Pesticide fates in the environment can be affected by its sorption onto the
soil colloids. The batch equilibrium method is frequently used to determine
the soil sorption capacity of pesticide. Determination of soil sorption capacity
of a pesticide is vital in evaluating its fate in the environment. Napropamide
[N, N-Diethyl-2-(1-naphthalenyloxy) propionamide] is a quite polar herbicide
and fairly soluble in water, used to control several grasses and broadleaf weeds
in many agricultural cultivation. The analytical methods for determination of
napropamide concentration are numerous and the most frequently used method are
the Gas Chromatography (GC-NPD) (Antonious and Patterson,
2005; Antonious et al., 2005; Kim,
2004) and the High Performance Liquid Chromatography (HPLC) (Biswas
et al., 2007; Lu et al., 2002). However,
not all researchers have access to these two instruments especially in the less
developed countries. Furthermore, these methods require extraction and cleaning
procedures to remove the unwanted constituents from the soil extract that may
interface with subsequent analysis (Eagle et al.,
A cheaper, less complicated instrument that can be used as an alternative to
the GC and HPLC for the determination of pesticide in soil is an UV-spectrophotometer
(Jakczyk, 1977). The UV-spectroscopy method is routinely
used in the quantitative determination of transition metal ions and highly conjugated
organic compounds in solution. The UV-spectrophotometry method has been successfully
used to measure the concentration of triazine herbicides in soils (Jakczyk,
1977). Rodriguez-Rubio et al. (2006) measured
the 2, 4-D on natural and organic amended soil using the UV-spectrophotometry
method. However, in literature there is no study on the use of UV-spectrophotometry
method to measure napropamide in solution. The objective of this study was to
compare the HPLC with the UV-spectrophotometry in measuring the concentration
of napropamide in soil solution supernatant used for batch equilibrium sorption
MATERIALS AND METHODS
Chemicals: An analytical grade napropamide (99% purity) was supplied
by Sigma-Aldrich (Seelze, Germany). Napropamide is a polar nonionic herbicide
and has an aqueous solubility of 74 mg L-1; vapor pressure of 1.7x10-7
mm Hg; K0C of 700 L kg-1 and degradation half-life of
70 days (Wauchop et al., 1992). The chemical
structure of napropamide is shown in Fig. 1. Due to the low
solubility of napropamide in water, stock solution of napropamide (5000 mg L-1)
was prepared in methanol. Then the stock solution was added to a distilled water
containing 0.01 M CaCl2 and 200 mg L-1 HgCl2
and this solution was later used for our sorption study. Total methanol concentration
in the solution did not exceed 0.1 % (v/v) to avoid any changes in the solution
Napropamide determination using UV-UV-spectrophotometry method: The
UV-visible dual beam spectrophotometer (Shimadzu, UV-1650 PC) was used for determination
of napropamide. The solution containing napropamide was scanned to find the
absorption wavelength of napropamide. The wavelength for maximum absorbance
of napropamide was determined to be 288 nm and this wavelength was used throughout
the study. A blank solution contained 0.01 M CaCl2, 200 mg L-1
HgCl2 and methanol in distilled water but without any napropamide.
The detection and quantification limits for both HPLC and UV methods were defined
as the concentration of napropamide that gave signals 3 and 10 times than the
noise, respectively. The detection limit for UV-spectrophotometer method was
0.05 mg L-1 while and the quantification limit was 0.15 mg L-1.
|| Chemical structure of napropamide
Figure 2 illustrate the UV-spectrophotometer chromatograms
for the background solution with and without the addition of 20 mg L-1
Napropamide determination using HPLC: The concentration of napropamide was determined using a HPLC equipped with a UV detector (Model 1050, Hewlett Packard, USA). The stationary phase was a ZORBAX 300SB-C18 (4.6 μmx250) column. The analyses were done under the following conditions: flow rate of 0.6 mL min-1; mobile phase was water/acetonitrile (45/55 v/v); detection wavelength was 288 nm and injection volume of 20 μL. Limits of detection and quantification were 0.005 and 0.016 mg L-1, respectively. The retention time was 10.2 min. Figure 3 shows the HPLC-UV chromatograms for the background solution with and without 20 mg L-1 napropamide.
||The UV spectra o f (a) background solution and (b) background
solution added with 20 mg L-1 napropamide
||The HPLC chromatograms of (a) background solution and (b)
background solution added with 20 mg L-1 napropamide
Linearity of absorbance against concentration of napropamide: The linearity of absorbance with different napropamide concentrations was determined using 0.5, 1, 5, 10, 15, 20, 30 and 40 mg L-1 of napropamide. Calibration curves for both UV-spectrometry and HPLC methods were plotted and linear regressions were used for both plots.
