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GEOCHEMICAL MODULATION OF BIOAVAILABILITY AND
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MICHAEL GENE ROBERTS

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GEOCHEMICAL MODULATION OF BIOAVAILABILITY AND TOXICITY OF
NITROAROMATIC COMPOUNDS TO AQUATIC PLANTS

By

MICHAEL GENE ROBERTS

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Crop & Soil Science

2006

ABSTRACT
GEOCHEMICAL MODULATION OF BIOAVAILABILITY AND TOXICITY OF
NITROAROMATIC COMPOUNDS TO AQUATIC PLANTS
By

MICHAEL GENE ROBERTS

Nitroaromatic compounds (NACS) are a prominent class of environmental toxic
contaminants that pose a threat to human and ecosystem health. Nitroaromatic
compounds demonstrate a high affinity with smectite clays which are widely distributed
in nature as components of soils and sediments, and in geological deposits. The
magnitude of NAC sorption by smectites is highly dependent on the exchangeable cation.
For example, smectites saturated with K+ have a very high affinity for NACs while those
saturated with Ca2+ do not. In this study the ability of K+-saturated smectite to attenuate
the bioavailability and hence toxicity of 2,4-dinitrotoluene (2,4-DNT) to the aquatic plant
duckweeds (Lemna gibba and Lemna minor) was evaluated. 2,4—DNT at the
concentration of 7.5 mg/L was highly toxic to duckweeds. However, the presence of
small amounts of K-smectite in aqueous growth media reduced this toxicity substantially
because the sorption of 2,4-DNT by the added smectite reduced bioavailability. This
reduced bioavailability could be modulated by simple K+/Ca2+ cation exchange reaction
occurring on mineral surfaces. In the subsequent experiments, CaClz was added and the
Ca2+ replaced K+ associated with smectite surfaces. Such reaction released clay-sorbed
NACs to aqueous solution, which is readily available to target plants. This study proves

the concept that we can modulate the type and fraction of cations occupying the cation-

exchange-capacity sites of smectite clays to control the sorption/desorption hence
bioavailability of NACs in smectite-rich soils and sediments. This information is needed
in developing strategies for control of NAC mobility and bioavailability to target
organisms such as plants and microorganisms utilized in in—situ bioremediation

technologies.

DEDICATION

This work is dedicated to my daughter, Alyssa Marie, wife Jennifer, who have support
and patienc has made this goal possible and my mother Annie Mae Roberts and family,
who always sacrificed for my education. In the memories of my father, Clarence Roberts
Sr. (July 16, 2005) and my grandmother, Susie Robinson Callier (January 23, 2006).

ACKNOWLEDGEMENTS

I would like to express my appreciation to my advisor, Dr. Stephen Boyd, for his
guidance, support, and encouragement for my study. This dissertation could not have
been completed without his help and support. I thank my guidance committee, Dr. Brian
Teppen, Dr. Clayton Rugh, and Dr. Thomas Voice for their insight and helpful
comments.

Special thanks is given to numerous people, especially, Dr. Hui Li for his sincere
friendship and valuable assistance with all aspects of my research, Vaneet Aggarwal,
Sarah Kinder, Senthil Subramanian, Tanya Pereira, Cun Liu for their friendship and
several others, who have encouraged and supported me. I give thanks to Dr. James Jay,
Dr.Robert Taylor, Dr. Senwo and Dr. Loston Rowe for their sincere friendship.

I want to acknowledge the College of Agriculture and Natural Resources and
Crop and Soil Sciences for their financial support. This research was supported in part by
National Research Initiative Competitive Grant no. 2003-35107-12899 and 2005-35107-
15237 fi'om the USDA Cooperative State Research, Education, and Extension Service,
and the Michigan Agricultural Experiment Station. We thank Dr. Thomas Pinnavaia at
Department of Chemistry, Michigan State University for measuring mineral surface area.
Finally, appreciation is extended to my daughter, wife, family, friends and professors for

their support and patience throughout this program.

TABLE OF CONTENTS

CHAPTER I: SORPTION OF NITROAROMATICS BY AMMONIUM- AND
ORGANIC AMMONTUM -EXCHANGED SMECTITE SHIFTS FROM
ADSORPTION/COMPLEXATION TO A PARTITION-DOMINATED

ABSTRACT .................................................................................................................. 2
INTRODUCTION ................................................................................ 4
MATERIALS AND METHODS ..................................................................................... 9
RESULTS AND DISCUSSION ..................................................................................... 12
SUMMARY ............................................................................................................... 27
REFERENCES ........................................................................................................... 28

CHAPTER II: REDUCING BIOAVAILABILITY AND PHYTOTOXICITY OF
NITROAROMATIC COMPOUNDS BY SORPTION ON K-SMECTITE

ABSTRACT .............................................................................................................. 32
INTRODUCTION ...................................................................................................... 33
MATERIALS AND METHODS ................................................................................. 38
RESULTS AND DISCUSSION ................................................................................. .42
REFERENCES ......................................................................................................... 53

CHAPTER III: GEOCHEMICAL MODULATION OF BIOAVAILABILITY AND
TOXICITY OF NITROAROMATIC COMPOUNDS TO AQUATIC

PLANTS ......................................................................................... 57
ABSTRACT ............................................................................................................. 58
INTRODUCTION ...................................................................................................... 59
MATERIALS AND METHODS ................................................................................. 64
RESULTS AND DISCUSSION .................................................................................. 70
REFERENCES ........................................................................................................ 85

vi

LIST OF TABLES

CHAPTER I

Table 1. Properties of reference smectite (SWy-2) and SWy—2 exchanged with
ammonium, trimethylammonium (TMA), trimethylphenylammonium (TMPA)
and hexadecyltrimethylarnmonium (HDTMA) ................................... 13

Table 2. Freundlich sorption isotherm parameters Kr (mg 1'” LN/ kg), N and r2 for 2,4-
dinitrotoluene, 1,3-dinitrobenzene and naphthalene by smectite clay (SWy-2)
exchanged with ammonium, tetramethylammonium (TMA),
trimethylphenylammonium (TMPA) and hexadecyltrimethylammonium
(HDTMA). Standard errors are reported in parentheses ......................... 19

Table 3. Freundlich sorption isotherm parameters K; (mg "N LN/ kg), N and r2 for 1,3-
DNB and 2,4-DNT by tetramethylammonium (TMA)- and
hexadecyltrimethylarnmonium (HDTMA)-smectite (SWy—2) in binary solute
systems. The standard errors are reported in parentheses ........................ 26

