- Email: [email protected]
A spectrofluorimetric study of the binding of carbofuran, carbaryl, and aldicarb with dissolved organic matter
Analytica Chimica Acta 373 (1998) 139±151
A spectro¯uorimetric study of the binding of carbofuran, carbaryl, and aldicarb with dissolved organic matter Feng Fang1,a, So®an Kananb, Howard H. Pattersona,b,*, Christopher S. Cronana a
Graduate Program in Ecology and Environmental Science, University of Maine, Orono, ME 04469, USA b Department of Chemistry, University of Maine, Orono, ME 04469, USA Received 16 March 1998; received in revised form 20 May 1998; accepted 26 May 1998
Abstract This study examined the binding of carbamate pesticides with dissolved organic matter (DOM) using ¯uorescence quenching and synchronous scan ¯uorescence spectroscopy (SSFS). Fluorescence spectra of the three pesticides were characterized as follows: carbofuran and carbaryl ¯uoresce at 305 and 330 nm, respectively, upon excitation at 276±279 nm, whereas, aldicarb shows broad emission at 350±380 nm upon excitation at 326 nm. A ¯uorescence quenching technique was used to obtain conditional binding constants for the carbamate pesticides with Aldrich humic acid under ®xed conditions of 228C and pH 6. The binding constant of carbofuran with humic acid is greater than the binding constants of both carbaryl and aldicarb. Estimates were also obtained for the binding of carbofuran with DOM samples from a coniferous forest soil O horizon, a deciduous forest soil O horizon, a sedge marsh wetland, and a stream in the drainage sequence and their molecular weight (MW) fractions. Those conditional binding constants were used to predict the potential transport of carbofuran in the drainage sequence. When binding constants and DOM concentrations were both taken into account, it was found that DOM from the coniferous forest O horizon had the largest capacity to bind and to transport carbofuran in the drainage sequence. SSFS was used to probe the binding mechanisms of DOM with carbofuran. Overall, the potential mobility of carbofuran in the upland± wetland±stream drainage sequence was signi®cantly enhanced via binding with DOM. # 1998 Published by Elsevier Science B.V. All rights reserved.
1. Introduction Pesticides are one of the major organic contaminants in the environment. The carbamate pesticides, carbofuran, carbaryl, and aldicarb are highly toxic to cold and warm water ®sh, freshwater invertebrates, and to birds [1]. Carbofuran is commonly used in *Corresponding author. Tel.: +1-207-581-1178; fax: +1-207581-1191; e-mail: [email protected] 1 Current address: 500 Pillsbury Dr. SE, Civil Engineering Building, University of Minnesota, Minneapolis, MN 55455
potato and rotation crop farms to control Colorado potato beetle, ¯ea beetles and leafhoppers [2]. It acts as a cholinesterase inhibitor after insects contact a treated surface and/or ingest treated plant tissue. Carbaryl is used in cotton, fruit, forests, nuts, and other crops, and is inherently toxic to humans by skin contact, inhalation, and/or ingestion [3]. Aldicarb is one of the most acutely poisonous pesticides ± the oral LD50 value for rats is 0.95 mg/kg [4]. It is widely used to control mites, nematodes, and aphids in cotton and soybean crops. The chemical structures of carbofuran, carbaryl, and aldicarb are given.
0003-2670/98/$19.00 # 1998 Published by Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00392-4
140
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Understanding the transport and fate of pesticides in the environment is of great importance for their application and regulation. The interaction between dissolved organic matter (DOM) and carbamate pesticides not only changes the solubility and mobility of the pesticides in the environment, but also affects the photodegradation and hydrolysis rate of the pesticides [5]. Therefore, it is important to understand the binding tendency between DOM and carbamate pesticides if the transport and fate of these pesticides in the environment are to be predicted. A classical approach for examining the binding reaction of a pesticide with DOM is to measure the concentration of the free pesticide before and after its binding with DOM in an aqueous reaction system. From the difference between the two concentrations, an association constant can be calculated. As a requirement for this approach, an accurate analytical method for detecting the free pesticide concentration is essential. In addition, before the concentration of free pesticide is determined, the free pesticide must be separated from the DOM-bound pesticide, because the presence of DOM and DOM-bound pesticide in solution can cause errors in the measurement. The drawback of separation processes is that they may be incomplete or may disrupt established equilibria and lead to inconsistent estimates of binding constants. Fluorescence quenching, however, does not require that absolute concentrations of contaminants be known, and does not require a separation step. Therefore, ¯uorescence spectroscopy has unique advantages in the study of contaminant binding to DOM [6±9]. In this paper we report the ¯uorescence properties of the carbamate pesticides (carbofuran, carbaryl, and aldicarb), and demonstrate that the ¯uorescence
quenching technique can be used to study the binding af®nity of the three carbamates with a sample of Aldrich humic acid. We also compare the binding interaction of carbofuran with DOM separated from an upland±wetland±stream sequence using ¯uorescence quenching and synchronous scan ¯uorescence spectroscopy (SSFS). We believe this is the ®rst paper to show that ¯uorescence spectroscopy can be used to probe pesticide±DOM interactions. 2. Experimental 2.1. Reagents Carbofuran, carbaryl, and aldicarb crystals (purity: 99%) were purchased from Chem Service and were used as received. The humic acid was purchased from Aldrich Chemicals. Analysis performed at Aldrich showed the sample to be 40.7%C. A concentrated pH 6.0 phosphate buffer was prepared according to a modi®ed method from the CRC Handbook of Chemistry and Physics [10]. Fifty ml of 1.00 M KH2PO4 and 5.6 ml of 1.00 M NaOH were mixed and diluted to 100 ml in a volumetric ¯ask. This solution was diluted to a 1 in 50 ratio to adjust all of the carbamate solutions, distilled and deionized H2O blanks, and DOM dilutions to a pH of 6. Distilled and deionized water (dd H2O) was made by Barnstead ion exchange cartridges. Carbofuran, carbaryl, and aldicarb crystals were dissolved in methanol (HPLC grade, EM Science) to make stock solutions of 3.55910ÿ4 M in a 25 ml volumetric ¯ask. 1.91 ml of the stock solutions were transferred to three 50 ml volumetric ¯asks into which 1 ml of the pH 6 phosphate buffer had been
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
141
added. The ¯asks were then ®lled to the mark with dd H2O, giving a set of pH 6.0 aqueous carbamate solutions of about 1.3610ÿ3 M for the quenching experiments. Although the hydrolysis half-life of these pesticides at 258C and pH 6 is very insigni®cant (see Extension Toxicology Network. World Wide Wet site: http://ace.ace.orst.edu/info/extoxnet/pips/ghindex.html), the solutions to be studied were always made within 1 day before the quenching experiments and were stored in the dark to minimize hydrolysis. 2.2. Environmental DOM sample collection and preparation Environmental DOM samples were collected from sites at the Penobscot Experimental Forest, Bradley, Maine, in October, 1996. A sampling ®eld consisting of two types of upland forests (deciduous and coniferous), a sedge marsh wetland, and a stream draining the forests and wetland was selected for the upland± wetland±stream environmental drainage gradient. A shovel was used to collect 10±15 cm thick samples of organic horizon material from the forest ¯oors in each forest stand. The wetland sample was collected from a sedge marsh bordering Blackman stream. A hole of about 20 cm in diameter and 10 cm in depth was dug using a shovel. The hole promptly ®lled with sediment laden ground water which was collected into one-liter Nalgene1 sample bottles. Approximately 3 l of water was taken from Blackman stream to represent surface water in the drainage system. One-liter Nalgene1 sample bottles were submerged completely into water when sampling. Samples were delivered to the laboratory immediately after collection and were stored in a refrigerator at a temperature of 48C. To obtain DOM solutions and their molecular weight (MW) fractions from these raw samples, the procedures shown in Fig. 1 were followed. For ultra®ltration, Dia¯o1 ultra®lters and a model 8400 Amicon1 stirred ultra®ltration cell were used. Ultra®ltration membranes were type YM10 and YM1, which have nominal MW cutoffs of 10 000 and 1000 daltons, respectively. YM membranes have exceptionally low non-speci®c protein binding properties and are recommended where maximum solute recovery is of utmost importance [11]. The operating pressure for the YM10 and YM1 ultra®ltration membrane was 55 psi N2 (3.7 atm) and 65 psi N2 (4.4 atm),
Fig. 1. DOM sample processing protocol (the coniferous and deciduous forest floor raw samples started from the first step with water extraction; the sedge marsh wetland sample started from Whatman 41 filter paper filtration; and the Blackman stream sample started from VacuCap1 filtration).
