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ELSEVIER

Analytica Chimica Acta 327 (1996) 191-201

ANALYTICA

CHIMICA

ACTA

The interaction of some pesticides and herbicides with humic

substances

Nicholas Hesketh, Malcolm N. Jonesa**, Edward Tippingb

hool o Biological Science, University of Manchestel; Manchester Ml3 9PT, UK

bInstitute of Freshwater Ecology, Ambleside, Cumbria LA.22 OLE UK

Received 13 November 1995; revised 25 January 1996; accepted 29 January 1996

Abstract

The interaction of the pesticides, chlordimeform and lindane and the herbicides paraquat, 2,4-dichlorophenoxyacetic acid

and atrazine with humic substances (humic and fulvic acids) has been studied. Binding isotherms were measured by

equilibrium dialysis and used to derive the Gibbs energies of interaction of the biocide ligands with the humic substances.

Detection of binding at very low ligand concentrations (50 PM) was demonstrated by ultracentrifugation. Microcalorimetry

was used to measure the enthalpies of interaction as a function of ligand concentration. The data were interpreted using a

Langmuir-type model to obtain the enthalpies of interaction at saturation and the association constants. Both equilibrium

dialysis and microcalorimetry gave comparable specific Gibbs energies (Ag, J gg

)

of interaction for those systems (paraquat

and chlordimeform) where a complete thermodynamic analysis was possible. The specific Gibbs energies of binding of

paraquat and chlordimeform to aquatic fulvic acid were of the order of -5 Jg- and an order of magnitude larger than for

binding to peat humic acid.

Keywords: Pesticides; Herbicides; Humic substances

1 Introduction

Humic substances are the break-down products

of

plant material found in almost all terrestrial and

aquatic environments on the earths surface. They are

the major organic components of soils and sediments

and play a part in many of the physical, chemical and

geochemical processes in

the

natural environment,

including the binding and transport of herbicides and

pesticides. There have been several investigations on

the interactions of humics with biocides [l-13].

* Corresponding author. Fax: 0161 275 5082.

0003-2670/96/ 15.00 0 1996 Elsevier Science B.V. All rights reserved

PIZ SOOO3-2670(96)0008 l-5

This study is concerned with the interactions of the

pesticides chlordimeform [N-(4-chloro-o-tolyl)-N,N-

dimethylformamidine] and lindane [hexachlorocyclo-

hexane], and the herbicides atrazine [2-chloro-4-

(ethylamino)-6-(isopropylamino)-s-triazine ], 2,4-D

[2,4-dichlorophenoxyacetic acid] and paraquat

[methyl viologen-dichloride hydrate] with an aquatic

fulvic acid (WBFA2) and a peat humic acid

(WPHAl). The structures of these compounds are

shown in Fig. 1.

In aqueous solution paraquat is a divalent cation

which binds to humics by an ion exchange mechan-

ism [4,6] and a similar mechanism has been proposed

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192

N.

Hesketh et al./Analytica Chimica Acta 327 (1996) 191-201

cl

cl

0

u

cl

cl

cl

(II)Lindane

(m)A&

( IV ) 2,4-D

( v >Paraquat

Fig, 1. Structures of pesticides (I and II) and herbicides (III-V)

used in this study.

for the binding of chlordimeform, which ionises to

give a monovalent cation [8]. The weakly acidic 2,4-

D probably binds by hydrophobic interaction [6] as

has been proposed for the binding of lindane [2,3,7],

Cation exchange [ 13, hydrogen bonding [93 and

charge transfer interactions [lo] have been proposed

for the binding of atrazine to humic substances.

