Analysis of Pesticide Raid® in Feed of Wistar Rat by High-Pressure Liquid Chromatography (HPLC)

doi:10.4236/ajac.2011.228121

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American Journal of Anal yt ical Chemistry, 2011, 2, 32-36

doi:10.4236/ajac.2011.228121 Published Online December 2011 (http://www.SciRP.org/journal/ajac)

Analysis of Pesticide Raid® in Feed of Wistar Rat by

High-Pressure Liquid Chromatography (HPLC)

Albert C. Achudume

Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria

E-mail: [email protected]

Received November 21, 2011; revised December 24, 2011; accepted December 31, 2011

Abstract

The distribution of pesticide by-product in tissues of wistar rats were analyzed using high pressure liquid

chromatography. The limit of detection of the HPLC was 0.1 µg. Results show bioaccumulation factor of

pesticide “Raid®” in lipid, up to three times that of the feed at the first concentration and gradually de-

creased as the concentration increased in the muscle > (0.7), brain > (0.5) and liver > (0.3) as indicated in the

text. At higher concentration of 961 µg/g, bioaccumulation factor decreased in the lipid to 1.2 and 0.6 in the

muscle, 0.03 in the brain and 0.08 in the liver respectively. High Pressure Liquid Chromatography (HPLC)

analysis of raid extract suggests the presence of micprothrin and palethrin. The implications are numerous,

but simply put that accidental ingestion of chlorinated hydrocarbon as in “Raid®” may involve convulsions,

collapse and coma after only brief excitation and ataxia at the onset.

Keywords: High Pressure Liquid Chromatography, Pesticide, Raid®, Chlorinated Hydrocarbon,

Bioaccumulation

1. Introduction

Pesticides have been used to boost food production to a

considerable extent and to control vectors of disease [1].

However, these advantages of great economic benefits

sometimes come with disadvantages when subjected to

critical environmental and human health considerations.

The chronic effects of pesticides from food intake on

human health are not well defined, but there is increasing

evidence of carcinogenetic [2], teratogenic [3] and ge-

netotoxic [4], as well as disruption of hormonal functions

[5]. Analysis of these pesticides and their residues h ad in

the past aided objective re-evaluation and reassessment

of these substances on a benefit-risk analysis basis and

their subsequent withdrawal from use when found to be

hazardous to human health and the environment.

Residues of pesticides and heavy metals enter the food

system. This occurs more often than one might think.

Additionally, residues of pesticides found in the air in-

side the home and on floors and other interior surfaces

contribute to non-occupational pesticide exposure [6].

For example, any potentially harmful substances that

have no set tolerance thresh may not necessarily be

harmful. This means that if there is any toxin in food

supply or water, such food cannot be ban unless it exists

in quantities over th e tolerance threshold no matter what.

There are few studies of compliance to pesticide use

[7,8].

The quality and sophiscation of analysis have grown

and very minute quantities of these pesticides and their

residues can be analyzed with high degree of specify,

precision and accuracy. Therefore, it is important and

utmost necessary to develop methods for analysis of pes-

ticides residues in environmental samples. In the light of

the above, and in view of pesticide related adverse reac-

tions, the study, presents chromatographic and absorp-

tion spectra from a common pesticide Raid® in tissu e of

rat as it affects basal metabolism using high pressure

liquid chromatograph.

2. Materials and Methodology

2.1. Test Facility

Tests with Wistar rats were conducted in a room tem-

perature exposure facility (23˚C). Thirty-five animals

weighing between 180 - 210 were caged in perforated

aluminum chambers (38 × 55 × 35 cm). Five animals

were placed in each of the chambers containing sawdust

shaves, and a light: dark cycle of approximately 12 h.

A. C. ACHUDUME33

Water was given ad libitum, temperature was maintained

at 24˚C ± 1˚C. Air temperature was ±2˚C of the water

temperature. Protocols describing the use of animals in

accordance with National Research Council (NRC) [8]

and World Medical Association (WMA) [9] guide on the

care and use of laboratory animals.