Recovery, inter and intra-day precision: The recovery study was conducted by using known concentrations of napropamide (5, 10, 15, 20, 25, 30, 35 and 40 mg L-1) in solution containing 0.01 M CaCl2 and 200 mg L-1 HgCl2. The solution was shaken for 24 h, centrifuged at 7000 rpm for 10 min and the supernatant was analyzed using both the HPLC-UV and UV-spectrophotometer.
The concentration of napropamide in the supernatant was calculated from the calibration curve. The percent recovery was calculated based on the initial concentration and the concentration recovered after the shaking and centrifugation processes.
The intra-day precision was assessed using 0.5, 1, 5, 10, 15, 20, 30 and 40 mg L-1 of napropamide and the solution were analyzed seven times on the same day but at different times. For the inter-day precision solution samples of 0.5, 1, 5, 10, 15, 20, 30 and 40 mg L-1 of napropamide were analyzed at different times.
Recovery in spiked soil samples: Five gram of sample was taken from each of four different soil types and placed into 50 mL centrifuge tubes separately and fortified with 0.5 mL of 50 μg mL-1 analytical grade napropamide to give a final concentration of 5 μg napropamide g-1 of soil. The soil samples were left at room temperature for 24 h and then each of them were mixed thoroughly and left for another 1 h after which a 15 mL mixture of methanol and water (3:1 v/v) was added, followed by shaking on a reciprocal shaker at 250 rpm for 2 h. The samples were then centrifuged at 7000 rpm for 10 min. The supernatant was transferred to a round bottomed flask and evaporated on a rotary evaporator to evaporate the methanol. The concentration of napropamide in the remaining water solution was measured by the HPLC-UV and UV-spectrophotometer.
The effects of Dissolved Organic Matter (DOC) on the napropamide absorbance
using UV-spectrophotometer: The DOC was extracted from sandy soils receiving
0, 10, 20, 30, 40, 50, 60, 70 and 80 Mg ha-1 Chicken Dung (CD). The
extracting solution was 0.01 M CaCl2 and the soil: CaCl2
ratio was 1:10 (w/v). The mixture of soil and solution was shaken for 24 h then
it was centrifuged at 7,000 rpm for 10 min. The supernatant was filtered using
0.45 μm membrane filter and the filtrate was labeled as solution of DOC.
The DOC in the filtrate was measured using a total organic carbon analyzer (ANA
TOC Series II). The Dissolved Organic Carbon (DOC) concentration from soils
receiving 0, 10, 20, 30, 40, 50, 60, 70 and 80 Mg ha-1 CD were 3,
7, 11, 15, 19, 26, 30, 35 and 39 mg L-1, respectively. The absorbance
of this solution was measured at 288 nm using the UV-spectrophotometer. Then,
12.6 mg L-1 (the concentration which usually added to soil for controlling
the weeds) of napropamide was added to the filtrate and the absorbance was again
measured using the UV-spectrophotometer. Since DOC interfered with napropamide
absorption at 288 nm, addition of DOC in a solution of napropamide will increase
the UV sorption by the napropamide. Therefore, in this study we assumed that
the UV wave absorption in solution containing DOC only was the same as the napropamide
sorption. The concentration of napropamide in solution was calculated using
the napropamide standard curve of absorbances against its concentrations. The
concentrations of napropamide in the solutions containing different amount of
DOC and the same solution of DOC but with the addition of 12.6 mg L-1
napropamide were plotted against the amount of chicken dung added to the soil
Fig. 4. Finally, the concentration of napropamide in DOC solutions
containing 12.6 mg L-1 napropamide was calculated by the differences
between the concentrations of napropamide in DOC solutions containing 12.6 mg
L-1 napropamide and the DOC solutions without napropamide.