The weight average molecular masses (fi,) of the

humic substances used here have previously been

studied by the sedimentation equilibrium and

approach to equilibrium (Archibald) methods of

analytical ultracentrifugation [14,151. To study the

interactions of the herbicides and pesticides with the

humics, the techniques of equilibrium dialysis, micro-

calorimetry and the Archibald method of ultracen-

tifugation were used, which have not previously

been used to study these particular interactions,

although equilibrium dialysis has been used to

measure the binding of DDT to dissolved humic

materials [l l] and the binding and dissociation

interactions between polycyclic aromatic hydrocar-

bons and dissolved humic material [12]. Studies of

the fate of atrazine in alluvial sediments [ 131 and its

interactions with clay-minerals [14] and lindane with

fulvic acid [15] have been reported and the interac-

tions of pesticides with soils have been reviewed by

Gamble et al. [16]. Microcalorimetry has been used

to study the adsorption of prometryne, a member of

the triazine family of herbicides, to humic acid 1171.

2. Experimental

2 1 Extraction procedures

Surface water was taken from a stream draining

a peat-dominated hill-slope (Whitray Fell in NW

England, ordinance survey sheet SD66/76, High

Bentham and Clapham, grid reference 6090 North

to South, 6835 East to West). Peat was also taken

from this location. The isolation and characterisation

of the fulvic acid (WBFA2, molecular mass 2300)

from this source has been previously described [ 181.

The peat humic acid sample, which has a molecular

mass of 40500 [19], was isolated by Reid et al. [20].

The methods of isolation were an adaptation of

the International Humic Substances Society (IHSS)

method for extracting soil and aquatic humic sub-

stances [21]. The samples were stored freeze-dried in

a vacuum desiccator.

2.2. Materials

All solutions were made up in doubly distilled or

de-ionised water and all salts (sodium chloride,

sodium hydroxide, hydrochloric acid, Tris and

phosphate buffer salts) were of analytical grade.

Chlordimeform, 2,4-D, lindane and paraquat were

purchased from Aldrich. Atrazine was purchased

from Riedel-de-Haen.

2.3. Equilibrium dialysis

A solution of the peat humic acid WPHAl (0.2%

w/v) was made in 1 mM NaOH, and sonicated for

several hours. The pH of aliquots of the solution were

adjusted to 5, 7 and 10 using 10M HCl. This

procedure was also used for aquatic fulvic acid

(WBFA2) to give a 0.1% (w/v) solution. Solutions of

paraquat (O-50 mM), chlordimeform (O-50 mM), and

2,4-D (U.8 mM, maximum solubility) were made

up in 1 mM NaOH adjusted to the required pH (5,7

and 10) by addition of 10 mM HCl.

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N. Hesketh et al./Andytica Chimica Acta 327 (1996) 191-201

193

SpectraPor CE dialysis tubing (molecular weight

cut-off 500) was washed with doubly distilled water

to remove the sodium azide preservative. Humic/

fulvic solutions (2 ml) were pipetted into the dialysis

bags made using SpectraPor dialysis clips. Each

dialysis bag was placed in a glass flask along with

12ml of NaCl solution at the concentration required

and pre-dialysed for 24 h [22], to allow the system to

equilibrate with solvent. The NaCl outside the bag

was replaced with pesticide solution (12 ml) and the

samples placed in a water bath at a temperature of

25C. It was found in separate experiments that

dialysis equilibrium was attained after 4 days for

paraquat, 2 days for 2,4-D and 4 days for chlordime-

form. For each experiment a control (no pesticide)

was also set up. To check that there was no leakage of

humic solution the UV absorbance of the blank

dialysate was measured against NaCl solution of the

appropriate concentration. No leakage was detected

in any of the experiments.

Analysis of the samples was achieved using a

Cary 219 spectrophotometer at a wavelength of

262 nm for paraquat, 285 nm for 2,4-D and 244 nm

for chlordimeform. Aliquots (1 ml) of the pesticide

solution from outside the dialysis bags were taken,

diluted using NaCl solution and their absorbances

measured.

A calibration experiment was performed using the

pesticide solutions covering the required concentra-

tion range. Each dialysis experiment was done in

triplicate at the different conditions of pH.

2.4. Ultracentrifugation (Archibald approach to

equilibrium)

Measurements were made using a Beckman L8-70

ultracentrifuge fitted with a UV scanner covering an

absorbance range of O-l at 280nm. The samples

were contained in 12mm aluminium filled Epon

double sector centre-pieces with quartz windows. The

sample WBFA2 was diluted using 0.5 M NaCl plus

0.1 M phosphate buffer pH 7 so that the absorbances

were 0.2, 0.4 and 0.6, respectively. These absor-

bances correspond to concentrations to 0.0375,

0.0750 and 0.1125 mg ml-. The high salt concentra-

tion helped prevent convective mixing in the cell.

