-phenothrin. The house cricket, Acheta
domesticus, was the most susceptible species, and, based on the
results, this seems to be a good surrogate species for estimating
potential non-target terrestrial insect impacts from exposure to
pyrethroids used in public health applications.
Levels of concern (LOC) are tools that policy makers and
regulatory agencies use to assess the acceptability of risks.14
The ratio between the estimated environmental concentration
(EEC) and LC50, termed the risk quotient (RQ), gives an estimate
of the risk. The calculated RQ is compared with the respective
RQ LOC to determine if there is a need for regulatory action.14
The USEPA typically uses an acute RQ LOC of ≥0.5 for terrestrial
animals (i.e. the estimated exposure is 50% of the LC50). If it is
assumed that the EECs are equivalent to the field application rates
(assuming 100% even deposition of insecticide over 1 ha), daily
RQs for A. domesticus and H. convergens were >0.5 for resmethrin
and δ-phenothrin. RQs for S. frugiperda larvae did not exceed 0.5
for resmethrin or δ-phenothrin.
These results suggest that mortality risks would be greater
for resmethrin than for δ-phenothrin. Risk quotients for some
non-target terrestrial insects may exceed RQ LOCs. However, it is
highly unlikely that pyrethroid deposition on terrestrial surfaces
after ULV applications would be 100% of application rate. Small
droplets produced from ULV sprays are distributed by wind over
a wide area, and observed deposition rates have been low.15,24
Indeed, the weight of evidence suggests that surface deposition
ranges from approximately 1 to 10%.12,24,25 If terrestrial deposition
were 10% of the application rate, then the EECs from the maximum
ULV application rates for the insecticides in this study would result
in RQs that exceeded 0.5 only for A. domesticus and H. convergens
exposed to resmethrin on days 2 and 3. No other RQ LOCs would
be exceeded.
Toxicities as determined by LC50 values were lower on day 1 for
resmethrin and δ-phenothrin for all surrogate species compared
with those on days 2 and 3. The toxicities were greater on the
second and third days primarily because the insects had been
exposed continuously to the insecticides in the vials for 72 h. In
contrast to the exposure in the vial, exposure in the field most likely
is much different. Knepper et al.24 observed very low persistence
of malathion and permethrin, with only trace levels after 36 h.
Additionally, the adsorption of resmethrin and δ-phenothrin to
soil particles and vegetation in the terrestrial environment may
reduce their bioavailability, further limiting their effects on non-
target insects.
In a deterministic, reasonable worst-case risk assessment, Davis
et al.10 concluded that acute and chronic risks to ecological
receptors, including terrestrial insects, from ULV insecticides used
for mosquito management most likely are low. However, toxicity
data for non-target insects were not available at the time of their
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study. The present results provide LC50 values for surrogate, non-
target terrestrial insects and can be compared with actual and
estimated environmental insecticide concentrations to estimate
risks. Given the LC50 values that were estimated and what is
currently known about the fate of resmethrin and δ-phenothrin
after ULV application, the present results may be used to support
the conclusions of Davis et al.10
ACKNOWLEDGEMENTS
The authors thank J Schleier and R Davis (Montana State University)
for assistance. This study was funded by a grant from the US
Armed Forces Pest Management Board’s Deployed War Fighter
Protection Research Program, Montana State University, and the
Montana Agricultural Experiment Station.
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2008 Society of Chemical Industry www.interscience.wiley.com/journal/ps