Determination of soil characteristics and napropamide sorption study:
Four different soils were used for soil sorption study. Soils were labeled as
soil A, B, C and D (Table 1). The soil Total Organic Carbon
(TOC) was determined using the Walkley and Black method (Nelson
and Sommers, 1982), particle size distribution was determined by the pipette
method (Gee and Bauder, 1986) Cation Exchange Capacity
(CEC) was determined using 1 N ammonium acetate and soil pH and Electrical Conductivity
(EC) were determined using a soil to double-distilled water ratio of at a 1:2.5.
The batch equilibrium method was used to determine napropamide sorption in
soils. Two grams air-dried soil from each soil was added to 20 mL of background
solution containing 0, 5, 10, 15.0, 20, 25, 30, 35 and 40, mg L-1 napropamide.
The soil suspensions were shaken in 50 mL centrifuge tubes for 24 h in a rotating
shaker and which the samples were centrifuged for 10 min at 7000 rpm. Ten milliliter
of supernatant was removed to determine the napropamide concentration by the
HPLC-UV and UV-spectrophotometer. We have previously determined that equilibrium
time for all soils was reached within 24 h and no biodegradation occurred because
of HgCl2 efficiently inhibited the microbial growth.
||Plots of napropamide concentrations in solution containing
DOM extracted from soil receiving different rates of chicken dung and the
same solution added with 12.6 mg L-1 napropamide
|| Percentage of recovery by HPLC-UV and UV-spectrophotometer
| Values are Mean±standard error
Mass balance determination: The soil samples used for the sorption study were retained to determine the mass balance of napropamide in the sorption studies. The napropamide that remained in the soil sample after decantation of supernatant in the sorption study was extracted with 6 mL solution of methanol and water (3:1; v/v). The sample was shaken for 2 h and then centrifuged at 7000 rpm for 10 min. The methanol in the solution was evaporated then concentration of napropamide in the water was measured by the HPLC-UV and UV-spectrophotometer.
RESULTS AND DISCUSSION
The results showed that the linear regressions for absorbance/peak area for
both methods were high, exceeding 99.99% (graph not shown). This indicated that
both methods were highly reliable at concentration range between 0 to 40 mg
L-1 of napropamide which are the range usually used for napropamide
sorption study (Auger et al., 2000). The UV spectra
of background solution with and without 20 mg L-1 napropamide are
shown in Fig. 2 while the HPLC chromatograms are shown in
Fig. 3. The retention time of napropamide by the HPLC was
10.2 min and running time for each sample took 15 min whereas determination
by UV-spectrophotometer took only a few seconds. Therefore, UV-spectrophotometry
method is much faster than HPLC-UV method. Determining of napropamide using
HPLC-UV needs organic solvent (HPLC grad) as flushing (e.g., methanol) and mobile
phase (e.g., acetonitrile). Moreover, HPLC is more expensive than the UV-spectrophotometer.
Table 1 shows the percent recovery of napropamide at different
concentrations from the background solution using both the UV-spectrophotometer
and HPLC. The results indicated that the recovery of napropamide at all concentrations
were satisfactory for both UV-spectrophotometer and HPLC. However, the measurement
of napropamide recovery was from background solution without any interference.
When separation of target compound from impurities in sample matrix is needed,
HPLC offer better separation and therefore better sensitivities (Baskaran
and Bolan, 1998). In order to examine the matrix effect on the UV absorption
by napropamide using the UV-spectrophotometer, we carried out an experiment
to determine the effect of DOC in the background solution on napropamide absorption.
The UV absorption by the DOC at 288 nm increased linearly with the increasing
amount of DOC Fig. 4. The UV absorption of background solution
containing both the DOC and napropamide (12.6 mg L-1) also increased
linearly with the increasing amount of DOC. The UV wave absorption by the solution
without napropamide was lower than the solution with napropamide, at the same
DOC concentration. We converted the UV wave absorbance by the solution containing
DOC only to concentration of napropamide in order to calculate the concentration
of napropamide in solution of DOC plus napropamide. When we calculated the concentration
of napropamide in the solution of DOC plus napropamide by subtracting the value
obtained from the top plot in Fig. 4 with the value obtained
from the bottom plot, the mean concentration of napropamide that we got was
very close to 12.6 mg L-1. The results showed that the matrix effect
can be easily corrected by subtracting the absorbance of a sample with the absorbance
of a blank solution obtained by extracting similar soil but without napropamide.