CHAPTER II

Table 1. Growth rate constant (k) and doubling time (T2) of Lemna minor and Lemna
gibba exposed to several levels of aqueous phase 2 ,4-dinitrotoluene (2 ,4-DNT)
during 12 days of incubation ......................................................................... .46

Table 2. Growth rate constant (k) and doubling time (T2) of Lemna minor exposed to
aqueous-phase 2,4-dinitrotolune (2,4-DNT) in the presence and absence of k-
smectite during an 11 day exposure period ......................................... 51

CHAPTER 111

Table 1. Growth rates (k) and doubling times (T2) of Lemna minor during 12 day
incubations in 1/5 X DWM containing added cations K+ and Ca at different
molar concentrations. Doubling time were calculated using: T2 (days)= (ln
(2)/k) .................................................................................... 74

Table 2. Growth rate (R) and doubling times (T2) of duckweed Lemna minor during 17
day incubations; values derived from measurements of fronds and biomass
produced. Doubling time (days) calculated using: T2 (days) = (1n (2)/k).
Treatments on day 4 consisted of 0.01M KCl or 0.1M CaCl2 added to 1(-

vii

Table 3.

smectite suspensions containing, 2,4-DNT and equal number of fronds.
Controls were identical but did not contain 2,4-DNT. Addition of Ca2+ caused
release of sorbed 2,4-DNT into solution resulting in decreased frond numbers
and biomass .......................................................................... 82

Comparison of duckweed frond numbers (average of four samples) at different
times in K-smectite suspensions with 2,4-dinitrotoluene (2,4-DNT) and no 2,4-
DNT present. At day 4, KC] or CaCl2 solutions were added to the suspensions
and the incubation was continued until 17 days. The standard errors of the
means were all 5 1%. The AN OVA was applied to determine the statistical
significance (P<0.05) for differences in treatments .............................. 84

viii

LIST OF FIGURES

CHAPTER I

Figure 1. Sorption isotherms of nitroaromatic compounds (NACS) (a) 2,4-dinitrotoluene
(2,4-DNT) and (b) 1,3-dinitrobenzene (1,3-DNB) by smectite (SWy-2)
saturated with ammonium, tetramethylammonium (TMA),
trimethylphenylammonium (TMPA) or hexadecyltrimethylammonium
(HDTMA) .............................................................................. 16

Figure 2. Sorption isotherms of naphthalene (NAPH) by smectite (SWy-2) saturated with
ammonium, tetramethylammonium (TMA), trimethylphenylammonium
(TMPA) or hexadecyltrimethylammonium (HDTMA) ........................... 21

Figure 3. Sorption isotherms of nitroaromatic compounds (NACS) (a) 2,4-dinitrotoluene
(2,4-DNT) and (b) 1,3-dinitrobenzene (1,3-DNB) by
hexadecyltrimethylammonium (HDTMA)-smectite (SWy—2) in single- and
binary-solute systems ................................................................... 23

Figure 4. Sorption isotherms of nitroaromatic compounds (NACS) (a) 2,4-dinitrotoluene
(2,4-DNT) and (b) 1,3-dinitrobenzene (1,3-DNB) by tetramethylammonium
(TMA)-smectite (SWy-2) in single- and binary-solute system ....................... .25

CHAPTER 11

Figure 1. Growth of two species of duckweed over a 12 day incubation time as function
of different solution concentrations (0 to 20 mg/L) of 2,4-dinitrotoluene.. . ....43

Figure 2. Photograph showing reduced bioavailability and toxicity of 2,4-dinitrotolune
(2,4-DNT) to Lemna minor in the presence of homoionic K— montrnorillonite
(K-SWy-Z) clay (300 mg clay in 150 mL solution). The photograph was taken
after an incubation period of l ldays. Treatments consisted of various
combinations of 2,4-DNT and/or K-SWy-Z ........................................ 48

Figure 3. Effects of 2,4-dinitrotoluene (2,4-DNT) and clay on the growth rate of Lemna
minor in 1/5X DWM over a incubation period of 11 days. Treatments were:
7.5 mg/L 2,4-DNT; 7.5 mg/L 2,4-DNT (added) plus homoionic K-
montrnorillonite (K-SWy-Z); K-SWy-2; control containing neither 2,4-DNT
nor K-SWy-2 .................................................................................................... .50

CHAPTER 111

Figure 1.

Figure 2.

Figure 3.

Adsorption of 2,4-DNT by K-smectite suspensions in presence of
0.01M KC] or 0.005 M CaCl2 ......................................................... 71

Growth of duckweed after 12 day bioassay in presence of different electrolyte
solutions at concentrations ranging from 0.01 and 0.20M. The electrolytes
chosen contain cations that might be used to control binding (NHX, K”) and
release (CaZ+) of nitroaromatic compounds by smectite clay ..................... 75

The growth of Lemna minor in the presence of 2,4—dinitrotoluene (2,4-DNT)
and K-smectite as affected by addition (day 4) of KCl or CaCl2 solutions.
Growth and toxicity are indicated by fi'ond numbers (A) and biomass (B).
Aqueous concentrations of 2,4-DNT during the 17 day growth experiment are
show in panel (C) ...................................................................... 77

Figure 4. The effects of 2,4-dinitrotoluene (2,4-DNT) in K-smectite suspensions on

duckweed (Lemna minor) growth. Duckweed fiond numbers were equal in
presence (A) or absence (B) of 2,4-DNT on day 4 when either KCl or CaCl2
solution were added. The effects of these treatments after 17 day incubations
are shown in panel (C). Addition of CaCl2 caused release of 2,4-DNT into
solution where it inhibited growth .................................................... 79

“Images in this thesis/dissertation are presented in color.”