Samples were placed in one sector (well) of a two

sector cell (centre-piece) and solvent in the other. The

sectors are parallel to one another. The centre-piece is

located in the centrifuge rotor, and as it spins the

solute in the sample sediments towards the sector

base away from the solution meniscus. The absor-

bance measured by the instrument is the difference

between the absorbances of the two sectors so that the

movement of the solute away from the meniscus as it

sediments can be followed. The fluid column length

was approximately 1.1 cm.

The principle of the Archibald method depends on

the fact that equilibrium between the rate of

sedimentation and the rate of diffusion is attained at

all times at the meniscus (and the base of the sector).

Thus by following the concentration gradient at the

meniscus, (&/ar),, the weight average molecular

mass (ti,) can be calculated from the equation for

sedimentation equilibrium [23];

(1)

where m refers to the meniscus, c, is the concentra-

tion at the meniscus, R the gas constant, T the

absolute temperature, c2 the partial specific volume, p

the solution density, w the angular velocity, c the

concentration,

r

the radius from the centre of rotation

of the rotor, and (Ck/&), the concentration gradient

at the meniscus. The centrifuge was run at a number

of speeds (12 000-50 000 rpm), and once the required

speed was reached, scans of the solute distribution in

the cells were taken at 1Omin intervals for approxi-

mately 3 h. To calculate &f, it is necessary to

determine c, and (&l&),. In order to do this the

absorbance profiles were digitised and fitted to

second-degree polynomials of absorbance

A

as a

function of radius r. The parameters of the

polynomial were used to obtain absorbance at the

meniscus A, and the initial slope (dA/dr), since,

(2)

Plots of iii, as a function of time were extrapolated

to zero time, which was taken to be the time the

centrifuge reached the set speed, to give the weight

average molecular weights of the samples. The

method depends on the fact that the conditions of

sedimentation equilibrium are realised at the ends of

the fluid column at all times during the centrifugation

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194 N

Hesketh et al./Analytica Chimica Acta 327 (1996) 191-201

run. The advantage of the method when dealing with

humics is that measurements are made before any

material is lost from the concentration gradient and so

the weight average molecular mass should be close to

that of the whole sample. A value of 0.58 cm3 g-i was

used for the partial specific volume of the samples

[ 19,201.

2.5. Microcalorimetry

Enthalpy measurements were made at 25C using a

LKB-Produckter microcalorimetry system. Solutions

(0.05% w/v) of peat humic acid (WPHAl) or aquatic

fulvic acid (WBFA2) were made in Tris buffer (pH

9.61), and sonicated for 1 h. The pH of the Tris and

humic solutions were adjusted to pH 7 using 10 M

HCl. Before use the solutions were dialysed using

Spectra/Par CE dialysis tubing bags (molecular

weight cut-off 500) as described above. The bags

were placed in 11 glass measuring cylinders with 11

of Tris buffer and dialysed for 3 days, after which the

Tris was replaced with 11 of fresh Tris buffer and left

to dialyse for another 3 days. After the dialysis was

completed the humic solutions were vacuum filtered.

The filter paper (Whatman No. 1) was oven-dried

before filtration, and dried again immediately after

filtering the humic solution. The exact amount of

undissolved humic material could then be deter-

mined. Solutions of paraquat, 2,4-D, and chlordime-

form were prepared in 50mM Tris buffer.

The microcalorimeter had twin bicompartmental

cells. The sample cell was charged with 2f0.01 g of

pesticide solution of the required concentration and

2f0.01 g of humic/fulvic solution. The reference cell

was charged with 2f0.01 g of biocide solution

identical to the sample cell and 2f0.01 g of buffer

solution. On mixing the enthalpy of dilution of the

biocide solutions cancel. Solutions were then left

to equilibrate, usually for 4 h. The heats of dilution

of the humiclfulvic substances were measured

separately and used to correct for this enthalpy

effect.