We did not encounter any other UV wave absorption interferences at 288 nm other
than the DOC absorption.
The intra and inter day precisions by both methods are shown in (Table 2). The average Standard Deviation (SD) for intra day precision was 0.25 and 0.2 for UV-spectrophotometer and HPLC-UV, respectively which indicated the measurements using the UV-spectrophotometer method were as reproducible as the HPLC method. The average of SD for inter assay precision was 0.7 and 0.45 for UV-spectrophotometer and HPLC-UV, respectively. Although the SD for inter day precision of the UV-spectrophotometry method was slightly higher than the HPLC method, it was way below the acceptable value. The results indicated that the napropamide measurement using the UV-spectrophotometry method was consistent.
The percent recoveries of napropamide from spiked soils are shown in (Table 3) and the properties of the soils are shown in (Table 5). The percent recoveries for both methods were quite high, the lowest was 88.2% and the highest was 95.4%. The percent recoveries for both methods decreased with the increasing amount of clay and organic content of soil. Stronger sorption of napropamide in soils with high amounts of organic matter and clay may be the reason for lower recoveries in those soils.
The napropamide in supernatant solutions measured at equilibrium for the sorption
study determined using HPLC-UV and UV-spectrophotometer are shown in Fig.
5(a-d). There was no difference in napropamide concentrations
at all concentrations of napropamide added to the soil solution mixture between
the two methods at p<0.05 for all soils that we studied. At any given napropamide
concentration in the soil solution, the concentration of napropamide in the
supernatants decreased with soil types according to the following trend; soil
A>soil B>soil C>soil D. Again, the reason for the decreasing amount
of napropamide can be attributed to the differences in clay and organic matter
contents of the soils as well as the CEC. The results showed that UV-spectrophotometer
was a reliable method to determine napropamide concentration in the batch equilibrium
method for different soils which have different amount of OM and clay content.
The result is in agreement with the result of Rodriguez-Rubio
et al. (2006) which showed that the 2, 4-D concentration in soil
solution can be measured by UV-spectrophotometry method. However, Baskaran
and Bolan (1998) reported that the presence of water soluble organic matter
interfered with the measurement of the same pesticide using UV-spectrophotometry
method. We have shown that this can be corrected easily using a dual beam UV
spectrophotometer which was not done by Baskaran and Bolan
The napropamide mass balance calculations which involved the initial amount of napropamide added to the soil solution mixture, its concentration in the supernatant and the amount adsorbed by the soil are shown in Table 4. The percent recoveries of napropamide in the four soils are calculated from the mass balance and presented on the same (Table 4). In all four soils that we studied, the percent recoveries of napropamide for the sorption study using the UV-spectrophotometry method were as good as the HPLC-UV method. Similar to the sorption trend, percent recoveries of napropamide from soils with higher clay and organic matter contents were lower probably because napropamide was strongly adsorbed in those soils and were not completely released by extraction with the methanol/water mixture.
|| The inter and intra day precision of HPLC-UV and UV-spectrophotometer
|Values are Mean±standard deviation
|| The recovery of napropamide in spiked soils determined by
HPLC-UV and UV- spectrophotometer
| Values are Mean±standard error
|| Percent recovery of napropamide in the sorption study for
||The napropamide concentration in soil solution supernatant
used in the sorption study for different soils using HPLC-UV and UV-spectrophotometer
plotted againts the initial
The results showed that the percent recoveries of napropamide in background solution, spiked soil sample and in the sorption study were quite high for both methods. The intra and inter day precision indicated that napropamide measurements using both methods were highly reproducible and stable at different times of the day as well as at different days. There were no differences between napropamide concentration in the supernatant measured by UV-spectrophotometry and HPLC method. Although DOC interfered with napropamide UV wave absorption, it can be easily corrected on the dual beam UV-spectrophotometer. As the UV-spectrophotometry method is fast and a reliable method to determine napropamide concentration in the supernatant of batch equilibrium sorption study, it can be used as an alternative to the HPLC method.
This study was supported by Research University Grant Scheme from Universiti Putra Malaysia under Grant No. 01/01/0010RU. The first author thanks Universiti Putra Malaysia for financial support (Graduate Research Fellowship) throughout his study period.