The behavior of pesticide in agricultural produce is of great importance, since
the disappearance, persistence or partial transformations of such a compound
determine its usefulness or its potential effects to our environment (Bergmann
et al., 1989; Waxman, 1998), Currently organophosphates
(OP), carbamates and pyrethroids are mostly used while organochlorine (OC) insecticides
have been banned because of their toxicity, persistence and bioaccumulation
in the environment (Molto et al., 1991). Carbamates
and pyrethroids are of limited persistence as compared with organophosphates.
However, knowledge of withholding period becomes important even for less persistent
insecticides, specifically in fruits and vegetables since these crops are harvested
shortly after pesticide application. The problem of food contamination with
pesticide residues is a cause of concern for almost everyone and everywhere.
Many of the developed countries have established regular monitoring programmes
(Reed et al., 1987). These programmes determine
the contamination levels in food products and identify those possible occasions
in which pesticide residues exceed their tolerance levels due to incorrect agricultural
practices. Pesticide residues above the tolerance limits (MRL) in the crop at
harvest are a cause of great concern globally and nationally. The gravity of
the problem of residues is augmented by untimely, uneconomical and unscrupulous
spraying of pesticides. These residues make food commodities hazardous for human
consumption and export. They also pollute the environment.
There is a lack of published data in Bangladesh for the fate of the insecticide
on field-grown eggplant fruits and in the processed products. In addition,
local farming practices concerning application of pesticides and subsequent
harvest of treated crops have also raised concerns over the possibility
of excessive residues on crops sold in local markets. Therefore, the present
research was designed to study the residues of pesticides in eggplant
fruits. Emphasis on the safety periods for this insecticide in the tested
vegetable was considered.
Pesticide residue analysis can be carried out efficiently by various biological
and chemical techniques (immunoassay, thin layer chromatography and capillary
electrophoresis, etc.) but the most popular methods for this type of analysis
are Gas Chromatography (GC) and Liquid Chromatography (LC). A critical review
of literature showed that different solvents such as n-hexane, petroleum ether,
methylene chloride and acetone or ethyl acetate have been used for extraction
of pesticide residue from fruits and vegetables (Kovacicova
et al., 1975; Kearney and Philip, 1978). As
more polar pesticides, such as organophosphates and phenoxyacetic acid, came
into use, more polar solvents, such as chloroform, acetone, acetonitrile and
methanol were found to be good (Luke et al., 1981).
Kadenezki et al. (1992) and
Startin et al. (2000) found that ethyl acetate proved to be a good
solvent as compared to other solvents for the extraction of residues of several
pesticides from fruits and vegetables because its polarity is high and it is
a less volatile and thermally labile compound. In the present study, for the
extraction of pesticide residues from fruit and vegetable samples, the
Anderson and Palsheden (1998) method was followed with little modification
because it is faster, less laborious, friendly to environment and less expensive.
And a florisil clean up method was adapted in our analysis being necessary to
modify the preconditioning steps. The determination was carried out by reversed
phase HPLC with UV detection modifying the parameter to achieve a good separation
between the front and the pesticide peak.
Therefore, this study aimed to throw light on how to:
||Develop simplified conditions for the RP-HPLC analysis
to determine the residue levels of various insecticides on eggplant.
|| Study the rates of residue dissipation of three organophosphorus
insecticides (Diazinon, Malathion and Sumithion) after application
to eggplants in field experiments under agro climatic conditions of
MATERIALS AND METHODS
Sample material: For the present study, field experiments were
conducted during the spring of 2005 in an experimental field in the farm
of the Entomology Division, Bangladesh Agricultural University, Mymenshing.
Three pesticides under this study were sprayed on the fields when the
eggplants were matured for harvest. Untreated control plots were included
for each treatment. The plants were sprayed with Diazinon at the recommended
rate of 1.7 L ha-1, double of the recommended dose 3.4 L ha-1
and half of the recommended dose 0.85 L ha-1. Similarly six
samples for each of the pesticide Malathion and Sumithion were sprayed
at the same recommended dose of 1.12 L ha-1, double of the
recommended dose 2.24 L ha-1 and half of the recommended dose
0.56 L ha-1, respectively. All agricultural management practices
were made as usually practiced in commercial production of eggplant. Fruit
samples were randomly collected (500 g were sampled per replicate).
For each treatment, fruit samples were harvested on day 1 and 5 following
the pesticide application. The collected representative samples were placed
in plastic bags and frozen at -15°C until insecticide residue analysis.
Chemicals and reagents: The organic solvents, acetonitrile, ethyl
acetate, hexane, diethyl ether used were HPLC grade and were purchased
from E. Merck. Technical grade pesticide standards were obtained from
Bangladesh Agricultural Research Institute (BARI) with a purity of 95-99%.