CHAPTER I
Sorption of Nitroaromatics by Ammonium- and Organic Ammonium-Exchanged

Smectite Shifts from Adsorption/Complexation to a Partition-Dominated Process

Abstract
Nitroaromatic compounds (NACS) are components of munitions commonly found

as soil contaminants at military training sites and elsewhere. These compounds pose
adverse effects on human health and ecological systems. Recent studies indicate these
compounds are strongly retained by smectite clays. The adsorption mechanisms are not
fully reconciled, but it is known that the type of exchangeable cation strongly affects
NAC affinity for smectites. This study examined the sorption of 1,3-dinitrobenzene, 2,4-
dinitrotoluene and naphthalene from water by a smectite clay (SWy-2) saturated with
ammonium, tetramethylammonium (TMA), trimethylphenylammonium (TMPA) and
hexadecyltrimethylammonium (HDTMA). In all cases, we observed greater sorption of
2,4-dinitrotoluene compared with 1,3-dinitrobenzene. The sorption isotherms for 2,4-
dinitrotoluene and 1,3-dinitrobenzene displayed a concave-downward curve for NH..—
SWy-2 and TMA-SWy-2, whereas the isotherms for sorption of HDTMA-SWy-Z and
TMPA-SWy-2 were essentially linear. The magnitude of sorption (sorbed 2, 4-DNT
concentration at Ce = 40 mg/L in parenthesis) followed the order: NH4-SWy-2 (40,165
mg/kg) > TMA-SWy-2 (23,370 mg/kg) > TMPA-SWy-2 (8,922 mg/kg) > I-IDTMA-
SWy—2 (6,005 mg/kg) for both compounds. The greater affinity of NACs for NFL. -and
TMA-SWy-2 is due in part to complex formation between the exchangeable cation and —
N02 groups. These clays also provide near optimal interlayer distances that approximate
the molecular thickness of NACs hence promoting the simultaneous interaction of the
planar aromatic rings with opposing siloxane surfaces, and solute dehydration. Both
processes are energetically favorable. In HDTMA-SWy-Z, sorption of all solutes is via a

partition-dominated process. Solute competition (diminished uptake of the one solute in

the presence of a second one) was observed for TMA-SWy-2 but not HDTMA-
SWy—Z. This is consistent with an adsorptive mechanism for TMA-SWy-Z and a
partitioning mechanism for HDTMA-SWy-Z. This study demonstrates that the dominant
molecular mechanism of NAC sorption by smectite changes fundamentally from
complexation between —NO2 groups and the exchangeable cation (viz. NIL. and TMA) to
partitioning for a systematic series of ammonium- and quaternary- ammonium cations in
which the locus of positive charge (the central N atom) is progressively shielded by

organic moieties of increasing size.

Introduction

Smectites are layered 2:1 aluminosilicates with structural negative charges
that arise fiom isomorphous substitution. In nature, inorganic cations (e.g., Ca2+, Mg2+,
Al”, K+, and Na+) are commonly associated with the clay surfaces to balance these
negative charges. In the presence of water, inorganic exchangeable cations are hydrated
causing the interlayer to expand. Among the clay minerals commonly found in soils,
smectites are especially important because of their widespread occurrence, high surface
areas, large cation exchange capacities, and high expansibility. Smectite clays can be
modified through ion exchange reactions utilizing inorganic or organic cations. Previous
studies have shown that these simple modifications can dramatically alter the affinities of
smectites for organic contaminants and pesticides, and that this capability could enhance
the successful implementation of a variety of in-situ and ex-situ
remediation/immobilization technologies (Boyd et al., 1998a; 2001; Lee et al., 1998; Xu

et al., 1997; Sheng et al., 1998; Weissmahr et al., 1999; Li et al., 2004a).

Replacing the inorganic exchangeable cations of smectites with organic cations
typically causes an increase in the hydrophobic nature of the clay interlayer environment
(Barrer, 1961; Boyd et al., 1988b; McBribe, 1977; Mortland etal., 1986; Wolfe, 1985;
Xu and Boyd, 1995). For instance, as naturally occurring inorganic exchange cations of
clay minerals are replaced with organic cationic surfactants such as
hexadecyltrimethylammonium (HDTMA), the resultant organoclays are rendered more
effective sorbents for removing poorly water soluble organic compounds fi'om aqueous
solution (Boyd et al., 1988; Burris, 1992; Lee et al., 1989; Sheng et al., 1996). In

HDTMA-modified clays, the association of the C-16 alkyl carbon chains of HDTMA

creates an organic phase in the clay interlayer that covers most clay siloxane surface.
In the case of aromatic hydrocarbons, solute partitioning into this organic phase was
shown to be the primary sorptive process (J aynes and Boyd, 1991a). Increasing the
amount of HDTMA in the clay interlayer enlarges the partitioning domains as indicated
by an expansion of the clay basal spacing from 12 up to 18 and 23 A. The increased
interlayer distances of HDTMA-modified smectites correspond to the formation of
bilayers and paraffin complexes by the alkyl-carbon chains of HDTMA.

Lee et a1. (1989, 1990) showed that smectite clays exchanged with smaller
organic cations such as tetramethylammonium (TMA) or trimethylphenylammonium
(TMPA) cations display adsorptive properties as indicated by curvilinear isotherms for
sorption of benzene and other aromatic hydrocarbons. They concluded that TMA and
TMPA cations function as weakly hydrated pillars which prop open the smectite
interlayers providing access to adsorption domains on the siloxane surface. J aynes and
Boyd (1991b) and Lee et a1. (1990) studied the effect of clay surface charge density on
adsorption of organic contaminants by such organo-smectites. High-charged smectites
exchanged with a correspondingly greater amount of TMA or TMPA sorbed less benzene
compared to TMA- or TMPA-modified smectites derived fi'om clays with lower charge
densities. The authors attributed this observation as additional evidence for organic solute
adsorption by mineral siloxane surfaces between the exchangeable TMA or TMPA
cations. Also, sorption of benzene vapor by dry TMA-smectite was greater compared to
sorption of benzene from bulk water. Hydration of the TMA cations, albeit weak,
diminished the size of adsorption domains between exchangeable TMA ions. Thus there

are at least two primary mechanisms for sorption of organic solutes by organoclays, i.e.,

partitioning into organic phases derived from organic cations containing large alkyl
hydrocarbon chains (e.g. HDTMA-clay), and adsorption to the available siloxane
surfaces in organoclays comprised of smaller organic cations such as TMA.

So, for TMA-and TMPA-smectites the small, weakly-hydrated organic
ammonium cations serve to prop open the clay interlayers without the organic cation or
its hydration waters fully occupying the interlayer environment, hence rendering the clay
siloxane surfaces available for adsorption of organic solutes. Recent studies have shown
that several weakly-hydrated inorganic cations (e. g. K+ and (38+) can manifest similar
changes in the clay interlayer environment leading to higher adsorption of certain organic
solutes (Haderlein and Schwarzenbach, 1993; Haderlein et al., 1996; Boyd et al., 2001;
Sheng et al., 2002). For example, nitroaromatic compounds (NACS) demonstrate strong
affinities for smectites exchanged with K+ or Cs+, but much less adsorption by smectites
saturated with Na+, Ca2+, or Mg”. This was attributed to the comparatively stronger
hydration of Na+, Ca2+, and Mg“ cations which create an inhospitable (i.e. hydrophilic)
environment for organics in clay interlayers. This is, in part, because strongly bound
water is more difficult to displace, thus restricting access of the organic contaminants to
potential adsorption sites in smectite.