respectively. To avoid the breakdown of larger-molecular-size DOM solutes, the ®ltrate volume was never allowed to exceed 90% of the initial total volume [12]. To name each DOM sample and its MW fractions, Dec (or D), Con (or C), Sedge (or S), and Black (or B) were used to represent samples of deciduous forest soil DOM, coniferous forest soil DOM, sedge marsh DOM, and Blackman stream DOM, respectively. `P' and `R' were used to represent samples that either passed through or were retained on an ultra®ltration membrane, respectively. For instance, SR10 is the name for a sedge marsh DOM sample that has a nominal MW larger than 10 000. Three low pressure chromatography columns (internal diameter: 4.5 mm, length: 28 cm) ®lled with
142
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Rexyn 101 H form beads were used to perform cation exchange on the DOM samples. A model 700 Total Organic Carbon Analyzer (detection limit: 0.5 ppm) from O.I. Corporation, College Station, Texas, was used to conduct DOC (dissolved organic carbon) analysis. Instrumental baselines were set by using carbon-free water (0.1 ppm total carbon) obtained from the Sawyer Environmental Laboratory at the University of Maine. Two potassium hydrogen phthalate standard DOC solutions 20 and 50 mg C/l (ppm) were used to standardize the instrument. Sample Black (Blackman Stream sample after VacuCap1 ®ltering) was too dilute (DOC 8.5 ppm) for a ¯uorescence quenching experiment. The YM1 ultra®ltration membrane was employed to concentrate this sample. 2.3. Fluorescence spectra of carbamate pesticides and DOM Fluorescence data were collected on a computerdriven model QM-1 ¯uorescence spectrometer from Photo Technology International (PTI). The instrument was equipped with a model UXL - 75 XE xenon short arc lamp from Ushio. The ¯uorescence spectrometer had a cuvette holder with a built-in magnetic stirring setup. The instrument had two excitation monochromators, and, thus, had two excitation slits. In the carbofuran and carbaryl experiments the excitation wavelength was set at 279 and 276 nm, respectively and the emission scan was from 290 to 350 nm. In the aldicarb experiments the excitation was ®xed at 326 nm and the emission scan was from 340± 550 nm. Both excitation and emission slits were set at 5 nm. Fluorescence data collection and analysis TM were conducted with Oscar software from PTI. Fluorometry quartz cuvettes from Whatman1 with a light path length of 10 mm were used for both ¯uorescence and absorption measurements. Emission ¯uorescence spectra of each DOM sample were measured and collected under the same experimental conditions as the carbamate emission spectra. Since in this research, the carbamate pesticides were the ¯uorophore and DOM was the quencher, the ¯uorescence of DOM was the background component of the total ¯uorescence of carbamate after DOM was added to the solution. To obtain the quenched ¯uorescence intensity of carbamates, the ¯uorescence of DOM was
simply subtracted from the total ¯uorescence as part of the data analysis process [7]. 2.4. Absorption spectra of DOM solutions Absorption data were collected on a computerdriven model DU1 640 spectrophotometer from Beckman Instruments. Absorption of DOM solutions was measured from 260 to 400 nm for the inner ®lter effects correction. 2.5. Fluorescence quenching measurements Five dilutions of Aldrich humic acid were prepared after adjustment to pH 6 with phosphate buffer solution. Also, six dilutions of each of the environmental DOM samples, adjusted to pH 6 with phosphate buffer, were prepared and were stored in uniform glass vials from Fisher Science. For unfractionated and R10 DOM samples, a dilution series was prepared with DOC concentrations of 4, 8, 12, 16, 20, and 24 ppm. For the P10R1 samples and the CP1 sample, because of their smaller binding ability, the above concentration series was not high enough to signi®cantly quench the ¯uorescence of carbofuran to give Stern±Volmer plots with good linearity. Therefore, a DOC concentration series of 6, 12, 18, 24, 30, and 36 ppm, or 8, 16, 24, 32, 40, and 48 ppm, depending on the maximum concentration available from the stock DOM samples, was used instead for the P10R1 samples and the CP1 sample. A 2.00 ml aliquot of each dilution was pipetted to a cuvette. An absorption scan from 260 to 350 nm was recorded. The cuvette was then transferred to the ¯uorescence spectrometer and the background ¯uorescence of DOM was measured. A 25 mm Te¯on magnetic stirring bar from Thomas Scienti®c1 was put into the cuvette followed by 0.50 ml aliquot of the carbamate working solution. The cuvette was immediately put back to the cuvette holder in the ¯uorescence spectrometer and stirring was started. After 3 min the stirring was stopped. The solution was allowed to stand quiescent (without stirring) for an additional 1 min before the ¯uorescence spectrum was recorded. Since carbamates are strongly photodegradable under ultraviolet light [5], the shutter of the excitation monochromator was closed to protect the carbamate from ultraviolet light
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
during the 4 min reaction time. To improve the signal to noise ratio, the ¯uorescence signals were recorded twice for each dilution and average values were computed for the two scans. The average values were used to carry out the data analysis. The blank (Fo) was run twice: once in the beginning and again at the end of each dilution series. This step was to insure an accurate value of Fo because this number would be used six times in the Ksv calculation and the Stern±Volmer plot. Also, the blank replicates were run in such a manner-in the beginning and at the end of each dilution series-that the ¯uctuation of the ¯uorescence spectrometer within a series of dilutions could be cancelled out to some extent. Between two dilutions of each DOM sample, cuvettes were always rinsed with dd H2O at least six times and then were rinsed twice with acetone (>99.5%, EM Science). The next dilution was not added until acetone was totally dried. Kimwipes1 low-lint paper wipers were used to clean impurities and ®ngerprints from cuvette outside walls. Between two DOM samples, cuvettes were soaked in washing acid (K2Cr2O7 dissolved in concentrated sulfuric acid) for several minutes to clean out any carbamate and DOM residues on the cuvette walls. Before starting the dilution series of the next sample, background corrections for the ¯uorescence spectrometer and the UV-Vis spectrophotometer were made again to insure the accuracy of the data for each dilution series. Three replicates were run for each sample. The average of the binding constant results of these three replicates was used as the ®nal result. The standard deviation for each sample was also calculated from the three replicates.
143
Table 1 DOC concentrations in water samples and soil extracts used in this study Sample
DOC (mg C/l)
Coniferous O horizon Deciduous O horizon Sedge marsh soil water Blackman stream
173.6 31.8 32.3 8.5
increases. Fig. 3 shows the Stern±Volmer plots for the three carbamate pesticides with Fo/F plotted against increasing Aldrich humic acid concentrations after the correction of inner ®lter effects. The binding constant for carbofuran with Aldrich humic acid (8.75104 l/kg) is greater than the binding constants for aldicarb (7.21104 l/kg) and carbaryl (0.96104 l/kg). The second phase of our study involved analysis of carbofuran binding to natural DOM isolated from an environmental drainage gradient at Penobscot Experimental Forest. Concentrations of DOC in the initial aqueous isolates of the ®eld samples are presented in Table 1. For each sample or MW fraction, the quenching of carbofuran emission ¯uorescence was examined as a function of DOM sample concentration (Fig. 4), and conditional binding constants for the pesticide and DOM sample were calculated (Table 2). The binding strength of carbofuran with DOM samples from the drainage sequence decreased in the order of Sedge (the wetland) > BR1 (concentrated stream DOM) > Con (the coniferous upland) > Dec (the deciduous upland). Comparing the binding constants of the MW fractions for each DOM sample, it is apparent that for a given sample, the higher MW fractions exhibited the highest binding constants.
3. Results and discussion
3.1. Fluorescence quenching
Fluorescence analysis of the three pesticides indicated that carbofuran and carbaryl ¯uoresce at 305 and 330 nm, respectively, upon excitation at 276±279 nm, whereas, aldicarb shows broad emission at 350± 380 nm upon excitation at 326 nm. Fig. 2 displays an example of the carbaryl ¯uorescence emission spectra as a function of Aldrich humic acid concentrations. It shows that the emission intensity at 330 nm decreases as the concentration of humic acid
The application of ¯uorescence quenching is based on the Stern±Volmer equation that describes the static quenching of the ¯uorescence intensity of a ¯uorophore [13]. The Stern±Volmer equation can be presented as Fo =F 1 Ksv Q
(1)
where Fothe initial ¯uorescence intensity of a ¯uorophore, Fthe ¯uorescence intensity of the
144
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Fig. 2. An example of carbaryl fluorescence quenched by increasing concentrations of the Aldrich humic acid. (a) without the presence of humic acid; (b) with a DOC of 3.2 ppm; (c) with a DOC of 6.4 ppm; (d) with a DOC of 12.8 ppm; (e) with a DOC of 32 ppm.
¯uorophore which remains after its complexation with a quencher, Ksvthe association constant for the complexation process, and [Q]the concentrations of the quencher. In this research, DOM was initially used as the ¯uorophore and carbamate as the quencher. However, DOM did not show suf®cient ¯uorescence quenching to apply the Stern±Volmer equation quantitatively. After we discovered the ¯uorescence properties of the three carbamates, we successfully obtained suf®cient quenching using the carbamate pesticides (CP) as the ¯uorophore and DOM as the quencher. The reaction between them can be described as
3.2. Static and dynamic quenching mechanisms
CP DOM $ CPÿDOM
Fo =F 1 kq to Q 1 KD Q
(2)
and the binding constant is Ksv CPÿDOM=CPDOM
(3)
where [CP], [DOM], and [CP±DOM] are the concentrations of the uncomplexed or free carbamates, DOM, and CP±DOM complex, respectively.
