Forensic Science International 192 (2009) 14–18

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Forensic Science International

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Determination of Malathion levels and its effect on the development of Chrysomya

megacephala (Fabricius) in South China
Xiaoshan Liu, Yanwei Shi, Haiyang Wang, RunJie Zhang *
State Key Laboratory for Biocontrol Institute of Entomology, Sun Yat-Sen University, Guangzhou 510275, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Article history: The present study determines the concentration of Malathion in rabbit tissues and Dipteral larvae
Received 23 August 2008 feeding on those tissues. Malathion was found in all muscle and liver samples of the test rabbit, as well as
Received in revised form 28 June 2009 larvae fed on it. Samples from the control rabbit and pupae from all colonies were negative for Malathion.
Accepted 2 July 2009
Correlations were found between administered dosage, tissue concentrations and younger or
Available online 30 July 2009
prepuparial larvae. Effects of Malathion on the development rate of Chrysomya megacephala (Fabricius)
were also reported. C. megacephala is the most common fly species found on corpses in South China
Keywords:
during the early stages of decomposition. Significant differences in larval growth rate were both
Malathion
Forensic entomology
observed among the colonies fed on muscle and liver. The presence of Malathion in both muscle and liver
Chrysomya megacephala appears to retard the normal growth rate of C. megacephala in larval stage. Larvae from all colonies fed on
Postmortem interval tissues from rabbits treated with Malathion were smaller and attained maximum length later than those
from the control colony. Duration of the larval and pupal stages was both significantly prolonged for
larvae on tissues from rabbit receiving Malathion than those from the control colony. The difference of
the duration of the larval and pupal stages together from the muscle colonies would alter the
postmortem interval estimation by up to 36 h. As for liver colonies, it would alter the postmortem
interval estimation by up to 28 h. A significantly different duration of the larval and pupal stages from the
muscle colonies would alter a postmortem estimate by up to 28 h relative to the liver colonies.
ß 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction phenothiazines and benzodiazepines, as well as barbiturates and

Entomotoxicology is a specialized area of entomology and
forensic science that deals with the qualitative or quantitative
determination of toxic substances in insects or arthropods feeding on
human remains. Insects are useful sources for toxicological analysis
when the tissues or body fluids normally used are not available. The
study of insects, especially larvae found in cadavers, can contribute to
the qualitative identification of substances or illegal drugs present in
the corpse. The history of entomotoxicology is relatively short, the
toxin substances in earlier studies included environmental pollu-
tants, pesticides and drugs. Environmental pollutants such as metals
and mercury were first reported by Sohal and Lamb [1] and Nuorteva
and Nuorteva [2] in late 1970s. Goff and Kanthi [3] first reported the
detection of the pesticide Malathion, while Beyer et al. published the
first article about the drug Phenobarbital [4]. In entomological cases,
drug-related deaths are also detected through analyses of insects
feeding on the corpse: opiates such as morphine and codeine, cocaine
and benzoylecognine, amphetamines, tricyclic anti-depressants,
* Corresponding author. Tel.: +86 02084036901.
E-mail address: lsszrj@mail.sysu.edu.cn (R. Zhang).
several salicylates [5–9].
Previous studies have demonstrated no reliable correlation
between drug or toxin concentrations in larvae and its feeding
tissues. Several earlier studies have suggested such a correlation
might exist [10,11], while other studies showed no relevant
correlation [12,13]. Various results show that drug accumulation
in larvae is unpredictable and quantitative extrapolations are
unreliable, which is particularly true when dealing with more than
a single drug or concentration level [14]. It seems that while
Diptera larvae are useful as qualitative toxicological specimens,
they are of limited quantitative value.
Additionally, understanding the effect of drugs and toxins on
Dipteran development rate is paramount, before the use of
maggots for PMI determination can be qualified. Lord et al. [15]
gave an example of adjustment of the PMI estimation based on the
presence of cocaine in the remains. Works by Goff et al.
[10,11,16,17] have documented differences in growth rates for
two species of flesh fly maggots (Diptera: Sarcophagidae) fed on
decomposing tissues containing known amounts of different drugs
and their metabolites. These studies have shown that presence of
drugs would accelerate, retard or have no influence on the
development rates of different larvae.