The hydration of inorganic exchangeable cations strongly influences the interlayer
environment in smectites where NAC sorption predominantly occurs. Besides
determining the abundance of adsorptive sites of sufficient size (between exchangeable
cations), interlayer cation hydration is a major determinant of the distance between clay
layers in smectites. A door Spacing of ~12.5 A is commonly observed for K+ and Cs+-

smectite clays. This appears to provide an optimal interlayer spacing which allows the

planar aromatic rings of NACS to orient parallel to and interact directly with the
opposing clay sheets, thereby, minimizing contact with water molecules, which is
energetically favorable (Li et al., 2004b). This is consistent with the now common
observation that smectite clays saturated with relatively weakly-hydrated K+ or Cs+ ions
have a strong affinity for NACS (Boyd et al., 2001; Haderlein and Schwarzenbach, 1993;
Haderlein et al., 1996; Johnston et al., 2001; Johnston et al., 2002; Li et al., 2004b; Sheng
et al., 2002; Weissmahr et al., 1998). In contrast, those saturated with more strongly
hydrated cations, such as Na+ or Ca2+, are much less effective adsorbents for NACS. Such
an arrangement of NACS in the interlayers of K+-, Cs+- or NHf-smectites might also
allow the formation of electron donor-acceptor complexes involving the electron
deficient aromatic ring of NACS and the electron rich siloxane oxygens of smectites
(Haderlein and Schwarzenbach, 1993; Haderlein etal., 1996; Weissmahr et al., 1998).
Weakly-hydrated exchangeable cations on clay surfaces can also favorably interact with
the —N02 groups of NACS. Boyd et a1. (2001) demonstrated that one mechanism
responsible for the strong sorption of NACS is the formation of inner sphere and/or outer
sphere complexes with weakly-hydrated exchangeable cations in the clay interlayers.

Smectite clays are strong sorbents for many organic compounds. The dominant

sorption mechanism (3) on a given smectite varies depending on properties of the
exchangeable cations and the organic solutes. Previous studies indicated that organic
compounds could be retained by partition interactions with organic phases derived from
exchanged organic cations and by adsorption on clay siloxane surfaces. The latter process
results from additive processes including hydrophobic interactions between the solute and

the clay siloxane surfaces, solute dehydration in the clay interlayers, formation of

complexes between polar functional groups of the solute and exchangeable cations
and/or formation of electron—donor acceptor complexes. The size, structure, and
hydration properties of exchangeable cations associated with smectites strongly influence
the clay interlayer environment, hence determining the degree of sorption of organic
compounds and the operative sorption mechanism(s). To ftuther understand the
dominant sorptive mechanism(s) of smectite clays responsible for the retention of NACS
where multiple mechanisms are possible, we measured the sorption from water of 1,3-
dinitrobenzene (1,3-DNB) and 2,4-dinitrotoluene (2,4-DNT) and naphthalene in single-
and binary-solute systems by a reference smectite (SWy-2) saturated with ammonium
and a series of organic quaternary ammoniums, viz. TMA, TMPA and HDTMA, of
increasing size and hydrophobicity. A central goal of this study was to determine the
predominance of complex formation between the exchangeable cation and the —N02
groups of NACS (Boyd et al., 2001) as the locus of positive charge, viz. the central N
atom, is progressively shielded by organic moieties of increasing size (methyl, phenyl,
hexadecyl). XRD and surface area determination of clays were used to gain insight into
the effects of exchangeable cation on the nature of the clay interlayer environment and

hence sorption of NACS.

Materials and Methods

Chemicals

1,3-DNB and 2,4-DNT were purchased from Aldrich Chemical Company Inc.
(Milwaukee, WI) with a reported purity > 97%, and naphthalene was obtained from
Sigma Chemical Company Inc. (St. Louis, MO) with a reported purity of 99%. Methanol
(HPLC grade) was purchased from Millinckrodt Baker Inc. (Phillipsburg, NJ). The
HDTMA—bromide, TMA-bromide, TMPA-chloride, and NIL-chloride were purchased
from Aldrich with chemical purities > 98%. All chemicals were used as received.
Clay Preparation

A reference smectite clay (Wyoming montrnorillonite SWy-2) was obtained from
the Source Clays Repository of Clay Minerals Society at Purdue University (West
Lafayette, IN) and used throughout this study. The SWy-2 has a cation exchange
capacity (CEC) of 82 cmolclkg and a theoretical surface area of 750 m2/g (van Olphen
and Fripiat, 1979). The < 2-um particle-sized clay fraction was obtained by wet
sedimentation. The clay suspensions were then quick fiozen and freeze-dried. Cation
exchange reactions were used to prepare the homoionic clays. The naturally occurring
inorganic exchangeable cations were replaced by ammonium, TMA, TMPA and
HDTMA. The NIL-smectite was prepared by washing the clay three times with 0.2 M of
NH4CI solution. The TMA- and TMPA clays were prepared by adding TMPA chloride
and TMA bromide in excess of three times the clay CEC. HDTMA-saturated clays were
prepared by adding HDTMA bromide equal to the clay CEC. Then the clay suspensions
were washed four to five times with Milli-Q water to remove excess electrolytes; AgNO3

was used to indicate if the samples were free of chloride or bromide. The clay

suspensions were then quick-frozen and freeze—dried. The clay organic carbon
content was measured using combustion method and surface area was determined using

N2-BET method.

Single-Solute Sorption

The sorption of 1,3-DNB, 2,4-DNT and naphthalene by NH4-, TMA-, TMPA— and
HDTMA-SWy-Z was measured using a batch equilibration method. First 0.05 to 0.10 g
clay samples depending on the type of solute and prepared clay were placed in glass
centrifuge tubes. Then added a series of initial solute concentrations in water ranging
fiom 5 to 200 mg/L for 1,3-DNB and 2,4-DNT and 1.0 to 25 mg/L for naphthalene. The
tubes were closed immediately with Teflon-lined screw caps and shaken end-over-end at
40 rpm for 24 h at room temperature (23 °C). Previous studies demonstrated that sorptive
equilibrium in such systems has been reached within 18 h (Sheng et al., 2002). The tubes
were centrifuged at 2600 g for 30 min to separate liquid and solid phases. Concentrations
of 1,3-DNB and 2,4-DNT and naphthalene in supematants were measured by a high-
perforrnance liquid chromatography (HPLC) system consisting of a Perkin-Elmer Binary
250 LC pump, a Series 200 autosampler and a Series 200 UV-visible detector. The
optimal wavelength was set at 270 nm for 1,3-DNB, 250 nm for 2,4—DNT and 260 nm for
NAPH. The mobile phase composition was a mixture of methanol and water, and
optimized for each compound. Controls consisted of the aqueous organic solutes in the
absence of clay. The sorbed amount was calculated from the concentration difference
between the aqueous solute concentrations in the control and supematants in the clay
suspensions. Sorption isotherms were obtained by plotting sorbed vs. equilibrium

aqueous phase concentrations. No changes in solute concentrations were detected in the

10

control tubes, therefore solute mass lost fiom the aqueous phase in the presence of

clay was assumed to be sorbed by clay.

