a spectrofluorimetric study of the binding of carbofuran, carbaryl, and aldicarb with dissolved...

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, USAb 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 contami-

nants 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

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.

Analytica Chimica Acta 373 (1998) 139±151

*Corresponding author. Tel.: +1-207-581-1178; fax: +1-207-

581-1191; e-mail: [email protected] address: 500 Pillsbury Dr. SE, Civil Engineering

Building, University of Minnesota, Minneapolis, MN 55455

0003-2670/98/$19.00 # 1998 Published by Elsevier Science B.V. All rights reserved.

P I I S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 3 9 2 - 4

Understanding the transport and fate of pesticides in

the environment is of great importance for their appli-

cation and regulation. The interaction between dis-

solved 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 pes-

ticides [5]. Therefore, it is important to understand the

binding tendency between DOM and carbamate pes-

ticides 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 solu-

tion can cause errors in the measurement. The draw-

back of separation processes is that they may be

incomplete or may disrupt established equilibria

and lead to inconsistent estimates of binding con-

stants. 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 ¯uores-

cence 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 Chem-

istry 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 3.559�10ÿ4 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

140 F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151

added. The ¯asks were then ®lled to the mark with dd

H2O, giving a set of pH 6.0 aqueous carbamate

solutions of about 1.36�10ÿ3 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/ghin-

dex.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 con-

iferous), 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 sam-

ples 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 Nal-

gene1 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 Ami-

con1 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 proper-

ties and are recommended where maximum solute

recovery is of utmost importance [11]. The operating

pressure for the YM10 and YM1 ultra®ltration mem-

brane was 55 psi N2 (3.7 atm) and 65 psi N2 (4.4 atm),

respectively. To avoid the breakdown of larger-mole-

cular-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 (inter-

nal diameter: 4.5 mm, length: 28 cm) ®lled with

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).

F. Fang et al. / Analytica Chimica Acta 373 (1998) 139±151 141

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. Sam-

ple Black (Blackman Stream sample after VacuCap1

®ltering) was too dilute (DOC 8.5 ppm) for a ¯uor-

escence quenching experiment. The YM1 ultra®ltra-

tion membrane was employed to concentrate this

sample.

2.3. Fluorescence spectra of carbamate pesticides

and DOM

Fluorescence data were collected on a computer-

driven 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 monochro-

mators, 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

were conducted with OscarTM

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 mea-

sured 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 ¯uores-

cence 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 computer-

driven 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 solu-

tion. 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 ¯uor-

escence of DOM was measured.

A 2�5 mm Te¯on magnetic stirring bar from Tho-

mas 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 ¯uores-

cence 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


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 begin-

ning 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, cuv-

ettes 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 ace-

tone was totally dried. Kimwipes1 low-lint paper

wipers were used to clean impurities and ®ngerprints

from cuvette outside walls. Between two DOM sam-

ples, 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 spectro-

photometer 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.

3. Results and discussion

Fluorescence analysis of the three pesticides indi-

cated 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 concen-

trations. It shows that the emission intensity at 330 nm

decreases as the concentration of humic acid

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

(8.75�104 l/kg) is greater than the binding constants

for aldicarb (7.21�104 l/kg) and carbaryl (0.96�104

l/kg).

The second phase of our study involved analysis of

carbofuran binding to natural DOM isolated from an

environmental drainage gradient at Penobscot Experi-

mental 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 quench-

ing of carbofuran emission ¯uorescence was exam-

ined 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 sam-

ples 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.

3.1. Fluorescence quenching

The application of ¯uorescence quenching is based

on the Stern±Volmer equation that describes the static

quenching of the ¯uorescence intensity of a ¯uoro-

phore [13]. The Stern±Volmer equation can be pre-

sented as

Fo=F � 1� Ksv�Q� (1)

where Fo�the initial ¯uorescence intensity of a

¯uorophore, F�the ¯uorescence intensity of the

Table 1

DOC concentrations in water samples and soil extracts used in this

study

Sample DOC (mg C/l)

Coniferous O horizon 173.6

Deciduous O horizon 31.8

Sedge marsh soil water 32.3

Blackman stream 8.5


¯uorophore which remains after its complexation

with a quencher, Ksv�the 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

CP� DOM$ CPÿDOM (2)

and the binding constant is

Ksv � �CPÿDOM�=�CP��DOM� (3)

where [CP], [DOM], and [CP±DOM] are the concen-

trations of the uncomplexed or free carbamates, DOM,

and CP±DOM complex, respectively.