0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2009.07.005
 X. Liu et al. / Forensic Science International 192 (2009) 14–18
Malathion, belongs to a group of chemical compounds
commonly referred to as organophosphorous pesticides (Ops),
which is considered to be one of the safest Ops for its
comparatively low mammalian toxicity, is also one of the Ops
mostly associated with unexpected deaths caused by either
accidental or intentional poisoning in China. Although, Goff
et al. [4] have identified Malathion in human cadavers and two
kinds of flies fed on it, data are lacking on the effect of this
substance on fly larvae.
The purpose of this study was to detect the concentration of
Malathion in rabbits and fly larvae samples using gas-chromato-
graphy–mass spectrometry (GC–MS), and analyze their correla-
tion. It was also to determine the effect of Malathion on the
development of larvae and to verify the time of emergence of
adults, which could affect the estimation of the postmortem
interval.
2. Materials and methods
2.1. Necrophagous fly experiment
Three domestic rabbits (2.25–2.45 kg in weight), Oryctolagus cuniculus L., were
used in each experiment. In order to obtain equivalent dosages, the human lethal
dose of Malathion 60 g/60 kg was converted to a rabbit lethal dose of 1.53 g/kg. The
90% Malathion provided by the Agriculture Office of Guangdong Province was
diluted to 25% with saline before use. Three rabbits were given 0.5, 1.0 and 1.5 times
the lethal dose of diluted Malathion respectively by enema. The fourth one was
given nothing and was therefore used as a control. After 1 h of treatment with
pesticide, the animals were all sacrificed via infusing air into the ear vein.
Immediately following death, muscle and liver tissue were taken from each
rabbit and frozen for later food source and analysis of the Malathion. The muscle
samples were designated as M0 for muscle from the control rabbit, while M1, M2, M3
identified muscle from rabbits that received 0.5, 1.0, 1.5 times the lethal dose of
Malathion respectively. L0 for liver from the control rabbit, and L1, L2, L3 for liver
samples obtained from the rabbits that received 0.5, 1.0, and 1.5 times the lethal
dosage respectively.
Flies used in this study came from a stock colony of Chrysomya megacephala
established from specimens collected from decomposing rabbit remains on the
campus of Sun Yat-Sen University in Guangzhou, China. The colony was reared in
the laboratory for three generations prior to use. Pork was exposed to the colony for
oviposition. From the oviposition, 400 larvae were obtained and placed onto each
muscle and liver sample to initiate the test colonies. Colonies established were
maintained in the laboratory at 25 8C in environmental chamber with a 12 h light
and 75% humidity. At 12 h intervals, length was recorded from random samples of
20 larvae from each colony to identify growth rate. At the second and third instar
stages, a sample of 100 larvae were removed from each colony and frozen for
analysis of Malathion concentration. Pupae were observed after 72 h and adult
emergence recorded.
SPSS 13.0 was used to analyze the fly growth rate data. Analysis of variance
(ANOVA) tests were performed using the general linear models (GLM). The means
for each factor and each species were compared using the Duncan multiple
comparisons tests.
2.2. Toxicological methods: base extraction
2.2.1. Muscle and liver samples and analytical procedure
In this study, approximately 10 g of muscle and liver samples were weighed and
grinded together with 10 g anhydrous sodium sulphate. To this, a 30 ml volume of
acetone was added and the sample shaken for 30 min on a shaking table at 25 8C,
with a rotating speed of 250 r/min. Then 30 ml of dichloromethane and 60 ml of 20%
sodium sulphate solution were added to the filtered solvent and the sample was
shaken for 2 min. Thirty milliliters of dichloromethane was again added to the
extracted liquid and concentrated using a rotary-evaporator at 60 8C to a 2 ml
volume. Finally, the concentrated solution was passed through the glass
chromatography column, which had been packed with anhydrous sodium sulphate
and neutral alumina, elution was with 30 ml hexane, the eluent was concentrated
by a rotary-evaporator at 60 8C to a 2 ml volume.
2.2.2. Fly samples and analytical procedure
The extraction procedures of fly sample were the same as for the rabbit tissues.
2.2.3. GC–MS
The GC–MS analysis was performed using gas chromatograph (Agilent
Technologies, Model 6890 N GC) coupled with a mass spectrometer (Agilent
Technologies, Model 5973N MSD). A HP-5 MS capillary column (30 m  0.25 mm,