Binary—Solute Sorption

To evaluate the competitive sorption of NACS by clays, we conducted binary-
solute sorption experiments using the batch equilibration method. A series of initial
solutions were prepared by mixing 2,4-DNT and 1,3-DNB with the molar ratios of 1:1
and 1:3 in aqueous solution. These initial solutions were added to centrifuge tubes
containing HDTMA-SWy-2 or TMA-SWy-Z. The equilibration and clay-solution
separation procedures were the same as described above. The supematants were sampled
and subjected to simultaneous measurements of 1,3-DNB and 2,4-DNT by HPLC. The
mobile phase was an isocratic mixture of methanol and water (50:50) with the flow rate
at 1.0 ml/min. The wavelength of UV-vis detector was set at 270 nm.
X-ray Difl‘raction Analysis

Afier the supematants were removed for HPLC analysis, approximately 2 mL of
clay suspension remained in the tubes. The clay slurries were mixed using a vortex
mixer, dropped onto glass slides using a disposable glass pipette, air-dried to produce
oriented clay films, then subjected to X-ray diffiaction (XRD) analysis. XRD patterns of
clay films were recorded using a Philips APD 3720 automated X-ray diffiaction meter
equipped with Cu—Ko. radiation, an APD 3521 goniometer and a diffracted-beam
monochromator. The scanning angle (20) ranged from 3 to 15° at steps of 002°, and the

scanning time was 2 seconds per step.

11

Results and Discussion

The properties of the clays used in this study are presented in Table 1. Reference
SWy-2 and NH4-SWy-2 clays each contained ~ 0.1 % organic carbon, presumably
present as an impurity. The organic carbon values of the organoclays increased in the
expected order: TMA-SWy-Z < TMPA-SWy-Z < HDTMA-SWy—Z. Comparisons of
measured organic carbon content with the calculated organic carbon percentages at fiIll
saturation indicated that 76.5, 83 and 83% of the cation exchange sites were occupied by
organic ammonium cations for TMA-SWy-2, TMPA-SWy-Z and HDTMA-SWy—Z,
respectively. The basal spacing (d001) increased with the size of the predominant
exchangeable cation: NIL-SWy-Z < TMA-SWy-2 < TMPA-SWy-Z < HDTMA-SWy-2.
For the organoclays, the measured N2 BET surface areas were inversely proportional to
the size of the organic ammonium, i.e., HDTMA-SWy-Z < TMPA-SWy-2 < TMA-SWy-
2. This ordering results fi‘om the fact that the larger organic ammonium cations occupy a
greater portion of the interlayer volume and obscure a larger portion of the siloxane
surfaces where N2 adsorption occurs. This also accounts for why the measured N2 BET

surface areas of the organo-clays are less than the theoretical surface area of SWy-2.

12

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I3

Sorption of Nitroaromatic Compounds

Sorption isotherms of 2,4-DNT and 1,3-DNB by HDTMA-, TMPA-, TMA- and
NH4-SWy-2 from water are shown in Figure l with amount sorbed per unit mass of clay
plotted vs. the equilibrium aqueous concentration of the solute. All sorption isotherms
were fit to the Freundlich equation (shown as solid lines in Fig.1):

Q = KfCeN
where Q (mg/kg) is the sorbed concentration, Ce (mg/L) is the equilibrium aqueous
concentration, Kr (mgl'NLN/kg) is the Freundlich sorption coefficient and N is an
empirical constant (unitless) that describes isotherm nonlinearity. The Freundlich
equation fitting parameters are listed in Table 2.

Sorption isotherms of 2,4-DNT and 1,3-DNB by the clays were nonlinear except
that representing sorption of 2,4-DNT by HDTMA-SWy—Z. The magnitude of sorption
on clays was greater for 2,4—DNT than for 1,3-DNB. The higher affinity of clays for 2,4-
DNT compared to 1,3-DNB is likely due in part to the lower water solubility of the
toluene derivative. Our previous studies of NAC sorption by K-SWy-2, and aromatic
hydrocarbon sorption by HDTMA-smectites, have shown that solute uptake fi'om water is
favored by lower water solubility (Boyd et al., 2001; Li et al., 2004, J aynes and Boyd,
1991a). The sorption of 1,3-DNB and 2,4-DNT by clays followed the order of NH4-
SWy-2 > TMA-SWy-2 > TMPA—SWy-2 > HDTMA-SWy-Z. The strong affinities of the
NACS for NH4-SWy-2 and TMA-SWy-Z, along with the sorption nonlinearity, are
characteristics of adsorption on the basal surfaces of smectite clays. Compared to
naturally occurring inorganic exchangeable cations commonly associated with smectites

(e. g. Na+, Ca2+, Mg“), the relatively low hydration fi'ee energies for ammonium (-292

14

kJ/mol) (Marcus, 1985) and TMA (-219kJ/mol)(Ford and Wang, 1992) result in less
water surrounding the cations rendering more mineral siloxane surface available for
adsorption (Boyd et al., 2001). The NH4-SWy-2 was at least 1.5 times more effective (on
a unit mass basis) than TMA-SWy-Z for sorption of 2,4-DNT and 1,3-DNB over the
concentration range studied. The higher affinity of NIL-SWy-Z for these solutes may
result fiom the formation of complexes between exchanged ammonium and the -N02
groups of the NACS, as in the case of NAC sorption by K-smectites (Boyd et al., 2001).
The larger molecular size of TMA than that of ammonium would inhibit direct
interactions between -NO2 and TMA ions since the locus of positive charge in TMA is
buried inside four methyl groups. Thus -N02 complexes with ammonium would be
expected to be stronger than those with TMA, leading to greater NAC sorption by NH4-

SWy-2.

15

50000

 

2,4-DNT (a) o 0
o

 
 
    

c)<)

40000 - 0

30000 -

  
 

 

 

 

 

 

3 20000 -
5 o NH4-SWy-2
3‘ 10000 i 0 TMA-SWy-Z
O A HDTMA-SWy-2
a El TMPA-SWy-2
o o . .
§ 0 20 40 60 80 100 120 140
fi
3 50000
C
5
O
5 1,3-DNB (b)

40000 -

30000 -

20000 .