The microcalorimeter was calibrated electrically at

frequent intervals during the course of the study. On

the most sensitive range used for the measurements

(3OuV), the mean sensitivity of the detectors in the

heat sinks of the two vessels was 14.66f0.32 pW/pV

~241.

3. Results

3 1

Binding isotherms

The binding isotherms for chlordimeform, 2,4-D,

and paraquat binding to peat humic acid WPHAl and

aquatic fulvic acid WBFA2, as measured by equili-

brium dialysis, are shown as the average number of

ligand molecules bound per humic molecule (V)

plotted as a function of log[ligand]r,,, in Figs. 2-4.

The term ligand used here and below refers to the

biocide molecules, since these are of much lower

molecular masses than the humic substances to which

they are binding, the concept of ligands binding to a

focal large macromolecule seems appropriate. The

data relate to solutions of low ionic strength (1 mM

NaCl). Very similar data (not shown) were obtained

at higher ionic strength (10 mM NaCl) for the peat

humic acid WPHAl with the three biocides. The

higher binding levels at a given free ligand concen-

tration for the peat humic acid reflect the higher

molecular mass. In terms of moles of ligand bound

per gram of humic/fulvic the levels are comparable

for both materials and in the ranges up to 2-7 mmol g-

(WPHAl) and 2-10mmol g- (WBFA2). For the

binding of the cationic chlordimeform, the isotherms

are shifted to lower free ligand concentrations at pH

10 for both humic and fulvic acids. In contrast, for

2,4-D, only the fulvic acid isotherms are affected by

pH, the pH 10 isotherm being shifted to higher free

ligand concentration, consistent with weaker binding

of the 2,4-D anion when the fulvic acid is most

negatively charged. Paraquat behaves similarly to

chlordimeform, binding most strongly to the humic

acid at pH 10 but there is no dependence of binding

on pH for the fulvic acid.

3.2. Ultracentrifugation

The Archibald method of ultracentrifugation was

used to estimate the levels of binding of the ligands

atrazine, chlordimeform, 2,4-D, lindane and paraquat

to aquatic fulvic acid WBFA2. An example of the

measurements of molecular mass as a function of

time for WBFA2 and WBFA2 plus paraquat is shown

in Fig. 5 and the data for all the ligands are

summarised in Table 1. The ligand concentrations

used were 50 pM in all cases, and the fulvic acid

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N. Hesketh et aI./Analytica Chimi ca Acta 327 1996) 191-201

195

120 -

A)

100 -

15 ao-

A

pH5

A

60 -

n

pH7

/

40

A pH10

A

20

0

j ,:,d,

1

10

a

6

I>

4

2

0

I

I

I I

-5 -4 -3 -2

-1 0

-5

4

-3

-2

-1 0

Log IChlordimeform] mbl)

Log

Chlordimeform] mM)

Fig. 2. Binding isotherms (Y is the average number of Iigands bound per molecule of humiclfulvic) for the binding of chlordimeform to: (A)

peat humic acid WPHAl (2 gl-I); and (B) fulvic acid WBFA2 (1 gl-) in aqueous sodium chloride (1 mM) at 25C.

30

25

20

IS 15

IO

5

0

(A)

i

I I I

6

5

4

IS 3

2

1

0

-5

-4

-3 -2 -1

-5

-4 -3 -2

-1

Log PA-D1 mM) Log [2.4-D] mM)

Fig. 3. Binding isotherms (Y is the average number of ligands bound per molecule of humic/fulvic) for the binding of 2,4-dichloro-

phenoxyacetic acid [2,4-D] to: (A) peat humic acid WPHAl (2 g 1-l); and (B) fulvic acid WBFAZ (1 g 1-l) in aqueous sodium chloride (1 mM)

at 25C.