0.25 mm) was used. The injector was equipped with a 4 mm-I.D. glass liner. For
qualitative determination, the MS system was operated in the full-scan mode from
15
m/z 50–550, and the SIM mode was used for quantitative determination. The carrier
gas was helium and the inlet was operated in a constant flow rate of 1.0 ml/min. The
operating condition for the analyses were: initial temperature 50 8C held for 3 min
and programmed at 20 8C/min to 130 8C and then 30–190 8C/min held for 10 min.
The injector temperature was 280 8C and the detector one was 250 8C. About 2 ml
extract ample was injected in the splitless-injector of the GC–MS system by the
Multipurpose-Autosampler from Gerstel, and the splitter was opened after 60 s. The
MS was operated in the electron impact ionization mode with electron energy of
70 eV. To increase the selected-ion-monitoring (SIM) mode and 125, 90, 153 m/z
were selected for quantification and qualification.
2.2.4. Calibration curves
Calibration curves were used to quantity the Malathion concentration obtained
from rabbit and fly samples in GC–MS. 5 mg of Malathion was dissolved in 50 ml of
acetone, the solution was diluted to 5, 1, 0.5, 0.1, 0.05 mg/ml, from which, 2 ml were
collected and injected into the GC–MS. Calibration curves were obtained by plotting
the peak-area ratio of Malathion. The curve obtained was y = 2E + 06x 153744,
R2 = 0.9994.
2.2.5. Method validation
Recovery experiments were carried out by adding known volumes of Malathion
standards in acetone to samples. After solvent evaporation the samples were
analyzed according to the proposed method. The recovery values were calculated
from calibration graphs that were constructed from the concentration and peak
area of the chromatograms obtained with standards of Malathion. Blank analyses
were performed in order to check interference from the sample. The detection and
quantification limits of Malathion were determined after spiking samples at lower
concentration levels. The analyze identification was based on relative retention
times, ion chromatograms and intensity ratios of the monitored ions for GC/MS. A
deviation of the ion intensity ratios within 20% of the mean values of the calibration
standards was considered acceptable. The precision of the method was expressed
by the relative standard deviation (RSD). To obtain the RSD values, 6 replicate
analyses of each of the 3 quality control samples were performed. The limit of
detection (LOD), defined as the concentration that corresponds to three times the
standard deviation of the blank, was measured by integrating blank peak area for
each analyze in 10 independent performances. The limit of quantification (LOQ) was
the lowest concentration that could be quantified in a sample with acceptable
relative standard deviation under the stated operational conditions of the method.
LOQ was determined as the analyte concentration corresponding to a signal/noise
ratio of 10.
3. Results
3.1. Validation of the method
Under the optimal experimental conditions, calibration curves
were obtained by plotting the peak-area ratio of Malathion. The
regression equation for Malathion was: y = 2E + 06x 153744, the
coefficient of determination (R2) was greater than 0.999. Spiked
experiments at levels of 1 and 10 mg/g showed that recoveries
ranged between 75% and 85%. Six replicate measurements of the 3
quality control samples were used to calculate the RSD value. The
RSD value of Malathion was between 4.7% and 12.8%. Analysis of a
standard solution with low concentration revealed a low LOD
(0.1 mg/ml) and LOQ (0.3 mg/ml) value in this method.
3.2. Concentration and correlation
The analyzed results showed that Malathion was present in
muscle and liver samples in the rabbit that received Malathion.
However, the sample from the control rabbit was negative for the
insecticide (Table 1). Qualitative analyses were made on the
samples of larvae from each colony at second and third instar,
prepuparial and pupal stage. Malathion was positive in all larvae
samples but negative in both the pupae and control samples.
Correlations were found between administered dosage, tissue
concentrations (Table 2). While the correlation between concen-
trations in tissues and larvae were exist at the early time, and
disappeared as the day increased. For the larvae of C. megacephala
reared on meat, the significant correlation was exist in second
instar stage, but it disappeared in third and prepuparial stage
(Table 3). While, for that reared on liver, significant correlation
16 X. Liu et al. / Forensic Science International 192 (2009) 14–18