10000 -

+ A
o I I I I

 

 

 

 

0 20 40 60 80 100 120 140 160
Aqueous Equilibrium Concentration, Co (mg/L)

Figure. 1. Sorption of 2,4-dinitrotoluene (2,4-DNT) and 1,3-dinitrobenzene (1,3-
DNB) by smectites saturated with ammonium (N H4 ),
tetramethylammoniumflMA), trimethylphenylammonium (TMPA) or
hexadecyltrimethylammonium (HDTMA).

l6

2,4-DNT and 1,3-DNB molecules differ by only a methyl group. The weaker
sorption by NH4- and TMA-SWy-2 observed for the smaller molecule 1,3-DNB than 2,4-
DNT cannot be explained by steric hindrance or surface-filling arguments. Rather, it
likely results from stronger complexes between 2,4-DNT forms and surfaces and/or
cations. The methyl group is expected to donate electron density to the ring, which
should decrease the strength of electron-donor-acceptor complexes with the surface as
proposed by Haderlein et a1. (1996). In fact, much of the donated electron density would
probably flow to the nitro groups in para- and ortho-positions (Boyd etal., 2001), which
may enable stronger formation of complexes with interlayer cations. Another factor that
helps to account for the stronger sorption of 2,4-DNT over 1,3-DNB is that hydrophobic
effects make a contribution to the sorption of NACS (Boyd et al., 2001; Li et al., 2004b).
2,4-DNT is less water-soluble than 1,3-DNB so that there should be more impetus to
remove it fi'om water.

Sorption of NACS fi'om water by HDTMA-and TMPA-clays exhibited sorption
properties distinct from those of NH..- and TMA-clays. Sorption of NACS by HDTMA-
or TMPA-clays was lower than sorption by NH4- or TMA-SWy-2 clays and the
isotherms were more linear (Table2 and Fig 1). In HDTMA-SWy-Z, the association of
alkyl chains creates an organic partitioning phase that covers most clay siloxane surfaces;
solute partitioning into this organic phase is believed to be the primary process occurring
in this clay for aromatic hydrocarbons (Boyd et al., 1988; J aynes and Boyd 1991a). The
linear isotherms and inverse relationship between sorption and solute water solubility are
consistent with a partition-dominated process (Chiou, 2002). The basal spacing of

HDTMA-SWy—Z used herein was 17.5 A, corresponding to the formation of a bilayer of

17

alkyl chains (J aynes and Boyd, 1991a). In this configuration, the densely packed alkyl
chains adopt a horizontal orientation relative to clay layers forming an organic partition
phase in which mineral surfaces are largely covered and hence contribute minimally to
the sorption of organic compounds. Sorption of NACS by TMPA-smectite is slightly
higher than that by HDTMA-clay, which may be due to some contribution of interlayer
surfaces to adsorption. Overall, sorption by the TMPA-clay is intermediate between
HDTMA-clay where NAC sorption is predominantly via partitioning and TMA-clay
which functions as an adsorbent. The underlying mechanism(s) for NAC sorption by

TMPA-clay is uncertain.

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l9

Naphthalene Sorption

In contrast to the sorption order of NACS, the magnitude of naphthalene sorption
followed the order: TMPA-SWy-2 > I-IDTMA-SWy-Z > TMA-SWy—Z > NH4-SWy-2
(Figure 2). As we observed previously (J aynes and Boyd, 1990) TMPA- SWy—2
appeared to have an unusually high affinity for naphthalene. In all cases, sorption of
naphthalene by clays produced upward curvature in the isotherms (Figure 2 and Table 2)
indicating that the increased concentration of naphthalene promotes further sorption in
the interlayers. Hundal et al. (2001) have suggested that sorption of phenanthrene by
smectites was primarily via a process of capillary condensation in interlayer nanopores
and micropores; phenanthrene was physically entrapped in the nanopores and micropores
of the clay driven primarily by hydrophobic effects. Zhu et a1. (2004) have shown a
strong correlation between F reundlich sorption constants and indices of cation-1t bonding.
They suggested that cation-1t bonding occurred between polycyclic aromatic
hydrocarbons and inorganic exchangeable cations in smectite interlayers. A comparison
of sorption between the two types of compounds in this study (naphthalene and NACS)
by organoclays suggests the prepared organoclays possess different sorption
sites/mechanisms for retention of the test compounds. The much stronger affinity of
NH4-, and TMA-SWy—Z for NACS compared to naphthalene indicates that interlayer
adsorption on the siloxane surfaces and complex formation involving the —N02 groups
are the predominant sorptive interactions for polar NACs; the latter mechanism is not
available for adsorption of nonpolar naphthalene. In contrast, TMPA-SWy—Z has a
particularly high affinity for naphthalene with upward curvature of the isotherm,

suggesting capillary condensation as a possible mechanism. Regardless of sorption

20

mechanisms, it seems plausible that the aromatic groups on TMPA would prop the

smectite interlayer at a d-spacing (14.2 A, Table2) that might be optimal for naphthalene.

 

 

 

 

60000
’6
X
E. A HDTMA-SWy-2
‘31 o TMA-SWy-2
E o NH4-SWy-2
O
(I)
I
3
'6
‘E
3
E
.e
15 20

 

NAPH Equilibrium Aqueous Concentration, Ce (mg/L)

Figure 2. Sorption isotherms of naphthalene (N APH) by smectite (SWy-Z) saturated
with ammonium, tetramethylammonium (TMA), trimethylphenylammonium
(TMPA) or hexadecyltrimethylammonium (HDTMA)
Sorption from binary solute-mixtures

Sorption of NACS from binary solute mixtures by clays was measured to evaluate
solute competition as a means of further revealing the operative sorption mechanisms.
Isotherrns representing sorption of a binary mixture of 1,3-DNB and 2,4-DNT from water

by HDTMA-SWy-Z and TMA—SWy-Z are shown in Figures 3 and 4 along with the

corresponding single solute sorption isotherms. The Freundlich fitting parameters are

21

reported in Table 3. The binary solutions of 2,4-DNT and 1,3-DNB were prepared at
initial molar ratios of 1:1 and 1:3. Sorption

isotherms of 2,4-DNT from the binary solute mixtures by HDTMA-SWy-Z were
coincident with the single solute isotherm demonstrating a total lack of solute
competition. Sorption of 1,3-DNB in binary solute mixtures by I-IDTMA-SWy-2
manifested somewhat higher sorption compared to the single solute sorption at Ce > 50
mg/L, indicating a cooperative sorption process. The lack of a negative effect on sorption
in binary solute systems is consistent with a partition-dominated process involving the

organic phase formed by HDTMA (Chiou, 2002)

22

 

14000

(a)
12000 .