2.2. Test procedures

Wistar rats were obtained from the animal breeding fa-

cility of College of Health Sciences, Obafemi Awolowo

University, Ile-Ife, Nigeria. Technical grade “RAID”

(Johnson Wax, Nigeria), was dissolved in corn oil and

with (in a rotary mixer) 2:98 (w/w) the co mmercial feed,

Purina 5000, for 10 min: the control diet contained corn

oil in the same proportion. Animals were fed raid-spiked

commercial feed at measured concentrations of 25.0 ±

2.4, 54.0 ± 9.2, 108.0 ± 12.5, 216.0 ± 14.6, and 430.0 ±

20.2 and 961.0 ± 80.6 µg/g “Raid” for 10 days, increas-

ing 5 - 10 g/day and ending with 96 g/rat. Two rats from

different chambers receiving the same concentration of

raid in the food were selected and tissue samples were

dissected out (lipid, muscle, brain and liver) at test ter-

mination. The symptom-li mited sensation tests were per-

formed after an overnight fast [10]. Mean values were

calculated from the two rats sampled from each concen-

tration of insecticide-raid.

2.3. Test Extraction

In the toxicity persistence test, “Raid” was extracted with

1:1 diethyl ether-petroleum ether [11]. This compound

was identified and quantified by high-pressure liquid

chromatography (HPLC), using UV1-202nm@01, Base

(Amersham Pharmacia Biotech), serial number 90042,

mobile phase was Methanol: H2O (1:1). The limit of de-

tection of the HPLC was 0.1 µg .The dimension of the

column C18, 4.6 × 250 mm and particle size 5 micron.

Flow rate was 1 ml/min, the injection volume 100 µl and

a wavelength of 202 nm was used for detection. The

values were quantified by standard calibration curve.

2.4. Analytical Procedures

The brain, muscle and liver homogenate fractions were

prepared by the method described by [12] and resus-

pended in 0.15 M KCl to an appropriate final concentra-

tion of 20 mg of protein per millimeter (approximately)

0.5 g of tissue. Liver tissue lipid s were extracted by ch lo-

roform/methanol ratio (2:1) following the method of

Folch [13]. Fractions of each tissue homogenate were

used for the determination of Glucose-6-phosphatase

(G-6-Pase) and Lactate dehydrogenase respectively, to

test for inhibition of metabolic enzymes required to aid

digestion. Glucose-6-phospatase was determined using

aliquot of each homogenate (20%), incubated with 1 ml

of P-nitrophenyl phosphate (NPP) for 15 min and the

reaction was stopped by addition of 2 ml of 0.2 N NaOH;

absorbance was measured at 450 nm as described by [14].

Lactate dehydrogenase was determined by the method of

[15]. Protein was determined by [16] method. Experi-

ments were conducted in a completely randomized man-

ner with five replicates. These were repeated and mean

data of two experiments is presented. Data were sub-

jected to one-way analysis of variance and significance

of treatments from control was tested at 5% level of sig-

nificance followed by Dunnet’s test. Further, data were

also subjected to determination of correlation coefficient

between raid concentration and the observed response.

The statistical analysis was performed using SPSS soft-

ware version 10.0.

3. Results and Discussion

Bioaccumulation factor of pesticide “Raid®” was ob-

served in lipid, up to three times that of the feed at the

first concentration and gradually decreased as the con-

centration increased (Table 1) while accumulation factor

in the muscle (0.7), brain (0.5) and liver (0.3) was about

the indicated number times that of the feed. At higher

concentration of 961 µg/g, bioaccumulation factor de-

creased in the lipid to 1.2 and 0.6 in the muscle, 0.03 in

the brain and 0.08 in the liver. Using the mean of insecti-

cide in feed, the tissues accumulate the insecticide in

ascending order: brain < liver < muscle < lipid. The ef-

fects increased with increasing insecticide concentration

in the feed an d showed a significant deference compared

to control (P < 0.05) at a concentration of 430 µg/g.

Table 2 indicates the estimated detectable levels of

toxicity in rat tissues exposed to the insecticide “Raid®”.

Tissue insecticide concentration increased in lipid, mus-

cle, brain and liver, respectively. The insecticide accu-

mulation in these organs may therefore indicate that the

active ingredients are also absorbed resulting in the de-

creased enzymes formation, which should accelerate

immediate metabolic processes, but instead, remained

complexed in the tissue organs. Additionally, the reten-

tion time (within 10 min.) may be increased because of it

easy solubility and lower excretion rate leading to bio-

accumulation. The brain shows insignificant decreases in

the enzymes Glucose-6-phosphatase and Lactic acid de-

hydrogenase, while significant decreases were noticeable

in the muscle and liver. The tissues concentrations were

the sums of the two components.