Table 1
Concentration of Malathion in muscle and liver tissues from rabbit treated with
Malathion and larvae of Chrysomya megacephala fed on those tissues.

Rabbit Second instar Third instar Prepuparial Pupal (ng)

sample (ng) larvae (ng) larvae (ng) larvae (ng)

M0 ND ND ND ND ND
M1 57.80 4.89 8.53 3.58 ND
M2 335.48 22.77 35.96 20.58 ND
M3 556.38 25.36 28.90 11.05 ND
L0 ND ND ND ND ND
L1 98.44 8.49 12.81 3.53 ND
L2 115.60 11.11 20.61 13.58 ND
L3 140.72 18.03 20.84 11.23 ND

ND = not detected. Fig. 1. Larval body length of C. megacephala fed on meat tissues (mean  SE).

Table 2 exposed to 0.5 times the lethal dosage. From 72 h through to 108 h,
Correlation between concentration in rabbit tissues and administrated dosage.
significant differences were observed between the Malathion
Liner regression equation r p receiving and control colonies. Larvae from colonies grown on
muscle of rabbits receiving Malathion were smaller and attained at
Meat y = 498.58x 182.03 0.99 0.04
Liver y = 42.28x + 75.98 0.98 0.04 maximum length later than the control colonies, with the
maximum length 16.1 mm received at 96 h for control colony
and the smallest maximum length 15.37 mm received at 108 h
Table 3 from the colony treated with 1.5 times the lethal dosage (Fig. 1).
Correlation between concentration in meat and larvae of C. megacephala.
There was a significant difference among colonies in the duration
Liner regression equation r p of larvae stage, with a mean duration of 136.3 h for the control
colony, 144.3, 150.6, 153.6 h for the colonies receiving 0.5, 1.0, 1.5
R1 y = 0.05x + 2.04 0.92 0.03
R2 y = 0.06x + 5.04 0.73 0.14 times the lethal dosage respectively. Pupal stage duration was also
R3 y = 0.02x + 2.99 0.48 0.30 longer in treated colonies than in the control, the difference
between larvae and pupal stage duration together would prolong
R1: correlation between concentration in meat and second instar larvae.
R2: correlation between concentration in meat and third instar larvae. the estimation of PMI up to 36 h. Length and weight of the pupae
R3: correlation between concentration in meat and prepuparial instar larvae. for control and treated colonies are significantly different, with
lighter and shorter pupae in the treated colonies (Table 5). The
relationship between the dosage and adult emergency among
Table 4 colonies received Malathion was reversed.
Correlation between concentration in liver and larvae of C. megacephala.

Liner regression equation r P 3.4. Development fed on liver

R1 y = 0.12x 0.87 0.92 0.04
R2 y = 0.15x 0.17 0.95 0.02 For larvae reared on liver, at the 24 h mark, development rates
R3 y = 0.09x 0.58 0.70 0.90 of larvae were significantly different between control and treated
colonies (Fig. 2). From 36 to 108 h, significant differences were
R1: correlation between concentration in liver and second instar larvae.
R2: correlation between concentration in liver and third instar larvae. also found between colony treated with 0.5 times the lethal
R3: correlation between concentration in liver and prepuparial instar larvae. dosage and colonies treated with 1.0 and 1.5 times the lethal
dosage. Larvae from all liver fed colonies from toxic rabbits were
smaller and attained at maximum length later than the control
were exist in both second and third instar stages, and disappeared colony. The maximum length was 17.31 mm received at 84 h from
in prepuparial stage (Table 4). the control colony, and 16.25 mm received at 96 h from the colony
treated with 1.5 times the lethal dosages. For colonies found
3.3. Development of larvae fed on muscle growing on muscle, the different larval and pupal stage durations
were both significant and would prolong PMI estimation up to
The development rate was indicated by the total length 28 h (Table 6). Significant differences in length and weight of
measurements of larvae. The presence of Malathion in tissue pupae were observed between pupae receiving 0.5 times the
resulted in the decreased growth rate of C. megacephala larvae. lethal dosage as well as pupae receiving 1.0 and 1.5 times the
From 24 to 72 h, the development rates of larvae were significantly lethal dosages. A reverse relationship between the dosage and
different for the 1.0 and 1.5 lethal dosages, no significant adult emergency was also observed among colonies that received
differences were found between the control colony or the colony Malathion.