Dynamic quenching refers to the attenuation of the ¯uorescence resulting from the collisional encounters between the ¯uorophore and dynamic quencher, such as an oxygen molecule. In dynamic quenching, the quencher diffuses to the excited ¯uorophore. Upon contact, the ¯uorophore returns to the ground state without emission. Dynamic quenching thus reduces the average lifetime of the ¯uorophore, while static quenching does not have the same effect. Like static quenching, dynamic quenching can be described by the Stern±Volmer equation [11] (4)
where kq, to, and KD, respectively, are the bimolecular quenching constant, the lifetime of the ¯uorophore in the absence of the quencher, and the Stern±Volmer constant for dynamic quenching. Since both dynamic and static quenching can be described by linear Stern± Volmer plots, it is possible that a dynamic quenching
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
145
Fig. 3. Stern±Volmer Plots of carbofuran, carbaryl, aldicarb quenched by Aldrich humic acid after the correction of inner filter effects.
146
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Fig. 4. An example of carbofuran fluorescence quenched by increasing DOC concentrations of DOM (sample BR1). (a, b) without the presence of DOM, 2 replicates; (c) with a DOC of 3.2 ppm; (d) with a DOC of 6.4 ppm; (e) with a DOC of 9.6 ppm; (f) with a DOC of 12.8 ppm; (g) with a DOC of 16.0 ppm; (h) with a DOC of 19.2 ppm.
process was involved in the carbofuran quenching experiment. A data analysis was conducted as follows to determine which type of quenching was the primary one that accounted for the carbofuran ¯uorescence quenching by DOM samples. For the CP1 sample, a binding constant of Ksv0.94104 l/kg (Table 2) was obtained from the quenching experiments. Since the CP1 sample includes all the DOM molecules with a MW less than 1000 daltons, we can assume an average MW of about 500 g/mol for the CP1 sample. Therefore, the Ksv becomes 0.47104 l/mol (Mÿ1). From Eq. (4), we have KD kq to
3.3. Correction for the inner filter effects
(5)
The inner ®lter effects due to the absorption of DOM at both the excitation and emission wavelengths were corrected by equations developed by MacDonald [14]
(6)
Fcor cf Fobs
and kq KD =to
Considering that a ¯uorescence lifetime to at room temperature is typically near 10ÿ8 s [13], a simple calculation tells us that, if carbofuran ¯uorescence quenched by DOM was dynamic quenching, kq would be 4.71010 Mÿ1 sÿ1. It is unreasonably high for DOM molecules whose MW are much higher than oxygen. Therefore, it is safe to assume the primary quenching mechanism in the DOM±carbofuran system is static quenching.
The value of the bimolecular quenching constant kq of a quencher cannot exceed 11010 Mÿ1 sÿ1 in aqueous solutions [11]. This value is based on the diffusion rate of oxygen, an extremely ef®cient quencher. A kq of a certain quencher that exceeds this value indicates the dominance of static quenching. If dynamic quenching was the primary process in the DOM±carbofuran quenching reaction, then KsvKD0.47104 Mÿ1.
(7)
where Fcor, cf, and Fobs are the corrected ¯uorescence intensity, the correction factor and the observed inner®lter-effect-quenched ¯uorescence intensity, respectively. The correction factor is given by cf
2:3Aex x10Aex x1 2:3Aem y10Aem y1 1 ÿ 10ÿAex x 1 ÿ 10ÿAem y
(8)
where Aex and Aem are the absorbance of the DOM
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151 Table 2 Binding constants of carbofuran with DOM
Stream R1 (MW>1000) R10 (MW>10 000) P10R1 (10 000>MW>1000) Wetland Sedge (Unfractionated) R10 (MW>10 000) P10R1 (10 000>MW>1000) Coniferous O horizon Con (Unfractionated) R10 (MW>10 000) P10R1 (10 000>MW>1000) P1 (MW<1000) Deciduous O horizon Dec (Unfractionated) R10 (MW>10 000) P10R1 (10 000>MW>1000)
Average Ksv (104 l/kg)
Standard deviation (104)
1.65
0.09
1.79
0.05
1.52
0.10
1.76
0.06
1.86
0.04
1.73
0.06
1.45
0.03
1.68
0.17
1.64
0.07
0.94
0.06
1.40
0.04
1.58
0.12
1.67
0.03
solution at the excitation and emission wavelengths, respectively. The geometric parameters (x, x1, y, and y1) in the equation were determined as reported by Feng [13]. The excitation light beam was strong enough to cause a visible light spot on the cuvette wall, and so y1 and y were simply measured by a ruler when an excitation light beam of 500 nm (green in color), with the excitation slits set on 5 nm, was shined on the cuvette. By this means y10.14 cm and y0.64 cm were obtained. Dif®culties were encountered when x1 and x were to be determined. First, there was not a clearly de®ned emission light beam that one could observe. Second,
147
as a matter of fact, the detector actually picked up all the light, including the emission light and the scattered light that reached the collection focus lens. To solve this problem, a mask made of cardboard and electrical tape was pasted on the emission side of the cuvette holder. A slit that had a width of 0.5 cm was cut on the mask. Doing so, one actually imposed a x of 0.5 cm on the ¯uorescence spectrometer. Also, x1 was set as long as the mask position was ®xed. The slit on the mask was cut in such a position that it gave a x of 0.5 cm and an x1 of 0.15 cm. To test the validity of these parameter values, DOM ¯uorescence intensities at 305.5 nm (excited at 279 nm) were recorded from a series of the sedge DOM dilutions. Fig. 5 displays the plots of ¯uorescence intensity of these Sedge DOM dilutions vs. DOC concentrations of the dilutions before and after the application of Eq. (8). The linearity of the two plots are also shown in the ®gure. An excellent R2 value of 0.9970 from the linear regression of the plot after the correction indicates a good correction of the inner ®lter effects caused by DOM self-absorption quenching. 3.4. Synchronous scan fluorescence spectroscopy as a probe of DOM±carbamate binding mechanisms Synchronous scan ¯uorescence spectroscopy (SSFS) has special advantages in studying the chemical structures and properties of DOM or humic substances [15±17]. We measured the SSFS spectra of our DOM samples and their MW fractions by a procedure previously described by Cronan et al., [16], and found that our spectra (Fig. 6) closely resemble the spectra reported in our earlier study. According to Cronan et al., the SSFS peak around 350 nm is related to the fulvic acid components of DOM and the 395 nm peak is from humic acid components. Since fulvic acid is generally more hydrophilic than humic acid, a ¯uorescence intensity ratio of the 395 nm to the 350 nm peaks (F395/F350) can qualitatively show the relative hydrophobicity of each sample (i.e., the higher the ratio, the greater the hydrophobicity of the sample). Table 3 lists the (F395/F350) estimates for the four DOM samples and their MW fractions, together with the carbofuran±DOM binding constants.
148
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Fig. 5. DOM (Sedge) fluorescence intensities at 305.5 nm (excited at 279 nm, for carbofuran quenching) vs. DOC concentrations of the DOM before and after the correction of inner filter effects.
Fig. 6. An example of a synchronous scan fluorescence spectroscopy (SSFS) spectrum of a DOM sample and its MW fractions isolated from a deciduous forest floor; all the samples have a DOC of 16 mg/l, a pH of 6, 18 nm, excitation slits5 nm, and emission slits10 nm.