High Pressure Liquid Chromatography (HPLC) analy-

sis of raid extract suggests the presence of micprothrin

A. C. ACHUDUME

Table 1. Tissue total raid concentrations and bioaccumulation factors (BAF) in Wister Rats exposed to insecticide “Raid”.

Mean ± SD insecticide “Raid” in feed (µg/g)

Raid concentration in Wister rats (µg/g) a and bioaccumulation factor (BAF)b

Lipid Muscle Brain Liver

0.00 - - - -

25.0 ± 2.4 72.5 17.5 12.5 7.5

(2.9) (0.7) (0.5) (0.3)

54.0 ± 9.2 86.4 21.7 16.4 9.4

(1.6) (0.4) (0.3) (0.2)

108.2 ± 12.5 172.8 30.4 19.5 10.8

(1.6) (0.3) (0.2) (0.10)

216.2 ± 14.6 280.8 45.8 22.9 19.8

(1.3) (0.2) (0.1) (0.09)

430.0 ± 20.6 324.0 86.4 25.8 37.3

(0.8) (0.2) (0.06) (0.09)

961.2 ± 70.5 1153.2 576.6 28.8 76.9

(1.2) (0.6) (0.03) (0.08)

aMean raid concentration at test termination (~steady state value); bBAF = bioaccumulation factor ‘Raid’ in tissue (µg/g)/feed raid concentration (µg/g).

Table 2. Index of toxicity in rat tissues exposed to insecticide “Raid”.

Indicators Brain IU/L Muscle IU/L Liver IU/L

Control 0.28 ± 0.02 1.15 ± 0.20 2.54 ± 0.1

Glucose-6-phospatase 0.25 ± 0.05 0.35 ± 0.09* 0.15 ± 0.03*

Control 0.22 ± 0.04 0.10 ± 0.01 1.94 ± 0.02

Lactate dehydrogenase 0.19 ± 0.08 0.15 ± 0.11* 0.07 ± 0.08*

*Significantly different P < 0.05 from control.

and palethrin. Figure 1(a) shows the chromatographic

profile of the raid insecticides. Figure 1(b) shows the

chromatographic profile of standards (0.025%). The high-

est peak obtained from “Raid®” extract is most likely

micprothrin. This peak possessed a retention time (9.25)

min. For the identification of raid, co-chromatography

with authentic standards in different chromatographic

conditions was used. Two different peaks were eluted

within 10 min and were well resolved (Figure 1(a)), but

unidentified peak was eluted within a retention time of

early eluting of 2 min. The HPLC analysis failed to de-

tect the unidentified component in “Raid®”. The reason

is not clear, but raid may have undergone a structural

change because of combination, hence different absorb-

ance property [17]. The fine tuning of the mobile phase

composition depended on the choice of the internal stan-

dard. Most internal standard compounds were rejected

because of interfering components. Only micprothrin and

palethrin had acceptable chromatographic characteristics.

The implications are numerous, but simply put that ac-

cidental ingestion of chlorinated hydrocarbon as in

“Raid®” may involve convulsions, collapse and coma

after only brief excitation and ataxia at the onset.

Other workers have contrasted neuritic outgrowth of

chlorpyrifos, diazinon and organophosphate pesticides

including parathion and concluded that all have adverse

effects on brain development and systemic toxicity [18].

In recent study, Walz, [19], shows that glyphosate, the

active ingredient in Roundup, causes birth defects in

frogs and chicken embryos at far lower levels than used

in agricultural and garden applications. The malforma-

tions primarily affected the skull, face, midline and spi-

nal cord. Other independent scientific research has also

found that glyphosate causes endocrine disruption, de-

velopmental and reproductive toxicity, DNA damage,

neurotoxicity and cancer [3,18]. Many of these effects

A. C. ACHUDUME35

(a)

(b)

Figure 1. Chromatograms and absorption spectra from analysis of “Raid” extract in tissue of rat. Analysis of micprothin and

palethrin (a) and standard peak (b).

were apparent at much lower doses than the typical lev-

els of pesticide residues found in food [20,21]. Yet de-

spite the evidence [which strongly support the National

Campaign for Sustainable Agriculture, (a diverse part-

nership of individuals and organizations), cultivating grass

roots efforts to engage in policy development processes

that may result in food, agricultural systems and rural

communities that are healthy, environmentally sound, pro-

fitable, humane and just [22], pesticides continue to be

found in agr icultural produce and lurking in tap water.

A. C. ACHUDUME

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