Table 5
The time of maximum larval length, duration of larval and puparial stage, pupurial weight, length, and adult emergency for colonies of C. megacephala on meat of rabbit
treated with Malathion (mean  SE).

Maximum larval Duration of Duration of Puparial Puparial Adult

length (mm)/ pupal stage (h) larval stage (h) weight (g) length (mm) emergency (%)
observed time (h)

M0 16.1/96 h 124.6  0.58a 136.3  1.52a 0.0455a 8.44  0.45a 89

M1 15.5/96 h 133.0  1.00b 144.3  1.52b 0.0387b 7.94  0.31b 88
M2 15.39/108 h 142.7  0.58c 150.6  2.08c 0.0355b 7.75  0.49b 76
M3 15.37/108 h 143.3  0.58c 153.6  2.17c 0.0397b 7.85  0.58b 78
 X. Liu et al. / Forensic Science International 192 (2009) 14–18 17

Table 7
Summary of best fit of body length over time for larvae of C. megacephala reared on
meat treated with Malathion.

Linear Second-degree Third-degree

polynomial polynomial

R2 P R2 P R2 P

M0 0.94 <0.0001 0.96 <0.0001 0.99 <0.0001

M1 0.94 <0.0001 0.97 <0.0001 0.99 <0.0001
M2 0.96 <0.0001 0.98 <0.0001 0.99 <0.0001
M3 0.94 <0.0001 0.96 <0.0001 0.99 <0.0001

Table 8
Summary of best fit of body length over time for larvae of C. megacephala reared on
Fig. 2. Larval body length of C. megacephala fed on liver tissues (mean  SE).
liver treated with Malathion.

Linear Second-degree Third-degree

polynomial polynomial
3.5. Curve fitting for body length over time
R2 P R2 P R2 P
Plots of length against time for larvae grown both on control or
L0 0.94 <0.0001 0.94 <0.0001 0.99 <0.0001
administrated rabbit tissues are shown in Tables 7 and 8. The L1 0.95 <0.0001 0.97 <0.0001 0.99 <0.0001
calculated R-square values for lines of fit for these plots are show in L2 0.94 <0.0001 0.97 <0.0001 0.99 <0.0001
Tables 7 and 8. The R-square values for linear lines of best fit are L3 0.93 <0.0001 0.96 <0.0001 0.99 <0.0001
comparable. A second-degree polynomial model improved pre-
diction. While, the calculated R-squared values and F-test for third-
degree polynomial model shows that for both the reared on control Different form the study of Introna et al. [18], where concentration
or administrated, liver or meat tissues, third-degree model would in larvae was very similar to those in the liver tissue, larval
be the best model, use this model, length would be a good predictor Malathion concentration this study was 10–30 times lower than
of time for these larvae. the tissue concentration. Other studies have also found the
concentration in larvae was significantly lower than the tissue
4. Discussion concentration [12].
Compared to previous studies [4,21,22], the concentration of
In the present study, Malathion concentrations in the larvae Malathion in our study was relatively low. These may be due to a
were significantly correlated to muscle and liver tissue concentra- number of factors. First, the quantity of Malathion consumed in
tions (Table 2). This is comparable to Introna et al. [18], which first each case was different. Also, variations in the time intervals
demonstrated this relationship. However, several earlier studies between ingestion and death, as well as the final laboratory
suggested this correlation might not exist [12,13]. This is probably preparation and analysis may also affect the concentration levels
because of maggots could eliminate a variety of drugs with varying found.
effectiveness. The presence of Malathion in both muscle and liver appears to
The concentration of Malathion in the tissues affected its retard the normal growth rate of C. megacephala in larval stage.
residual amount in larvae and puparia that fed on these tissues; Larvae fed on the carcass treated with the highest dosage
pesticide concentrations in larvae increased with increasing developed the slowest. For larvae reared on muscle, according
Malathion tissue concentration. Peak concentration was observed to the larval length, the administration of Malathion produced a
in the larvae fed on rabbits treated with 1.5 times the lethal dosage. developmental decrease of up to 17 h during the larval stage.
The concentration decreased in the prepuparial stage and was not Pupation occurred later, and once pupation occurred, the mean
even detected in puparial stage, which could be explained by the duration of the pupal stage was 9–19 h longer than the control. For
fact that the second and third instar undergoes rapid feeding larvae reared on liver, the administration of Malathion produced a
period prior to entering the nonfeeding prepuparial stage. decrease in development of up to 15 h during the larval stage, and
Following the cessation of feeding in the prepuparial stage, the the mean duration of pupal stage was 6–13 h longer than the
concentration of insecticide in the larvae decreased until the control.
puparial stage. This situation was also observed by Sadler et al. Differences in development between the colonies on muscle
[19], who demonstrated that larvae of Calliphora continuously fed and liver are significant. This corresponds to the recent study by
on armitriptyline laden muscle showed an increase in drug Clark et al. [23], in which the growth rates of flies on different
concentration, peaking at day 8, which diminished until day 16, at tissue were different. In this study, larvae reared on liver were
which time none could be detected. Other studies involving significantly larger than larvae from muscle. As well, the duration
different drugs and other fly species show this phenomenon [20]. of larval and pupal stages on liver was shorter than for muscle.