The standards were stored in a freezer at -15°C. Ultra high quality
water was obtained from Milli-Q water purification system (Millipore,
Bedford, MA, USA). Milli-Q Water and acetonitrile were degassed by vacuum
suction. All samples and solvents were filtered through Millipore membrane
filters (Polysulfone membrane and 0.45 μm pore size) before injection
on the column.
Likewise anhydrous sodium sulphate for residue analysis, 12-60 mesh,
was maintained at 300°C overnight. Florisil was activated at 300°C
overnight to get rid of any moisture adsorbed into it. A source of pure
nitrogen was used for evaporation to dryness in the extraction step.
Standard preparation: Stock solutions of Diazinon, Malathion and Sumithion
were prepared in acetonitrile and stored at -15°C. For the analysis of each
type of pesticides, the matrix-matched working standard solutions were prepared
immediately before injection. After the extraction and clean up procedures,
aliquots of a solution containing the target pesticides were added to the resulting
untreated sample matrix (Erney et al., 1993) to
give standards of the required concentration.
Extraction: Each vegetable sample (75 g) was homogenized by the use
of a kitchen blender. The blended material was extracted with ethyl acetate
(Anderson and Palsheden, 1998) (150 mL), anhydrous sodium
sulphate (60 g) in a conical flask using an Ulta-Turrax (IKA-WERK) for 4-5 min.
The content was allowed to settle down for about one hour and the ethyl acetate
extract was then filtered through a Buchner-funnel fitted with a filter paper
covered by 20 g of anhydrous sodium sulphate. After filtration, the extract
was evaporated to dryness and redissolved in 5 mL of Hexane and were then evaporated
using rotary vacuum evaporator until the volume was about 1.0 mL. The extract
was then transferred to graduated test tube and the final volume was adjusted
to 1.0 mL by adding hexane.
Isolation of residues/cleanup: All samples were cleaned up by Florisil
column before analysis by High performance liquid chromatography. According
to Lopez et al. (1989), florisil (20 g) in hexane
was allowed to settle in a chromatographic column (45 cm x 20 mm, i.d.) by tapping
the column. To the top of the florisil, a layer of 1 to 2 cm deep anhydrous
sodium sulphate was added. Then the column was pre-eluted with 60 mL of hexane
and the liquid was discarded. Concentrated eggplant extract (1 mL) in hexane
was transferred to the column. Then the column was eluted with 200 mL of 15%
Diethyl ether in hexane for the analysis of Diazinon. The organic phase thus
obtained was evaporated to complete dryness in a rotary evaporator at a temperature
of 40-45°C. The dry residue was redissolved in 1 mL of acetonitrile. For
the cleanup of the sample extract for Malathion the eluting solvent was 200
mL of 15% Diethyl ether in hexane followed by 200 mL of 50% Diethyl ether in
hexane. The elution was performed with 200 mL of 4% acetone in hexane to collect
the cleaned sample extract of Sumithion treatment.
HPLC systems: High Performance Liquid Chromatography was performed
on columns, SUPELCO Discovery Reversed Phase C18 (of 25 cm x 4.6 mm i.d.,
particle size 5 μ) using a Shimadzu SCL-10AVP, version 5.22 high
performance liquid chromatography equipped with a variable length UV/Visible
detector (SPD 10 AVP). The samples were injected manually through a Rheodyne
injector. HPLC working conditions were, Binary gradient, Eluent solvent
(Acetonitrile: water; 70:30) and flow rate 0.8 mL min-1 and
injection volume (loop size) 100 μL and the wavelength of the UV/visible
detector was fixed at 254 nm for the residual analysis of Diazinon and
230 nm for the analysis of other two pesticides (i.e., for Malathion and
Identification and quantification: The identification of target
pesticides were accomplished on the basis of the retention times of the
analytes by searching in the appropriate retention time windows. Quantification
was performed by external calibration. Sample analysis were run in triplicate
and in most, relative standard deviations of less than 10% were achieved.
Recovery: To examine the efficacy of extraction and clean up,
recovery studies were performed. Three samples for each type of pesticide
treatments were spiked with known concentration of the pure insecticide
standard solution and extraction and clean-up were performed as described
earlier. The concentration of each pesticide in the final extracts was
Statistical analysis: External calibration and recovery tests
were performed. The residue results were the means from three replicates
of each treatments and all data were analyzed using simple descriptive
statistics such as means, standard deviations, using Kaleidagraph version
4 for windows.
RESULTS AND DISCUSSION
Limit of detection: Limit of Detection (LOD) was calculated from
the peak intensity at 0.1 mg kg-1 and blank levels in recovery
tests. LOD was defined as S/N > 4 so that it is in the linear range
of the standard calibration. The LOD of Diazinon, Malathion and Sumithion
was 0.02 mg kg-1.
Recovery: Table 1 shows the recoveries which
were obtained by triplicate analysis of eggplant sample spiked with each
type of pesticide at one fortification level. These values were satisfactory
for residue analysis and are of the same order as those obtained by using
more complicated methodologies. Residues were corrected according to the
average of recovery.