 
 
 
  
 
 
 
 
 
  

10000 4

8000 -

6000 -

2.4-DNT 1.3-DNB
4000 .
Single solute

DNT:DNB (1:1) molar ratio
DNTzDNB (1:3) molar ratio

DUO
DI.

2000 -

 

 

 

I l r I

80 100 120

 

4000 ‘

Amount of MAC Sorbed, Q (mglkg)

O)

O

O

O
O
N
O
A
O
O)
O

3000 -

2000 e

1000 -

 

 

 

 

0 50 100 150 200

NAC Aqueous Equilibrium Concentration, Ce (mg/L)

Figure 3. Sorption isotherms of nitroaromatic compounds (NACs) (a) 2,4-
dinitrotoluene (2,4-DNT) and (b) 1,3-dinitrobenzene (1,3-DNB) by
hexadecyltrimethylammonium (HDTMA)-smectite (SWy-2) in single- and binary-
solute systems.

23

Sorption of 1,3-DNB and 2,4-DNT from binary solute mixtures by TMA-SWy—Z
are compared to the sorption of the single solutes in Figure 4. Sorption isotherms of 1,3-
DNB and 2,4-DNT in both single and binary solute systems are nonlinear indicating
adsorption to clay surfaces rather than a partitioning process, as described above. In both
cases, the presence of a second solute depressed the uptake of the primary solute clearly
demonstrating that 1, 3-DNB and 2,4-DNT were competing for adsorptive sites on TMA-
clay. The strong competitive effect on sorption of NACS by TMA-clay is characteristic
of an adsorptive process wherein 1,3-DNB and 2,4-DNT compete with each other for the
limited available sorptive sites. The greater amount of competitive solute present in
aqueous phase was observed to more substantially suppress the sorption of the primary
solute. For example, at Ce = 80 mg/L, binary sorption of 1,3-DNB was reduced ~ 50%
with the DNT: DNB molar ratio of 1 :3, and ~70% at a molar ratio of 1:1. In comparing
the affinity of 1,3-DNB and 2,4-DNT with TMA-SWy-Z, note that 2,4-DNT suppressed
sorption of 1,3-DNB by up to 75%, while 1,3-DNB suppressed 2,4-DNT sorption by less
than 30%. The greater competitive inhibition on sorption caused by 2,4-DNT is a logical

manifestation of its higher affinity for TMA-SWy—2 compared to 1,3-DNB (Fig 1).

24

 

35000

30000 -

25000 -

20000 -

15000 *

10000 .

 

 

(a)

2,4-DNT 1,3-DNB

Single solute 0 O
DNT:DNB (1:1) molar ratio 0 I
DNT:DNB (1 :3) molar ratio A ‘

 

 

I I I I I

20 40 60 80 100 1 20 140 160 1 80

 

20000 -

15000 1

10000 .

5000 .

Amount of NAC Sorbed, Q (mglkg)
O

 

(b)

 

 

I I I I

20 40 60 80 100 120 140 160
NAC Aqueous Equilibrium Concentration, Ce (mg/L)

Figure 4. Sorption isotherms of nitroaromatic compounds (NACS) (a) 2,4-
dinitrotoluene (2,4-DNT) and (b) 1,3-dinitrobenzene (l ,3-DNB) by
tetramethylammonium (TMA)-smectite (SWy-Z) in single- and binary-solute syste

25

 

 

 

 

 

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26

Summary

Sorption of two NACS by smectite exchanged with ammonium and a series of
quaternary ammonium ions (TMA, TMPA, HDTMA) revealed a shift in sorption
mechanism fiom a predominately adsorptive process in the case of NI-I4- and TMA-
smectite to a partition-dominated process for HDTMA-smectite. Nonlinear sorption
isotherms and competition between NACS for sorption sites indicated an adsorptive
mechanism. Adsorption of NACS was a manifestation of the small size and low
hydration energies of ammonium and TMA. The primary adsorptive interaction
appeared to be the formation of complexes between exchangeable cation and the —
N02 groups of the NACS. Adsorption domains are primarily the siloxane surface
between the exchangeable cations. The small size and weak hydration of these cations
(NI-14+, TMA) facilitates formation of the aforementioned complexes and makes
available the siloxane surface, which is largely unobscured by waters of hydration
associated with the exchangeable cations. Solute partitioning was the primary
sorption mechanism of clay exchanged with the much larger and hydrophobic cation
HDTMA. In this instance, the C-16 alkyl chains of exchanged HDTMA coalesce to
form a hydrophobic partition phase into which NACS dissolve. The siloxane surfaces
and interlayer volume is essentially fully occupied by exchanged HDTMA. Linear
isotherms and lack of solute competition in binary-solute systems provide evidence
for a partition-dominated process where degree of uptake from water is determined

mostly by the water insolubility of the solute.

27

REFERENCES

Barrer, R.M. and Perry, G. S. 1961. Sorption of mixtures, and selectivity in
alkylammonium montrnorillonite. Part II. Tetrarnethylammonium
montmorillonite: Journal of Chemistry Society, 850-858.

Boyd, S.A., Lee, J .F. and Mortland, M.M. 1988a. Attenuating organic contaminant
mobility by soil modification. Nature, 333, 345-347.

Boyd, S.A., Mortland, M.M. and Chiou, CT. 1988. Sorption characteristics of
organic compounds on hexadecyltrimethylammonium-smectite. Soil Science
Society of America Journal, 52, 652-657.

Boyd, S.A., Sheng, G., Teppen, BJ. and Johnston, CT. 2001. Mechanisms for the
adsorption of substituted nitrobenzenes by smectite clays. Environmental
Science and Technology, 35, 4227-4234.

Burris, DR. and Anthworth, CR 1992. In Situ modification of aquifer material by a
cationic surfactant to enhance retardation of organic contaminants. Journal of
Contaminant Hydrology, 10, 325-337.

Chiou, CT. 2002. Partition and Adsorption of Organic Contaminants in
Environmental Systems John Wiley & Sons, Inc., Hoboken, New Jersey.

Ford, GR, and Wang, B. 1992. The Optimized Ellipsoidal Cavity and Its Application
to the self-consistent reaction field calculation of hydration energies of cations

and neutral molecules. Journal of Computation Chemistry, 13, 229-239.

Haderlein, S.B., and Schwarzenbach, R.P. 1993. Adsorption of substituted
nitrobenzenes and nitrophenols to mineral surfaces. Environmental Science
and Technology, 27, 316-326.