In Table 3, the correlation between the ratios of the intensities of the two peaks and the carbofuran±DOM binding constants is obvious: the higher the ratio, the larger the binding constant. This result implies that hydrophobicity is a major factor that drives the carbo-
furan±DOM binding process. Carbofuran has a water solubility of 3.1610ÿ3 M or 700 ppm [1]. The carbofuran molecule includes two parts: the benzofuranol moiety and the methyl-carbamate moiety above the benzene ring. The benzofuranol moiety is hydropho-
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151 Table 3 SSFS (18) peak intensity ratios of the four DOM samples, their molecular weight fractions, and their carbofuran±DOM binding constants Samples
F395/F350
Ksv (104 l/kg)
Con CR10 CP10R1 CP1
1.040 1.116 1.118 0.652
1.44 1.68 1.64 0.94
Dec DR10 DP10R1
1.035 1.141 1.026
1.40 1.58 1.67
Sedge SR10 SP10R1
1.158 1.339 1.308
1.76 1.86 1.73
BR1 BR10 BP10R1
1.285 1.402 1.234
1.65 1.79 1.52
bic due to the benzene ring and aliphatic substitution on the furan side. The carbamate moiety is relatively hydrophilic due to the presence of ±C=O and >NH functional groups. However, as a whole, the water solubility of carbofuran is still very low due to its hydrophobic benzofuranol moiety, its large molecular size, and high carbon content. Therefore, hydrophobic adsorption and hydrophobic partitioning could be the major force that drives carbofuran binding to DOM. According to the concept of `like dissolves like', hydrophobic moieties of humic substances, such as condensed aromatic rings and aliphatic sidechains, can interact with the benzofuranol part of carbofuran. In addition, because humic acids have a higher MW, higher carbon content and a lower oxygen content than fulvic acids [18], they possess a lower polarity and hence a higher hydrophobicity than fulvic acids [19]. Therefore, if hydrophobic adsorption is the primary binding mechanism for carbofuran, higher MW fractions of DOM samples, which have a higher composition of humic acids, should have larger carbofuran binding constants and larger F395/F350 values. Carefully examining Table 3, we ®nd that carbofuran has larger binding constants with aqueous (wetland and stream) DOMs which also have a higher F395/
149
F350 value than upland (coniferous and deciduous forest ¯oors) DOMs. Also, larger MW fractions with higher F395/F350 values generally have a larger carbofuran±DOM binding constant than smaller MW fractions. The DP10R1 and Sedge samples are exceptions: the DP10R1 sample has a smaller F395/F350 value than the Dec and DR10 samples but a greater carbofuran binding constant than both of them; the Sedge sample has a smaller F395/F350 value than the SP10R1 sample but a greater binding constant. Because the relatively higher hydrophilicity of DP10R1 and Sedge samples, the exceptions may imply the existence of binding mechanisms other than hydrophobic adsorption and partitioning. From the molecular structures of carbofuran and DOM, theoretical explanations for the other possible binding mechanisms could be made. Since the carbofuran molecule has ±C=O, >NH, and >O groups, it is very likely that hydrogen bonds can be established between these groups and the numerous ± COOH and ±OH groups on DOM. Although water molecules are strong competitors for hydrogen bonding with these groups, this mechanism may well be responsible for the association of carbofuran with DOM samples such as DP10R1, Sedge and CP1, which have a high content of fulvic acids that contain more carboxyl (±COOH) groups. Hydrogen bonding provides a possible answer for the unusual carbofuran binding behavior of the DP10R1 and Sedge samples. Because these two samples are presumed to have more COOH groups (smaller F395/F350 values), they can form more hydrogen bonds with carbofuran and thus have greater binding constants. 3.5. Implication of DOM binding constants for water solubility enhancement of carbofuran Let Pf be the free pollutant, Pb be the bound pollutant, and DOC be the concentration of DOM, and then we can write: Pf DOM $ PbÿDOM
(9)
Ksv Pb =Pf DOC
(10)
Considering Pf Pb Pt the following relationship is obtained:
(11)
150
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Pb =Pt Ksv DOC= 1 Ksv DOC
(12)
To explain the application of Eq. (12), the DOM from the sedge marsh wetland is taken as an example. The wetland DOM had a DOC concentration of 32.6 ppm or 3.2610ÿ5 kg/l (Table 1). The Ksv for the wetland DOM and carbofuran is 1.76104 l/kg (Table 2). Substituting these two values in Eq. (12) yields a Pb/Pt ratio of 36.5%. If a carbofuran concentration of 20 ppb was detected in the wetland water, the total concentration of carbofuran in the wetland would be about 20/(1ÿ36.5%)31.5 ppb and the carbofuran that is bound to the DOM in the wetland would be 36.5%31.511.5 ppb. If a carbofuran contamination accident happened in the wetland, because the DOM adsorbed 36.5% of the carbofuran, the volume of polluted water would be reduced 36.5%. The binding ability of carbofuran to the DOM samples from the drainage sequence is in the order of sedge (the wetland) > BR1 (concentrated stream DOM) > Con (the upland) > Dec (the upland). It is the general pattern that aqueous DOM (wetland and stream) associates with carbofuran more effectively than DOM from the forest ¯oors. However, Eq. (12) tells us the DOC concentrations of DOM samples are as important as binding constants in determining the transport of carbofuran. Therefore, considering the upland forest ¯oor is a much larger DOM reservoir than the wetland and the stream, more carbofuran would be bound to the upland. The Pb/Pt values for the unfractionated DOM samples namely, Con, Dec, Sedge, and BR1 are 71.4, 30.9, 36.5, and 12.4, respectively. Overall, taking both binding constants and DOC concentrations into account, when a carbofuran contamination event occurs in the upland±wetland± stream drainage sequence, most of the contaminants will be bound in the upland forest soil and only a small portion can immediately reach the stream and be taken away from the system. However, because the mobility of DOM, rain or snow melting induced water leaching would very effectively carry the DOM bound carbofuran to the stream and eventually lead to the spreading of contamination to a larger river system. 4. Summary A ¯uorescence quenching technique was developed to obtain the conditional equilibrium binding con-
stants of the reactions between carbamate pesticides (carbofuran, carbaryl, and aldicarb) and Aldrich humic acid samples under conditions of pH 6 and 228C. Also, two upland forest soil DOM samples, a sedge marsh wetland DOM sample and a stream DOM sample from an upland±wetland±stream sequence were examined with a ¯uorescence quenching technique to determine the magnitude of binding with carbofuran. Three types of MW fractions were obtained: R10 (MW>10 000 daltons), P10R1 (10 000>MW>1000 daltons), and P1 (MW<1000 daltons). The major ®ndings and conclusions of this research can be summarized as follows: 1. It was found that carbofuran, carbaryl, and aldicarb have ¯uorescence properties. With carbamates as the ¯uorophore and DOM as the quencher, the ¯uorescence quenching technique can be applied to study the binding reaction between the carbamate pesticides and DOM. 2. The binding strength of the three pesticides with Aldrich humic acid decreases in the following order: carbofuran > aldicarb > carbaryl. 3. Aqueous DOM samples from wetland and stream environments had greater carbofuran binding constants than DOM from upland O horizon soil samples collected in coniferous and deciduous forest sites. The binding constants of carbofuran with DOM samples from the drainage sequence decreased in the order of sedge wetland > stream DOM > coniferous O horizon > deciduous O horizon. For all DOM samples from the four sampling sites in the drainage sequence, it was found that the R10 (MW>10 000) fraction of each sample generally had greater carbofuran binding constants than its unfractionated, P10R1, and P1 counterparts. 4. Taking the ratio of the SSFS intensity at 395 nm (F395) to the SSFS intensity at 350 nm (F350) as an indicator for the hydrophobicity of the DOM samples, it was found that aqueous DOM samples from stream and wetland environments were more hydrophobic than the upland forest soil DOM samples. It was also found that higher MW fractions were more hydrophobic than lower MW fractions. Hydrophobic adsorption and partitioning and hydrogen bonding appear to be the major
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
binding mechanisms for the association of carbofuran and DOM. 5. It was found that the DOC concentration of a particular sample was as important as the binding constant in determining the magnitude of the water solubility enhancement of carbofuran in a upland± wetland±stream drainage sequence. Taking both binding constants and DOC concentrations into account, it was found that the DOM from the coniferous forest floor had the largest capacity to bind carbofuran in the drainage sequence. As such, this DOM could potentially increase the mobility of carbofuran the most. Acknowledgements The authors thank the University of Maine Water Resources Program of the United States Geological Survey at the Department of the Interior for the ®nancial support received (USGS grant number 1408-G2023) for carrying out this research. References [1] EPA, Chemical Information Fact Sheet for Carbofuran, 25 June, 1984. [2] J.D. Dwyer, S.B. Johnson, L.S. Morro, E.S. Plissey, 1994 Maine Potato Pest Control Guide, University of Maine, Cooperative Extension, April 1994.
151
[3] Extension Toxicology Network, Oregon State University, URL: http://ace.ace.orst.edu/info/extoxnet/pips/carbofur.p93, 1997. [4] Y. Mason, E. Choshen, C. Rav-Acha, Water Res. 24 (1990) 12. [5] N. de Bertrand, D. Barcelo, Anal. Chim. Acta 254 (1991) 235. [6] J.F. McCarthy, B.D. Jimenez, Environ. Sci. Technol. 19 (1985) 1072. [7] T.D. Gauthier, E.C. Shane, W.F. Guerin, W.R. Seltz, Clarence L. Grant, Environ. Sci. Technol. 20 (1986) 1162. [8] D.A. Backhus, P.M. Gschwend, Environ. Sci. Technol. 24 (1990) 1214. [9] B.E. Herbert, P.M. Bertsch, J.M. Novak, Environ. Sci. Technol. 27 (1993) 398. [10] CRC Handbook of Chemistry and Physics, 66th ed., CRC Press, Cleveland, Ohio, 1985, D-145 pp. [11] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. [12] J. Buffle, P. Deladoey, W. Haerdi, Anal. Chim. Acta 101 (1978) 339. [13] F. Fang, Masters of Science Thesis in Ecology and Environmental Science, University of Maine, 1997. See also Reference [14]. [14] B. MacDonald, S.J. Lvin, H. Patterson, Anal. Chim. Acta 328 (1997) 155. [15] N. Senesi, Anal. Chim. Acta 232 (1990) 77. [16] C.S. Cronan, S. Lakshman, H.H. Patterson, J. Environ. Qual. 21 (1992) 457. [17] P. Patterson, C.S. Cronan, S. Lakshman, B.J. Plankey, T.A. Taylor, Sci. Total Environ. 113 (1992) 179. [18] S. Boggs Jr., D.G. Livermore, M.G. Seitz, JMS-Rev. Macromol. Chem. Phys. C25(4) (1985) 599. [19] D.W. Rutherford, C.T. Chiou, D.E. Kile, Environ. Sci. Technol. 26 (1992) 336.