Extraction/clean up: Sample preparation presents the advantages
of making extraction and liquid partitioning in organic phase in a similar
step. The use of blender allows intensive mixing, creating micro droplets,
which increase exchange surface. Thus, laborious conventional liquid-liquid
partitioning can be substituted by a rapid technique, since few minutes
are enough to obtain the final organic phases.
Large amount of coextractants results in complex chromatograms which
are difficult to interpret. Florisil sorbents have been used to eliminate
non-polar plant coextractants rapidly and efficiently. After passing the
extract through florisil column, most of the plant pigments were removed
and the eluent became pale yellow. The final extract contains very few
interfering peaks and the main benefit is lower limit of detection.
Calibration curve: The concentrations of the calibration levels
were selected for each pesticide according to the maximum residue limits.
The linearity of the calibration curve was studied and better, quantitation
results were obtained where peak area rather than peak height was considered.
The calibration data obtained are shown in Table 2.
As shown in Fig. 1, good linearity of response was found
for all pesticides at concentrations within the tested interval, with
linear determination coefficients higher than 0.990.
|| Summary of recovery data for each pesticide
||Retention Times Windows (RTWs) and typical calibration
parameters of the method in eggplant matrix
|| Residues of Diazinon, Malathion and Sumithion on eggplant
at various intervals after treatment
|AThe results are expressed on fresh weight
|| HPLC calibration curves for Diazinon, Malathion and
Sumithion prepared in crude eggplant extracts
Residues: The pesticide residues present in the eggplant samples
were identified and quantified with reference to standard pesticides.
The residue levels of pesticides found at different intervals on different
samples are listed in Table 3.
It was found that Malathion and Sumithion was present below the Maximum Residue
Limit (MRL) value (0.5 mg kg-1) (Anonymous, 2001;
Inger, 2004) in all the samples except one, which was sprayed
with double of the recommended dose and was harvested after 1 day of treatment.
Diazinon was detected and the amount was above the MRL value (0.02 mg kg-1)
(Anonymous, 1993) in the entire six samples.
The initial residues of Sumithion obtained by HPLC on the sample sprayed
with double of recommended dose and harvested on the following day was
2.39 mg kg-1. After 5 days, it was dissipated to 0.02 mg kg-1,
thus representing a loss of 99%. The samples did not contain any detectable
residues 5 days after application. However, the initial residues of Malathion
on samples treated with double of the recommended dose, analyzed by HPLC,
dissipated from 0.80 to 0.05 mg kg-1 (93.75%) within 5 days.
Subsequent samples had residues below MRL value. This indicates that the
detection of Malathion is not easy as it may rapidly vanish from the surface
of treated plants because of chemical and enzymatic decomposition and
evaporation and has low persistence in the environment. In plant`s tissues,
Malathion may intensively hydrolyze by phosphatases and carboxyesterases
along the P-X bond and ester to form dimethyl phosphorothiotioic and other
acids having a low toxicity.
But, in the case of Diazinon, residues were found above MRL value (0.02
mg kg-1) in all the samples, even at the sample treated with
half of the recommended dose. After 5 days of sampling, the dissipation
of Diazinon was only 83% of its initial value at double of the recommended
dose. The data indicates that the residues of Diazinon in eggplant decrease
with the longer time interval of sampling after spraying. That is the
deposition seemed to decrease with the increase of Pre-Harvest Interval
(PHI) showing lower persistence.
From the results, it was revealed that Malathion and Sumithion were safer
than Diazinon. It was found to be more persistent than Malathion and Sumithion.
Having the same treatment and similar conditions Diazinon showed more
residues even from lower dose of pesticide. Residues of these pesticides
showed a gradual declining trend and reached below the MRL between 5-12
days, depending on the chemicals nature of the pesticide. Based on the
data generated on the residues of these pesticides at different harvest
intervals, safe harvest period for the above mentioned pesticides may
be determined. Commercial eggplant samples obtained in local markets of
Bangladesh were analyzed by the proposed method. None of the studied pesticides
Since the organophosphorus and pyrethroids pesticide residues are not
degraded into non-toxic products in a short period of time, they persist
in the vegetable. Therefore, it is necessary to analyze more vegetable
samples to find out whether the pesticide residues are within the Acceptable
limit of Daily Intake (ADI) or not.
When the pre-harvest intervals between treatments and harvest are not
respected by the farmers, the risk of having higher pesticide levels is
not negligible. In this case, the higher levels of pesticides can involve
considerable economic losses if the maximum residue limits established
by FAO/WHO are surpassed. In conclusion, the present study may be useful
for establishing MRL and assessing the amount of pesticide residues in
vegetables under Bangladesh field conditions and suggests the need of
implementation of these safety intervals before harvesting and marketing
such crop fruit. Moreover, harvesting these vegetables at suitable intervals
has to be strictly considered by the farmers.