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196

N. Hesketh et al./Anulytica Chimi ca Acta 327 1996) 191-201

100

80

60

IZ

40

20

0 -r

. pH5

w pH7

A pHl0

.d 8

n

*/t

8

0 )

I

I

I

I

I

-1

-5

-4

-3

-2

-1

0

-5 -4 -3 -2

-1 0

Log [Paraquat] (mM)

Log [Paraquat] (mM)

Fig. 4. Binding isotherms (G is the average number of ligands bound per molecule of humic/fulvic) for the binding of paraquat to: (A) peat

humic acid WPHAl (2 g 1-r); and (B) fulvic acid WBPA2 (1 g lee) in aqueous sodium chloride (1 mM) at 25C.

j5m

20 -

15 -

1s

10 -

5-

(B)

0 pH5

= pH7

A PHlO

i

A

I

10000

-r

8000

WBFAZ

Cl WBFK2 + Paraquat

6000

12

4000

,

0

2000

0 /

I

I

I

I

/

0

50

100

150

200

250

300

Time minutes)

Fig. 5. The effect of paraquat on the weight-average molecular

mass of fulvic acid WBFAZ determined by the Archibald method.

The apparent molecular mass at the meniscus is extrapolated to

zero time to obtain the molecular mass of the fulvic acid in the

absence (&fw=2341~114) and presence of 50uM paraquat

(&,,=3341f156).

concentration was 0.0375 mg ml-. Although there

was scatter in the data as exemplified by Fig. 5, in all

cases an increase in molecular mass was detected in

the presence of the ligands. Low ligand concentra-

tions (50 PM) were used because, in the case of

atrazine and lindane, of their low solubilities, and

in the case of chlordimeform, 2,4-D, and paraquat

because their UV absorbance wavelengths are close to

that used to follow the distributions of the fulvic acid

in the ultracentrifuge. The results show that binding

for paraquat, a divalent cation, is larger than for the

other biocides.

3.3.

Microcalorimetry

Microcalorimetry was used to measure the

enthalpies of interaction between the ligands atra-

zine, chlordimeform, 2,4-D, lindane and paraquat

with peat humic acid WPHAl and aquatic fulvic acid

WBFA2. The enthalpies of interaction between the

ligands and the humic substances as a function of

total ligand concentration are shown in Figs. 6 and 7.

For chlordimeform and paraquat the enthalpies

reach a plateau value. For 2,4-D, enthalpy data could

only be obtained up to the 2,4-D maximum solubility.

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N. Hesketh et al./Analytica Chimica Acta 327 (1996) 191-201

197

Table 1

The binding levels of pesticides and herbicides to fulvic acid WBFA2 determined by the Archibald method of analytical ultracentrifugation

Ligand

WBFA2 .&I,

WBFA2&,

Increase in IV, Ligand Molecular

Molecules bound

Weieht (al

oer molecule

Atrazine

Chlodimefotm

2.4-D

Lindane

Paraquat

2341f114

2784f255

443f279 215.69

2.05k1.29

2314f114

2392f203

51f233 196.68 0.26zt1.18

2314f114

2857f266 516f289 221.04

2.33*1.13

2314f114

2631f238

290f264

290.83

10010.91

23146114

3341f156

lOOOf

257.15

3.89zt0.75

50

40

iij 30

3

B

2

5 20

10

l-

A)

2.4-D Enddhermic )

(B)

00

O 4

I

0 1 2 3 4

0 1 2 3

Total [Ligand] (mbl)

Total [Ligand] (mM)

Fig. 6. Enthalpies of interaction of 2,4dichlorophenoxyacetic acid with: (A) peat hunk acid WPHAl (0.25gl-I); and (B) fulvic acid

WBFA2 (0.25 gl-) in aqueous Tris buffer (47.6 mM) pH 7 at 25C.

This also applied to the interactions of atrazine and

lindane with the humic substances. However, for these

biocides the enthalpies of interaction were also small.

The enthalpies measured for atrazine and lindane

interactions (at maximum solubility) with WPHAl

being +129+23 and +147f17kJmol-, and with

WBFA2 -9f4 and +2&l kJ mol-, respectively.