A spectro¯uorimetric study of the binding of carbofuran, carbaryl, and aldicarb with dissolved organic matter Feng Fang1,a, So®an Kananb, Howard H. Pattersona,b,*, Christopher S. Cronana a
Graduate Program in Ecology and Environmental Science, University of Maine, Orono, ME 04469, USA b Department of Chemistry, University of Maine, Orono, ME 04469, USA Received 16 March 1998; received in revised form 20 May 1998; accepted 26 May 1998
Abstract This study examined the binding of carbamate pesticides with dissolved organic matter (DOM) using ¯uorescence quenching and synchronous scan ¯uorescence spectroscopy (SSFS). Fluorescence spectra of the three pesticides were characterized as follows: carbofuran and carbaryl ¯uoresce at 305 and 330 nm, respectively, upon excitation at 276±279 nm, whereas, aldicarb shows broad emission at 350±380 nm upon excitation at 326 nm. A ¯uorescence quenching technique was used to obtain conditional binding constants for the carbamate pesticides with Aldrich humic acid under ®xed conditions of 228C and pH 6. The binding constant of carbofuran with humic acid is greater than the binding constants of both carbaryl and aldicarb. Estimates were also obtained for the binding of carbofuran with DOM samples from a coniferous forest soil O horizon, a deciduous forest soil O horizon, a sedge marsh wetland, and a stream in the drainage sequence and their molecular weight (MW) fractions. Those conditional binding constants were used to predict the potential transport of carbofuran in the drainage sequence. When binding constants and DOM concentrations were both taken into account, it was found that DOM from the coniferous forest O horizon had the largest capacity to bind and to transport carbofuran in the drainage sequence. SSFS was used to probe the binding mechanisms of DOM with carbofuran. Overall, the potential mobility of carbofuran in the upland± wetland±stream drainage sequence was signi®cantly enhanced via binding with DOM. # 1998 Published by Elsevier Science B.V. All rights reserved.
1. Introduction Pesticides are one of the major organic contaminants in the environment. The carbamate pesticides, carbofuran, carbaryl, and aldicarb are highly toxic to cold and warm water ®sh, freshwater invertebrates, and to birds [1]. Carbofuran is commonly used in *Corresponding author. Tel.: +1-207-581-1178; fax: +1-207581-1191; e-mail: [email protected] 1 Current address: 500 Pillsbury Dr. SE, Civil Engineering Building, University of Minnesota, Minneapolis, MN 55455
potato and rotation crop farms to control Colorado potato beetle, ¯ea beetles and leafhoppers [2]. It acts as a cholinesterase inhibitor after insects contact a treated surface and/or ingest treated plant tissue. Carbaryl is used in cotton, fruit, forests, nuts, and other crops, and is inherently toxic to humans by skin contact, inhalation, and/or ingestion [3]. Aldicarb is one of the most acutely poisonous pesticides ± the oral LD50 value for rats is 0.95 mg/kg [4]. It is widely used to control mites, nematodes, and aphids in cotton and soybean crops. The chemical structures of carbofuran, carbaryl, and aldicarb are given.
0003-2670/98/$19.00 # 1998 Published by Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00392-4
140
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Understanding the transport and fate of pesticides in the environment is of great importance for their application and regulation. The interaction between dissolved organic matter (DOM) and carbamate pesticides not only changes the solubility and mobility of the pesticides in the environment, but also affects the photodegradation and hydrolysis rate of the pesticides [5]. Therefore, it is important to understand the binding tendency between DOM and carbamate pesticides if the transport and fate of these pesticides in the environment are to be predicted. A classical approach for examining the binding reaction of a pesticide with DOM is to measure the concentration of the free pesticide before and after its binding with DOM in an aqueous reaction system. From the difference between the two concentrations, an association constant can be calculated. As a requirement for this approach, an accurate analytical method for detecting the free pesticide concentration is essential. In addition, before the concentration of free pesticide is determined, the free pesticide must be separated from the DOM-bound pesticide, because the presence of DOM and DOM-bound pesticide in solution can cause errors in the measurement. The drawback of separation processes is that they may be incomplete or may disrupt established equilibria and lead to inconsistent estimates of binding constants. Fluorescence quenching, however, does not require that absolute concentrations of contaminants be known, and does not require a separation step. Therefore, ¯uorescence spectroscopy has unique advantages in the study of contaminant binding to DOM [6±9]. In this paper we report the ¯uorescence properties of the carbamate pesticides (carbofuran, carbaryl, and aldicarb), and demonstrate that the ¯uorescence
quenching technique can be used to study the binding af®nity of the three carbamates with a sample of Aldrich humic acid. We also compare the binding interaction of carbofuran with DOM separated from an upland±wetland±stream sequence using ¯uorescence quenching and synchronous scan ¯uorescence spectroscopy (SSFS). We believe this is the ®rst paper to show that ¯uorescence spectroscopy can be used to probe pesticide±DOM interactions. 2. Experimental 2.1. Reagents Carbofuran, carbaryl, and aldicarb crystals (purity: 99%) were purchased from Chem Service and were used as received. The humic acid was purchased from Aldrich Chemicals. Analysis performed at Aldrich showed the sample to be 40.7%C. A concentrated pH 6.0 phosphate buffer was prepared according to a modi®ed method from the CRC Handbook of Chemistry and Physics [10]. Fifty ml of 1.00 M KH2PO4 and 5.6 ml of 1.00 M NaOH were mixed and diluted to 100 ml in a volumetric ¯ask. This solution was diluted to a 1 in 50 ratio to adjust all of the carbamate solutions, distilled and deionized H2O blanks, and DOM dilutions to a pH of 6. Distilled and deionized water (dd H2O) was made by Barnstead ion exchange cartridges. Carbofuran, carbaryl, and aldicarb crystals were dissolved in methanol (HPLC grade, EM Science) to make stock solutions of 3.55910ÿ4 M in a 25 ml volumetric ¯ask. 1.91 ml of the stock solutions were transferred to three 50 ml volumetric ¯asks into which 1 ml of the pH 6 phosphate buffer had been
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
141
added. The ¯asks were then ®lled to the mark with dd H2O, giving a set of pH 6.0 aqueous carbamate solutions of about 1.3610ÿ3 M for the quenching experiments. Although the hydrolysis half-life of these pesticides at 258C and pH 6 is very insigni®cant (see Extension Toxicology Network. World Wide Wet site: http://ace.ace.orst.edu/info/extoxnet/pips/ghindex.html), the solutions to be studied were always made within 1 day before the quenching experiments and were stored in the dark to minimize hydrolysis. 2.2. Environmental DOM sample collection and preparation Environmental DOM samples were collected from sites at the Penobscot Experimental Forest, Bradley, Maine, in October, 1996. A sampling ®eld consisting of two types of upland forests (deciduous and coniferous), a sedge marsh wetland, and a stream draining the forests and wetland was selected for the upland± wetland±stream environmental drainage gradient. A shovel was used to collect 10±15 cm thick samples of organic horizon material from the forest ¯oors in each forest stand. The wetland sample was collected from a sedge marsh bordering Blackman stream. A hole of about 20 cm in diameter and 10 cm in depth was dug using a shovel. The hole promptly ®lled with sediment laden ground water which was collected into one-liter Nalgene1 sample bottles. Approximately 3 l of water was taken from Blackman stream to represent surface water in the drainage system. One-liter Nalgene1 sample bottles were submerged completely into water when sampling. Samples were delivered to the laboratory immediately after collection and were stored in a refrigerator at a temperature of 48C. To obtain DOM solutions and their molecular weight (MW) fractions from these raw samples, the procedures shown in Fig. 1 were followed. For ultra®ltration, Dia¯o1 ultra®lters and a model 8400 Amicon1 stirred ultra®ltration cell were used. Ultra®ltration membranes were type YM10 and YM1, which have nominal MW cutoffs of 10 000 and 1000 daltons, respectively. YM membranes have exceptionally low non-speci®c protein binding properties and are recommended where maximum solute recovery is of utmost importance [11]. The operating pressure for the YM10 and YM1 ultra®ltration membrane was 55 psi N2 (3.7 atm) and 65 psi N2 (4.4 atm),
Fig. 1. DOM sample processing protocol (the coniferous and deciduous forest floor raw samples started from the first step with water extraction; the sedge marsh wetland sample started from Whatman 41 filter paper filtration; and the Blackman stream sample started from VacuCap1 filtration).