Table 6
The time of maximum larval length, duration of larval and puparial stage, pupurial weight, length, and adult emergency for colonies of C. megacephala on liver of rabbit treated
with Malathion (mean  SE).

Maximum larval Duration of Duration of pupal Puparial Puparial length Adult

length (mm)/ larval stage (h) stage (h) weight (g) (mm) emergency (%)
observed time (h)

L0 17.31/84 120  1.52a 119.0  1.00a 0.0473a 8.69  0.29a 95

L1 16.92/96 129  0.57b 125.6  2.08b 0.0419b 7.77  0.56b 97
L2 16.82/96 134  2.00c 132.33  1.5c 0.0382c 8.23  0.71c 89
L3 16.25/96 135  2.08c 132.00  2.6c 0.0373c 8.11  0.52c 74
18 X. Liu et al. / Forensic Science International 192 (2009) 14–18
These duration differences on muscle and liver would lead to a 6–
33 h difference in PMI. These results contradict the study of
Kaneshraja and Turner [24], which suggests that for Lucilia sericata
growth rates also vary considerably on different tissues and
growth is poorest on liver as compared to other organs. Thus,
lacking data on the presence of Malathion in tissue as a food source
for developing larvae, as well as neglecting the differences between
muscle and liver, an estimate based on the normal development
pattern for C. megacephala can be significantly different from the
actual postmortem interval. If based on larval growth rates or
duration of larval and pupal stages, the estimate would be shorter
than the actual interval because of retardation of development
during these stages. If based on larval reared on muscle, the
estimate would be shorter than those raised on liver would.
Clearly, the tissue on which C. megacephala feed does have an
important effect on the growth rates and needs to be taken into
account more explicitly in the calibration of growth models for
estimation of postmortem interval.
According to previous studies, different species appear to have
different responses to different drugs. It is reasonable to assume
that similar variations also exist for other species of flies when
feeding on decomposing tissues containing Malathion. In like
manner, similar or different effects may exist when other
pesticides are present in tissues. Thus, further investigations of
the possible effects of pesticides in tissues on anthropod
development needed to be undertaken. Until appropriate baseline
data are available, care must be taken in the interpretation of
anthropod development and successional patterns in the case of
pesticides or toxicants.
Acknowledgements
We thank the following specialists for identifications or
confirmations of specimens: we thank M.L. Goff for his help in
revising this article, thank Chen Zhongshan for his help to the data
analysis. We also thank Xie Minnan and Luo ShuSheng for their
help with toxicological analysis. This study was supported by the
Science & Technology Brainstorm Project of China (Grant No.
2005BA529A06), and the Nature Science Foundation of China
(Grant No. 30671394).
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