4. Discussion

(Fig. 5, Table 1) demonstrate that small numbers of

biocide molecules bind to the humics at very low free

biocide concentrations much lower than can be

measured by the equilibrium dialysis technique. The

binding isotherms (Figs. 2-4) were analysed using

the binding potential concept proposed by Wyman

[25], in which the binding potential II(P,T,~,,~z. . .)

at pressure P and temperature T is related to the

binding (z?) and the chemical potential of the ligand

(p) as follows:

The technique of equilibrium dialysis has demon-

strated the binding of pesticides and herbicides to

humic substances. Also the ultracentrifugation data

drI

u = @

-)

P,T

(3)

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198 N. Hesketh et al./Analytica Chimi ca Acta 327 1996) 191-201

I

+

I/f

T

I

0 Paraqua exothermlc)

Chbrdtmefwm exothermic)

16

14

12

3

10

3

B

m

8

5

5

8

I

8)

I

0 Paaqwt UOthemllC)

m Chbrdlmeform exothermb)

0

5

10 15 20 25 30

0

IO

20

30

40

50

Total [Ligand] mM)

Total [Ligand] mM)

Fig. 7. Enthalpies of interaction of paraquat (0) and chlordimeform (B) with: (A) peat humic acid WPHAl (0.25 g 1-l); and (B) fulvic acid

WBFA2 (0.25 g 1-l) in aqueous Tris buffer (47.6 mM) pH 7 at 25C.

If the chemical potential of the ligand is given by

p=pO+RTln [L], the binding potential can be obtained

by integration of the binding isotherms, since from

(3) it follows:

ii

I I=RT

J

7ln

[L]

(4)

0

The integration was carried out after fitting the

isotherms to polynomials. The binding potential is

related to the apparent association constant (K,) at a

given ii by [26]

II = RTln(1 +Ka[L])

(5)

from which it is possible to calculate

K,

at a given V

and hence AG,, the Gibbs energy of binding per

ligand bound from,

AG, = -TlnK,

Fig. 8 shows the Gibbs energies per ligand bound

(AG,) as a function of ligands bound (V) for

paraquat binding to the humic substances, very

similar data were obtained for chlordimeform and

2,4-D.

The curves show initial high energy binding at low

V, the energies decrease with increasing V as the

humic substances bind more ligand. The range of

Gibbs energies are -21- -10 kJ mol- ligand

bound for chlordimeform and paraquat are -23-

-16kJ mol- ligand bound for 2,4-D.

The microcalorimetric results can be interpreted

assuming that an n:l complex is formed between

ligand [L] and humic substance [HI, so at equilibrium

nL+H HL,,.

(7)

Eq. (7) leads to the following Langmuir-type

equation [27] for AH,

AH =

AHsdLl

Kd + [L]

where AH is the enthalpy of interaction, AH,, the

enthalpy of interaction at saturation and Kd the

dissociation constant. The data were fitted to Eq. (8)

to determine AH,,, and

Kd

using the MULTIFIT

programme [28].

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199

-8

-18

(A)

PH 5

n

PH 7

A

pH 10

I

I I I

-a

-16

pH5

n

pH7

A

pH 10

0 20

40 60

80 100

0 5

10 15 20 25

molecules bound per molecule

molecules bound per molecule

Fig. 8. Gibbs energies per ligand bound as a function of binding level (V, molecules bound per molecule of humic/fulvic acid) for the binding

of paraquat to: (A) peat humic acid WPHAl; and (B) fulvic acid WBFA2 in aqueous solution (1 mM NaCl) at 25C.

Table 2

Thermodynamic parameters at saturation for the interactions of herbicides and pesticides with peat humic acid WPHAl and fulvic acid

WBFA2 [Tris buffer (47.6 mM), pH 7, 298 K]

Ligand

AZ?&, (kJ/mol

K. (M-l)

AC? (kJ/mol

TA.%,,

AS,, (J/K/m01

(mM) humic of fulvic) humic or fulvic) humic or fulvic)

WPHAl-Atrazine

WPHAl-Chlordimeform

WPHAl-2,4-D

WPHAl-Lindane

WPHAl-Paraquat

WPHAZAtrazine

WPHA2-Chlordimefomr

WBHA2-2,4-D

WBFA2-Lindane

WBFA2-Paraquat

+129~t23~

-611&105 107f44 -12fl -599f105 -2OlOf349

+1817f99a

+147*17

-547116

4308f588

-2lztl

-526f15 -1756f50

-9zt4

-59f33 21f18 -8f5

-5lf33 -171flll

+9fl

+2fla

-2lf2 821f287 -17+1 -4f2

-13f7

aEnthalpy of interaction at maximum solubility,

The Gibbs energies of association were calculated

from Kd (=1/K,) using

AC? = -RTlnK,.