respectively. To avoid the breakdown of larger-molecular-size DOM solutes, the ®ltrate volume was never allowed to exceed 90% of the initial total volume [12]. To name each DOM sample and its MW fractions, Dec (or D), Con (or C), Sedge (or S), and Black (or B) were used to represent samples of deciduous forest soil DOM, coniferous forest soil DOM, sedge marsh DOM, and Blackman stream DOM, respectively. `P' and `R' were used to represent samples that either passed through or were retained on an ultra®ltration membrane, respectively. For instance, SR10 is the name for a sedge marsh DOM sample that has a nominal MW larger than 10 000. Three low pressure chromatography columns (internal diameter: 4.5 mm, length: 28 cm) ®lled with
142
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Rexyn 101 H form beads were used to perform cation exchange on the DOM samples. A model 700 Total Organic Carbon Analyzer (detection limit: 0.5 ppm) from O.I. Corporation, College Station, Texas, was used to conduct DOC (dissolved organic carbon) analysis. Instrumental baselines were set by using carbon-free water (0.1 ppm total carbon) obtained from the Sawyer Environmental Laboratory at the University of Maine. Two potassium hydrogen phthalate standard DOC solutions 20 and 50 mg C/l (ppm) were used to standardize the instrument. Sample Black (Blackman Stream sample after VacuCap1 ®ltering) was too dilute (DOC 8.5 ppm) for a ¯uorescence quenching experiment. The YM1 ultra®ltration membrane was employed to concentrate this sample. 2.3. Fluorescence spectra of carbamate pesticides and DOM Fluorescence data were collected on a computerdriven model QM-1 ¯uorescence spectrometer from Photo Technology International (PTI). The instrument was equipped with a model UXL - 75 XE xenon short arc lamp from Ushio. The ¯uorescence spectrometer had a cuvette holder with a built-in magnetic stirring setup. The instrument had two excitation monochromators, and, thus, had two excitation slits. In the carbofuran and carbaryl experiments the excitation wavelength was set at 279 and 276 nm, respectively and the emission scan was from 290 to 350 nm. In the aldicarb experiments the excitation was ®xed at 326 nm and the emission scan was from 340± 550 nm. Both excitation and emission slits were set at 5 nm. Fluorescence data collection and analysis TM were conducted with Oscar software from PTI. Fluorometry quartz cuvettes from Whatman1 with a light path length of 10 mm were used for both ¯uorescence and absorption measurements. Emission ¯uorescence spectra of each DOM sample were measured and collected under the same experimental conditions as the carbamate emission spectra. Since in this research, the carbamate pesticides were the ¯uorophore and DOM was the quencher, the ¯uorescence of DOM was the background component of the total ¯uorescence of carbamate after DOM was added to the solution. To obtain the quenched ¯uorescence intensity of carbamates, the ¯uorescence of DOM was
simply subtracted from the total ¯uorescence as part of the data analysis process [7]. 2.4. Absorption spectra of DOM solutions Absorption data were collected on a computerdriven model DU1 640 spectrophotometer from Beckman Instruments. Absorption of DOM solutions was measured from 260 to 400 nm for the inner ®lter effects correction. 2.5. Fluorescence quenching measurements Five dilutions of Aldrich humic acid were prepared after adjustment to pH 6 with phosphate buffer solution. Also, six dilutions of each of the environmental DOM samples, adjusted to pH 6 with phosphate buffer, were prepared and were stored in uniform glass vials from Fisher Science. For unfractionated and R10 DOM samples, a dilution series was prepared with DOC concentrations of 4, 8, 12, 16, 20, and 24 ppm. For the P10R1 samples and the CP1 sample, because of their smaller binding ability, the above concentration series was not high enough to signi®cantly quench the ¯uorescence of carbofuran to give Stern±Volmer plots with good linearity. Therefore, a DOC concentration series of 6, 12, 18, 24, 30, and 36 ppm, or 8, 16, 24, 32, 40, and 48 ppm, depending on the maximum concentration available from the stock DOM samples, was used instead for the P10R1 samples and the CP1 sample. A 2.00 ml aliquot of each dilution was pipetted to a cuvette. An absorption scan from 260 to 350 nm was recorded. The cuvette was then transferred to the ¯uorescence spectrometer and the background ¯uorescence of DOM was measured. A 25 mm Te¯on magnetic stirring bar from Thomas Scienti®c1 was put into the cuvette followed by 0.50 ml aliquot of the carbamate working solution. The cuvette was immediately put back to the cuvette holder in the ¯uorescence spectrometer and stirring was started. After 3 min the stirring was stopped. The solution was allowed to stand quiescent (without stirring) for an additional 1 min before the ¯uorescence spectrum was recorded. Since carbamates are strongly photodegradable under ultraviolet light [5], the shutter of the excitation monochromator was closed to protect the carbamate from ultraviolet light
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
during the 4 min reaction time. To improve the signal to noise ratio, the ¯uorescence signals were recorded twice for each dilution and average values were computed for the two scans. The average values were used to carry out the data analysis. The blank (Fo) was run twice: once in the beginning and again at the end of each dilution series. This step was to insure an accurate value of Fo because this number would be used six times in the Ksv calculation and the Stern±Volmer plot. Also, the blank replicates were run in such a manner-in the beginning and at the end of each dilution series-that the ¯uctuation of the ¯uorescence spectrometer within a series of dilutions could be cancelled out to some extent. Between two dilutions of each DOM sample, cuvettes were always rinsed with dd H2O at least six times and then were rinsed twice with acetone (>99.5%, EM Science). The next dilution was not added until acetone was totally dried. Kimwipes1 low-lint paper wipers were used to clean impurities and ®ngerprints from cuvette outside walls. Between two DOM samples, cuvettes were soaked in washing acid (K2Cr2O7 dissolved in concentrated sulfuric acid) for several minutes to clean out any carbamate and DOM residues on the cuvette walls. Before starting the dilution series of the next sample, background corrections for the ¯uorescence spectrometer and the UV-Vis spectrophotometer were made again to insure the accuracy of the data for each dilution series. Three replicates were run for each sample. The average of the binding constant results of these three replicates was used as the ®nal result. The standard deviation for each sample was also calculated from the three replicates.
143
Table 1 DOC concentrations in water samples and soil extracts used in this study Sample
DOC (mg C/l)
Coniferous O horizon Deciduous O horizon Sedge marsh soil water Blackman stream
173.6 31.8 32.3 8.5
increases. Fig. 3 shows the Stern±Volmer plots for the three carbamate pesticides with Fo/F plotted against increasing Aldrich humic acid concentrations after the correction of inner ®lter effects. The binding constant for carbofuran with Aldrich humic acid (8.75104 l/kg) is greater than the binding constants for aldicarb (7.21104 l/kg) and carbaryl (0.96104 l/kg). The second phase of our study involved analysis of carbofuran binding to natural DOM isolated from an environmental drainage gradient at Penobscot Experimental Forest. Concentrations of DOC in the initial aqueous isolates of the ®eld samples are presented in Table 1. For each sample or MW fraction, the quenching of carbofuran emission ¯uorescence was examined as a function of DOM sample concentration (Fig. 4), and conditional binding constants for the pesticide and DOM sample were calculated (Table 2). The binding strength of carbofuran with DOM samples from the drainage sequence decreased in the order of Sedge (the wetland) > BR1 (concentrated stream DOM) > Con (the coniferous upland) > Dec (the deciduous upland). Comparing the binding constants of the MW fractions for each DOM sample, it is apparent that for a given sample, the higher MW fractions exhibited the highest binding constants.
3. Results and discussion
3.1. Fluorescence quenching
Fluorescence analysis of the three pesticides indicated that carbofuran and carbaryl ¯uoresce at 305 and 330 nm, respectively, upon excitation at 276±279 nm, whereas, aldicarb shows broad emission at 350± 380 nm upon excitation at 326 nm. Fig. 2 displays an example of the carbaryl ¯uorescence emission spectra as a function of Aldrich humic acid concentrations. It shows that the emission intensity at 330 nm decreases as the concentration of humic acid
The application of ¯uorescence quenching is based on the Stern±Volmer equation that describes the static quenching of the ¯uorescence intensity of a ¯uorophore [13]. The Stern±Volmer equation can be presented as Fo =F 1 Ksv Q
(1)
where Fothe initial ¯uorescence intensity of a ¯uorophore, Fthe ¯uorescence intensity of the
144
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Fig. 2. An example of carbaryl fluorescence quenched by increasing concentrations of the Aldrich humic acid. (a) without the presence of humic acid; (b) with a DOC of 3.2 ppm; (c) with a DOC of 6.4 ppm; (d) with a DOC of 12.8 ppm; (e) with a DOC of 32 ppm.
¯uorophore which remains after its complexation with a quencher, Ksvthe association constant for the complexation process, and [Q]the concentrations of the quencher. In this research, DOM was initially used as the ¯uorophore and carbamate as the quencher. However, DOM did not show suf®cient ¯uorescence quenching to apply the Stern±Volmer equation quantitatively. After we discovered the ¯uorescence properties of the three carbamates, we successfully obtained suf®cient quenching using the carbamate pesticides (CP) as the ¯uorophore and DOM as the quencher. The reaction between them can be described as
3.2. Static and dynamic quenching mechanisms
CP DOM $ CPÿDOM
Fo =F 1 kq to Q 1 KD Q
(2)
and the binding constant is Ksv CPÿDOM=CPDOM
(3)
where [CP], [DOM], and [CP±DOM] are the concentrations of the uncomplexed or free carbamates, DOM, and CP±DOM complex, respectively.