3.2. Static and dynamic quenching mechanisms

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]

Fo=F � 1� kqto�Q� � 1� KD�Q� (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

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.


Fig. 3. Stern±Volmer Plots of carbofuran, carbaryl, aldicarb quenched by Aldrich humic acid after the correction of inner filter effects.


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

Ksv�0.94�104 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 0.47�104 l/mol (Mÿ1). From Eq. (4), we

have

KD � kq � to (5)

and

kq � KD=to (6)

The value of the bimolecular quenching constant kq of

a quencher cannot exceed 1�1010 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 Ksv�KD�0.47�104 Mÿ1.

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 4.7�1010 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 sys-

tem is static quenching.

3.3. Correction for the inner filter effects

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]

Fcor � cf � Fobs (7)

where Fcor, cf, and Fobs are the corrected ¯uorescence

intensity, the correction factor and the observed inner-

®lter-effect-quenched ¯uorescence intensity, respec-

tively. The correction factor is given by

cf � 2:3Aex�x10Aexx1

1ÿ 10ÿAex�x� 2:3Aem�y10Aemy1

1ÿ 10ÿAem�y(8)

where Aex and Aem are the absorbance of the DOM

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.


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 y1�0.14 cm and �y�0.64 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,

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 ¯uores-

cence 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 com-

ponents. Since fulvic acid is generally more hydro-

philic 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 carbo-

furan±DOM binding constants.

Table 2

Binding constants of carbofuran with DOM

Average Ksv Standard deviation

(104 l/kg) (104)

Stream

R1 1.65 0.09

(MW>1000)

R10 1.79 0.05

(MW>10 000)

P10R1 1.52 0.10

(10 000>MW>1000)

Wetland

Sedge 1.76 0.06

(Unfractionated)

R10 1.86 0.04

(MW>10 000)

P10R1 1.73 0.06

(10 000>MW>1000)

Coniferous O horizon

Con 1.45 0.03

(Unfractionated)

R10 1.68 0.17

(MW>10 000)

P10R1 1.64 0.07

(10 000>MW>1000)

P1 0.94 0.06

(MW<1000)

Deciduous O horizon

Dec 1.40 0.04

(Unfractionated)

R10 1.58 0.12

(MW>10 000)

P10R1 1.67 0.03

(10 000>MW>1000)


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 3.16�10ÿ3 M or 700 ppm [1]. The car-

bofuran molecule includes two parts: the benzofuranol

moiety and the methyl-carbamate moiety above the

benzene ring. The benzofuranol moiety is hydropho-

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 slits�5 nm, and emission slits�10 nm.


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 side-

chains, 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 frac-

tions of DOM samples, which have a higher composi-

tion of humic acids, should have larger carbofuran

binding constants and larger F395/F350 values.

Carefully examining Table 3, we ®nd that carbo-

furan has larger binding constants with aqueous (wet-

land and stream) DOMs which also have a higher F395/

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 excep-

tions: 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, theore-

tical 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 bond-

ing 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� (11)

the following relationship is obtained:

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 1.040 1.44

CR10 1.116 1.68

CP10R1 1.118 1.64

CP1 0.652 0.94

Dec 1.035 1.40

DR10 1.141 1.58

DP10R1 1.026 1.67

Sedge 1.158 1.76

SR10 1.339 1.86

SP10R1 1.308 1.73

BR1 1.285 1.65

BR10 1.402 1.79

BP10R1 1.234 1.52


�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 3.26�10ÿ5 kg/l (Table 1). The Ksv for

the wetland DOM and carbofuran is 1.76�104 l/kg

(Table 2). Substituting these two values in Eq. (12)

yields a Pb/Pt ratio of 36.5%. If a carbofuran concen-

tration 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 car-

bofuran that is bound to the DOM in the wetland

would be 36.5%�31.5�11.5 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, respec-

tively. 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 carbo-

furan to the stream and eventually lead to the spread-

ing 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 tech-

nique 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 carba-

mates 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 con-

stants 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 hor-

izon. 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 gen-

erally had greater carbofuran binding constants

than its unfractionated, P10R1, and P1 counter-

parts.

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 sam-

ples, 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 frac-

tions were more hydrophobic than lower MW

fractions. Hydrophobic adsorption and partitioning

and hydrogen bonding appear to be the major


binding mechanisms for the association of carbo-

furan 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 14-

08-G2023) for carrying out this research.

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