(9)

The results calculated using Eqs. (8) and (9) are

shown in Table 2 together with the entropies of

interaction calculated from AGc=AH,,--TAS,,,.

The enthalpies of interaction of chlordimeform and

paraquat with WPHAl and WBFA2 are exothermic

and assist association. The enthalpies for 2,4-D and

lindane are endothermic, not favouring association.

Since 2,4-D is anionic in solution at pH 7, association

with humic substances is not favourable electrosta-

tically. The strength of binding of paraquat to both

WPHAl and WBFA2 is much greater than for

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200

Table 3

N. Hesketh et al./Analytica Chimica Acta 327 (1996) 191-201

Specific thermodynamic parameters (pH 7) of interaction between chlordimeform and paraquat with peat humic acid WPHAl and fulvic acid

WBFA2

Ligand (mM) Iw,,, (J/g humic AS,,, (J/K/g humic

or fulvic)

or fulvic)

Ag (calorimetry)

(J/g humic or fulvic)

Ag (equilbrium dialysis)

(J/g humic or fulvic)

WPHA 1 Chlodimerform

WPHA 1 Paraquat

WBFA2-Chlordimeform

WBFA2-Paraquat

-15.1f2.6 -0.052f0.010 -0.296+0.025 -0.338

-13.5f0.4 -0.044*0.003 -0.51910.025 -0.316

-25.1f14.1 -0.073f0.048 -3.42f2.14 -5.32

-9.1f0.7 -0.006zt0.001 -7.26f0.43 -4.72

chlordimeform as would be expected from its

divalency. In all cases the associations occur with a

decrease in entropy. Because of the large difference

in molecular mass of WPHAl and WBFA2

(approximately 40500 and 2300, respectively) it is

appropriate to compare the thermodynamic data in

terms of specific parameters i.e. per gram. Since the

values of K, calculated as discussed above are

average values over the ligand concentration range

in order to compare them with the data from the

binding isotherms at pH 7 a ii-average specific Gibbs

energy was calculated as defined by

(10)

The data in Table 3 show that both the calorimetric

and binding measurements give comparable results

for the specific Gibbs energies. It is seen that the

Gibbs energies of binding of both chlordimeform and

paraquat to the fulvic acid are significantly larger

than to the humic acid, this may well relate to the

high negative charge per unit mass for the fulvic acid

as compared to the humic acid [29].

5 Conclusions

Equilibrium dialysis in combination with micro-

calorimetry can be used to determine the thermo-

dynamic parameters for biocide interactions with

aquatic fulvic and peat humic acids. Ultracentifuga-

tion studies have demonstrated biocide binding at

very low biocide concentrations. The enthaplies of

interaction with the peat humic acid are endothermic

for atrazine, 2,4-D and lindane and exothermic for

chlordimeform and paraquat. The enthalpies of

interaction with aquatic fulvic acids follow a similar

patten but are much smaller and in the case of

atrazine almost athermal. Where a complete thermo-

dynamic analysis was possible (for the cations

chlodimeform and paraquat) interactions occurred

with a decrease in entropy. There was an order of

magnitude increase in the specific Gibbs energies of

interaction for binding of the biocides to aquatic

fulvic acid as compared to the peat humic acid. This

result suggests that in the environment retention of

biocides by fulvic acids would be greater, however,

aquatic fulvic acids being more mobile, could loose

biocide more easily on dilution in lakes and rivers.

Acknowledgements

We thank the NERC for a CASE studentship for

NH.

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