Dynamic quenching refers to the attenuation of the ¯uorescence resulting from the collisional encounters between the ¯uorophore and dynamic quencher, such as an oxygen molecule. In dynamic quenching, the quencher diffuses to the excited ¯uorophore. Upon contact, the ¯uorophore returns to the ground state without emission. Dynamic quenching thus reduces the average lifetime of the ¯uorophore, while static quenching does not have the same effect. Like static quenching, dynamic quenching can be described by the Stern±Volmer equation [11] (4)
where kq, to, and KD, respectively, are the bimolecular quenching constant, the lifetime of the ¯uorophore in the absence of the quencher, and the Stern±Volmer constant for dynamic quenching. Since both dynamic and static quenching can be described by linear Stern± Volmer plots, it is possible that a dynamic quenching
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
145
Fig. 3. Stern±Volmer Plots of carbofuran, carbaryl, aldicarb quenched by Aldrich humic acid after the correction of inner filter effects.
146
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Fig. 4. An example of carbofuran fluorescence quenched by increasing DOC concentrations of DOM (sample BR1). (a, b) without the presence of DOM, 2 replicates; (c) with a DOC of 3.2 ppm; (d) with a DOC of 6.4 ppm; (e) with a DOC of 9.6 ppm; (f) with a DOC of 12.8 ppm; (g) with a DOC of 16.0 ppm; (h) with a DOC of 19.2 ppm.
process was involved in the carbofuran quenching experiment. A data analysis was conducted as follows to determine which type of quenching was the primary one that accounted for the carbofuran ¯uorescence quenching by DOM samples. For the CP1 sample, a binding constant of Ksv0.94104 l/kg (Table 2) was obtained from the quenching experiments. Since the CP1 sample includes all the DOM molecules with a MW less than 1000 daltons, we can assume an average MW of about 500 g/mol for the CP1 sample. Therefore, the Ksv becomes 0.47104 l/mol (Mÿ1). From Eq. (4), we have KD kq to
3.3. Correction for the inner filter effects
(5)
The inner ®lter effects due to the absorption of DOM at both the excitation and emission wavelengths were corrected by equations developed by MacDonald [14]
(6)
Fcor cf Fobs
and kq KD =to
Considering that a ¯uorescence lifetime to at room temperature is typically near 10ÿ8 s [13], a simple calculation tells us that, if carbofuran ¯uorescence quenched by DOM was dynamic quenching, kq would be 4.71010 Mÿ1 sÿ1. It is unreasonably high for DOM molecules whose MW are much higher than oxygen. Therefore, it is safe to assume the primary quenching mechanism in the DOM±carbofuran system is static quenching.
The value of the bimolecular quenching constant kq of a quencher cannot exceed 11010 Mÿ1 sÿ1 in aqueous solutions [11]. This value is based on the diffusion rate of oxygen, an extremely ef®cient quencher. A kq of a certain quencher that exceeds this value indicates the dominance of static quenching. If dynamic quenching was the primary process in the DOM±carbofuran quenching reaction, then KsvKD0.47104 Mÿ1.
(7)
where Fcor, cf, and Fobs are the corrected ¯uorescence intensity, the correction factor and the observed inner®lter-effect-quenched ¯uorescence intensity, respectively. The correction factor is given by cf
2:3Aex x10Aex x1 2:3Aem y10Aem y1 1 ÿ 10ÿAex x 1 ÿ 10ÿAem y
(8)
where Aex and Aem are the absorbance of the DOM
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151 Table 2 Binding constants of carbofuran with DOM
Stream R1 (MW>1000) R10 (MW>10 000) P10R1 (10 000>MW>1000) Wetland Sedge (Unfractionated) R10 (MW>10 000) P10R1 (10 000>MW>1000) Coniferous O horizon Con (Unfractionated) R10 (MW>10 000) P10R1 (10 000>MW>1000) P1 (MW<1000) Deciduous O horizon Dec (Unfractionated) R10 (MW>10 000) P10R1 (10 000>MW>1000)
Average Ksv (104 l/kg)
Standard deviation (104)
1.65
0.09
1.79
0.05
1.52
0.10
1.76
0.06
1.86
0.04
1.73
0.06
1.45
0.03
1.68
0.17
1.64
0.07
0.94
0.06
1.40
0.04
1.58
0.12
1.67
0.03
solution at the excitation and emission wavelengths, respectively. The geometric parameters (x, x1, y, and y1) in the equation were determined as reported by Feng [13]. The excitation light beam was strong enough to cause a visible light spot on the cuvette wall, and so y1 and y were simply measured by a ruler when an excitation light beam of 500 nm (green in color), with the excitation slits set on 5 nm, was shined on the cuvette. By this means y10.14 cm and y0.64 cm were obtained. Dif®culties were encountered when x1 and x were to be determined. First, there was not a clearly de®ned emission light beam that one could observe. Second,
147
as a matter of fact, the detector actually picked up all the light, including the emission light and the scattered light that reached the collection focus lens. To solve this problem, a mask made of cardboard and electrical tape was pasted on the emission side of the cuvette holder. A slit that had a width of 0.5 cm was cut on the mask. Doing so, one actually imposed a x of 0.5 cm on the ¯uorescence spectrometer. Also, x1 was set as long as the mask position was ®xed. The slit on the mask was cut in such a position that it gave a x of 0.5 cm and an x1 of 0.15 cm. To test the validity of these parameter values, DOM ¯uorescence intensities at 305.5 nm (excited at 279 nm) were recorded from a series of the sedge DOM dilutions. Fig. 5 displays the plots of ¯uorescence intensity of these Sedge DOM dilutions vs. DOC concentrations of the dilutions before and after the application of Eq. (8). The linearity of the two plots are also shown in the ®gure. An excellent R2 value of 0.9970 from the linear regression of the plot after the correction indicates a good correction of the inner ®lter effects caused by DOM self-absorption quenching. 3.4. Synchronous scan fluorescence spectroscopy as a probe of DOM±carbamate binding mechanisms Synchronous scan ¯uorescence spectroscopy (SSFS) has special advantages in studying the chemical structures and properties of DOM or humic substances [15±17]. We measured the SSFS spectra of our DOM samples and their MW fractions by a procedure previously described by Cronan et al., [16], and found that our spectra (Fig. 6) closely resemble the spectra reported in our earlier study. According to Cronan et al., the SSFS peak around 350 nm is related to the fulvic acid components of DOM and the 395 nm peak is from humic acid components. Since fulvic acid is generally more hydrophilic than humic acid, a ¯uorescence intensity ratio of the 395 nm to the 350 nm peaks (F395/F350) can qualitatively show the relative hydrophobicity of each sample (i.e., the higher the ratio, the greater the hydrophobicity of the sample). Table 3 lists the (F395/F350) estimates for the four DOM samples and their MW fractions, together with the carbofuran±DOM binding constants.
148
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Fig. 5. DOM (Sedge) fluorescence intensities at 305.5 nm (excited at 279 nm, for carbofuran quenching) vs. DOC concentrations of the DOM before and after the correction of inner filter effects.
Fig. 6. An example of a synchronous scan fluorescence spectroscopy (SSFS) spectrum of a DOM sample and its MW fractions isolated from a deciduous forest floor; all the samples have a DOC of 16 mg/l, a pH of 6, 18 nm, excitation slits5 nm, and emission slits10 nm.
In Table 3, the correlation between the ratios of the intensities of the two peaks and the carbofuran±DOM binding constants is obvious: the higher the ratio, the larger the binding constant. This result implies that hydrophobicity is a major factor that drives the carbo-
furan±DOM binding process. Carbofuran has a water solubility of 3.1610ÿ3 M or 700 ppm [1]. The carbofuran molecule includes two parts: the benzofuranol moiety and the methyl-carbamate moiety above the benzene ring. The benzofuranol moiety is hydropho-
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151 Table 3 SSFS (18) peak intensity ratios of the four DOM samples, their molecular weight fractions, and their carbofuran±DOM binding constants Samples
F395/F350
Ksv (104 l/kg)
Con CR10 CP10R1 CP1
1.040 1.116 1.118 0.652
1.44 1.68 1.64 0.94
Dec DR10 DP10R1
1.035 1.141 1.026
1.40 1.58 1.67
Sedge SR10 SP10R1
1.158 1.339 1.308
1.76 1.86 1.73
BR1 BR10 BP10R1
1.285 1.402 1.234
1.65 1.79 1.52
bic due to the benzene ring and aliphatic substitution on the furan side. The carbamate moiety is relatively hydrophilic due to the presence of ±C=O and >NH functional groups. However, as a whole, the water solubility of carbofuran is still very low due to its hydrophobic benzofuranol moiety, its large molecular size, and high carbon content. Therefore, hydrophobic adsorption and hydrophobic partitioning could be the major force that drives carbofuran binding to DOM. According to the concept of `like dissolves like', hydrophobic moieties of humic substances, such as condensed aromatic rings and aliphatic sidechains, can interact with the benzofuranol part of carbofuran. In addition, because humic acids have a higher MW, higher carbon content and a lower oxygen content than fulvic acids [18], they possess a lower polarity and hence a higher hydrophobicity than fulvic acids [19]. Therefore, if hydrophobic adsorption is the primary binding mechanism for carbofuran, higher MW fractions of DOM samples, which have a higher composition of humic acids, should have larger carbofuran binding constants and larger F395/F350 values. Carefully examining Table 3, we ®nd that carbofuran has larger binding constants with aqueous (wetland and stream) DOMs which also have a higher F395/
149
F350 value than upland (coniferous and deciduous forest ¯oors) DOMs. Also, larger MW fractions with higher F395/F350 values generally have a larger carbofuran±DOM binding constant than smaller MW fractions. The DP10R1 and Sedge samples are exceptions: the DP10R1 sample has a smaller F395/F350 value than the Dec and DR10 samples but a greater carbofuran binding constant than both of them; the Sedge sample has a smaller F395/F350 value than the SP10R1 sample but a greater binding constant. Because the relatively higher hydrophilicity of DP10R1 and Sedge samples, the exceptions may imply the existence of binding mechanisms other than hydrophobic adsorption and partitioning. From the molecular structures of carbofuran and DOM, theoretical explanations for the other possible binding mechanisms could be made. Since the carbofuran molecule has ±C=O, >NH, and >O groups, it is very likely that hydrogen bonds can be established between these groups and the numerous ± COOH and ±OH groups on DOM. Although water molecules are strong competitors for hydrogen bonding with these groups, this mechanism may well be responsible for the association of carbofuran with DOM samples such as DP10R1, Sedge and CP1, which have a high content of fulvic acids that contain more carboxyl (±COOH) groups. Hydrogen bonding provides a possible answer for the unusual carbofuran binding behavior of the DP10R1 and Sedge samples. Because these two samples are presumed to have more COOH groups (smaller F395/F350 values), they can form more hydrogen bonds with carbofuran and thus have greater binding constants. 3.5. Implication of DOM binding constants for water solubility enhancement of carbofuran Let Pf be the free pollutant, Pb be the bound pollutant, and DOC be the concentration of DOM, and then we can write: Pf DOM $ PbÿDOM
(9)
Ksv Pb =Pf DOC
(10)
Considering Pf Pb Pt the following relationship is obtained:
(11)
150
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
Pb =Pt Ksv DOC= 1 Ksv DOC
(12)
To explain the application of Eq. (12), the DOM from the sedge marsh wetland is taken as an example. The wetland DOM had a DOC concentration of 32.6 ppm or 3.2610ÿ5 kg/l (Table 1). The Ksv for the wetland DOM and carbofuran is 1.76104 l/kg (Table 2). Substituting these two values in Eq. (12) yields a Pb/Pt ratio of 36.5%. If a carbofuran concentration of 20 ppb was detected in the wetland water, the total concentration of carbofuran in the wetland would be about 20/(1ÿ36.5%)31.5 ppb and the carbofuran that is bound to the DOM in the wetland would be 36.5%31.511.5 ppb. If a carbofuran contamination accident happened in the wetland, because the DOM adsorbed 36.5% of the carbofuran, the volume of polluted water would be reduced 36.5%. The binding ability of carbofuran to the DOM samples from the drainage sequence is in the order of sedge (the wetland) > BR1 (concentrated stream DOM) > Con (the upland) > Dec (the upland). It is the general pattern that aqueous DOM (wetland and stream) associates with carbofuran more effectively than DOM from the forest ¯oors. However, Eq. (12) tells us the DOC concentrations of DOM samples are as important as binding constants in determining the transport of carbofuran. Therefore, considering the upland forest ¯oor is a much larger DOM reservoir than the wetland and the stream, more carbofuran would be bound to the upland. The Pb/Pt values for the unfractionated DOM samples namely, Con, Dec, Sedge, and BR1 are 71.4, 30.9, 36.5, and 12.4, respectively. Overall, taking both binding constants and DOC concentrations into account, when a carbofuran contamination event occurs in the upland±wetland± stream drainage sequence, most of the contaminants will be bound in the upland forest soil and only a small portion can immediately reach the stream and be taken away from the system. However, because the mobility of DOM, rain or snow melting induced water leaching would very effectively carry the DOM bound carbofuran to the stream and eventually lead to the spreading of contamination to a larger river system. 4. Summary A ¯uorescence quenching technique was developed to obtain the conditional equilibrium binding con-
stants of the reactions between carbamate pesticides (carbofuran, carbaryl, and aldicarb) and Aldrich humic acid samples under conditions of pH 6 and 228C. Also, two upland forest soil DOM samples, a sedge marsh wetland DOM sample and a stream DOM sample from an upland±wetland±stream sequence were examined with a ¯uorescence quenching technique to determine the magnitude of binding with carbofuran. Three types of MW fractions were obtained: R10 (MW>10 000 daltons), P10R1 (10 000>MW>1000 daltons), and P1 (MW<1000 daltons). The major ®ndings and conclusions of this research can be summarized as follows: 1. It was found that carbofuran, carbaryl, and aldicarb have ¯uorescence properties. With carbamates as the ¯uorophore and DOM as the quencher, the ¯uorescence quenching technique can be applied to study the binding reaction between the carbamate pesticides and DOM. 2. The binding strength of the three pesticides with Aldrich humic acid decreases in the following order: carbofuran > aldicarb > carbaryl. 3. Aqueous DOM samples from wetland and stream environments had greater carbofuran binding constants than DOM from upland O horizon soil samples collected in coniferous and deciduous forest sites. The binding constants of carbofuran with DOM samples from the drainage sequence decreased in the order of sedge wetland > stream DOM > coniferous O horizon > deciduous O horizon. For all DOM samples from the four sampling sites in the drainage sequence, it was found that the R10 (MW>10 000) fraction of each sample generally had greater carbofuran binding constants than its unfractionated, P10R1, and P1 counterparts. 4. Taking the ratio of the SSFS intensity at 395 nm (F395) to the SSFS intensity at 350 nm (F350) as an indicator for the hydrophobicity of the DOM samples, it was found that aqueous DOM samples from stream and wetland environments were more hydrophobic than the upland forest soil DOM samples. It was also found that higher MW fractions were more hydrophobic than lower MW fractions. Hydrophobic adsorption and partitioning and hydrogen bonding appear to be the major
F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151
binding mechanisms for the association of carbofuran and DOM. 5. It was found that the DOC concentration of a particular sample was as important as the binding constant in determining the magnitude of the water solubility enhancement of carbofuran in a upland± wetland±stream drainage sequence. Taking both binding constants and DOC concentrations into account, it was found that the DOM from the coniferous forest floor had the largest capacity to bind carbofuran in the drainage sequence. As such, this DOM could potentially increase the mobility of carbofuran the most. Acknowledgements The authors thank the University of Maine Water Resources Program of the United States Geological Survey at the Department of the Interior for the ®nancial support received (USGS grant number 1408-G2023) for carrying out this research. References [1] EPA, Chemical Information Fact Sheet for Carbofuran, 25 June, 1984. [2] J.D. Dwyer, S.B. Johnson, L.S. Morro, E.S. Plissey, 1994 Maine Potato Pest Control Guide, University of Maine, Cooperative Extension, April 1994.
151
[3] Extension Toxicology Network, Oregon State University, URL: http://ace.ace.orst.edu/info/extoxnet/pips/carbofur.p93, 1997. [4] Y. Mason, E. Choshen, C. Rav-Acha, Water Res. 24 (1990) 12. [5] N. de Bertrand, D. Barcelo, Anal. Chim. Acta 254 (1991) 235. [6] J.F. McCarthy, B.D. Jimenez, Environ. Sci. Technol. 19 (1985) 1072. [7] T.D. Gauthier, E.C. Shane, W.F. Guerin, W.R. Seltz, Clarence L. Grant, Environ. Sci. Technol. 20 (1986) 1162. [8] D.A. Backhus, P.M. Gschwend, Environ. Sci. Technol. 24 (1990) 1214. [9] B.E. Herbert, P.M. Bertsch, J.M. Novak, Environ. Sci. Technol. 27 (1993) 398. [10] CRC Handbook of Chemistry and Physics, 66th ed., CRC Press, Cleveland, Ohio, 1985, D-145 pp. [11] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. [12] J. Buffle, P. Deladoey, W. Haerdi, Anal. Chim. Acta 101 (1978) 339. [13] F. Fang, Masters of Science Thesis in Ecology and Environmental Science, University of Maine, 1997. See also Reference [14]. [14] B. MacDonald, S.J. Lvin, H. Patterson, Anal. Chim. Acta 328 (1997) 155. [15] N. Senesi, Anal. Chim. Acta 232 (1990) 77. [16] C.S. Cronan, S. Lakshman, H.H. Patterson, J. Environ. Qual. 21 (1992) 457. [17] P. Patterson, C.S. Cronan, S. Lakshman, B.J. Plankey, T.A. Taylor, Sci. Total Environ. 113 (1992) 179. [18] S. Boggs Jr., D.G. Livermore, M.G. Seitz, JMS-Rev. Macromol. Chem. Phys. C25(4) (1985) 599. [19] D.W. Rutherford, C.T. Chiou, D.E. Kile, Environ. Sci. Technol. 26 (1992) 336.