Haderlein, S.B., Weissmahr, K.W. and Schwarzenbach, R.P. 1996. Specific
adsorption of nitroaromatic explosives and pesticides to clay minerals.
Environmental Science and T echnology, 30, 612-622.

Hundal, L.S., Thompson, M.L., Laird, D.A. and A.M. Carmo. 2001. Sorption of
phenanthrene by reference smectites. Environmental Science and T echnology,
35, 3456-3461.

Jaynes, W.F., and Boyd, S.A. 1990. Trimethylphenylammonium-smectite as an
effective adsorbent of water-soluble aromatic-hydrocarbons. Journal of Air
and Waste Management Association, 40, 1649-1653.

28

J aynes, W.F., and Boyd, S.A. 1991a. Clay mineral type and organic compound
sorption by hexadecyltrimethylammonium-exchanged clays. Soil Science
Society of America Journal, 55, 43-48.

J aynes, W.F., and Boyd, S.A. 1991b. Hydrophobicity of siloxane surfaces in
smectites as revealed by aromatic hydrocarbon adsorption from water. Clays
and Clay Minerals, 39, 428-436.

Johnston, C.T., Oliveria, M.F. de, Teppen, B.J., Sheng, G. and Boyd, S.A. 2001.
Spectroscopic study of nitroaromatic-smectite sorption mechanisms.
Environmental Science and Technology, 35, 4767-4772.

Johnston, C.T., Sheng, G., Teppen, B.J., Boyd, S.A. and Oliveira, M.F. de. 2002.
Spectroscopic study of dinitrophenol herbicide sorption on smectite.
Environmental Science and Technology, 36, 5067-5074.

Lee, J .F ., Crum, J .R. and Boyd, S.A. 1989. Enhanced retention of organic
contaminants by soils exchanged with organic cations. Environmental Science
and Technology, 23, 1365-1372.

Lee, J .F ., Mortland, M.M., Chiou, C.T., Kile, DE. and Boyd, S.A. 1990. Adsorption
of benzene, toluene, and xylene by two tetramethylammonium-smectites
having different charge densities. Clays and Clay Minerals, 38, 113-120.

Li, H., Teppen, B.J., Laird, D.A., Johnston, CT. and Boyd, S.A. 2004a. Geochemical
modulation of pesticide sorption on smectite clay. Environmental Science and
Technology, 38, 5393-5399.

Li, H., Teppen, B.J., Laird, D.A., Johnston, CT. and S.A. Boyd. 2004b.
Thermodynamics of nitroaromatic compounds adsorption from water by
smectite clay. Environmental Science and Technology, 38, 5433-5442.

Marcus, Y. 1985. Thermodynamic functions of transfer of single ions fiom water to
nonaqueous and mixed-solvents .2. Enthalpies and entropies of transfer to
nonaqueous solvents. Pure and Applied Chemistry, 57, 1103-1128.

McBride, M.B., Pinnavaia, T. J ., and Mortland, M.M. 1977. Adsorption of aromatic
molecules by clays in aqueous suspension. In Fate of Pollutants in the Air and
Water Environment, Part I, 8, 145-154.

Mortland, M.M., Sun, S. and Boyd, S.A. 1986. Clay-organic complexes as adsorbents
for phenol and chlorophenols. Clays and Clay Minerals, 34, 581-5 85.

Sheng, G., Xu, S., and Boyd, S.A. 1996. Mechanism(s) controlling sorption of neutral

organic contaminants by surfactant-derived and natural organic matter.
Environmental Science and Technology, 30, 1553-1557.

29

Sheng, G., Wang, X., Wu, S. and Boyd, S.A. 1998. Enhanced sorption of organic
contaminants by smectitic soils modified with a cationic surfactant. Journal of
Environmental Quality, 27, 806-814.

Sheng, G., Johnston, C.T., Teppen, B]. and Boyd, S.A. 2002. Adsorption of
dinitrophenol herbicides from water by montrnorillonites. Clays and Clay
Minerals, 50, 25-34.

van Olphen, H. and Fripiat, JJ. 1979. Data handbook for clay minerals and other non-
metallic minerals. Pergarnon Press, New York, NY.

Weissmahr, K.W., Haderlein, SB. and Schwarzenbach, R.P. 1998. Complex
formation of soil minerals with nitroaromatic explosives and other pi-
acceptors. Soil Science Society of America Journal, 62, 369-378.

Weissmahr, K.W., Hildenbrand, M., Schwarzenbach, R.P. and Haderlein, SB. 1999.
Laboratory and field scale evaluation of geochemical controls on groundwater

transport of nitroaromatic ammunition residues. Environmental Science and
Technology, 33, 2593-2600.

Wolfe, T.A., Demerirel, T. and Baumann, E. R. 1985. Interaction of aliphatic amines
with montmorillonite to enhance adsorption of organic pollutants. Clays and
Clay Minerals, 33, 301-31 1.

Xu, S., and Boyd, S.A. 1995. Cationic surfactant adsorption by swelling and
nonswelling layer silicates Langmuir, 11, 2508-2514.

Xu, S., Sheng, G. and Boyd, S.A. 1997. Use of organoclays in pollution abatemate.
Advances in Agronomy, 59, 25-61.

Zhu, D., Herbert, B. E., Schlautrnan, M.A., Carraway, E. R. and Hur, J. 2004.
Cation-1t bonding: a new perspective on the sorption of polycyclic aromatic
hydrocarbons to mineral surfaces. Journal of Environmental Quality, 33,
1322-1330.

30

CHAPTER II

REDUCING BIOAVAILABILITY AND PHYTOTOXICITY OF

NITROAROMATIC COMPOUNDS BY SORPTION ON K-SMECTITE CLAY

31

Abstract

Nitroaromatic compounds (NACS) are a prominent class of environmental
contaminants that are toxic to plants and microorganisms, and pose a threat to human
and ecosystem health. The NACS demonstrate high affinities for smectite clays which
are widely distributed in nature as components of soils and sediments, and in
geological deposits. The affinity of NACs for smectites is highly dependent on the
exchangeable cation. For example, smectites saturated with K+ have very high
affinities for NACS while those saturated with Ca2+ do not. In this study we evaluate
the ability of K-smectites to attenuate the bioavailability and hence toxicity of 2,4-
dinitrotoluene (2,4-DNT) to the aquatic plant duckweed (Lemna gibba and Lemna
minor). In the absence of K-smectite, 7.5 mg/L of 2,4-DNT was highly toxic to
duckweed. Small amounts of K-smectite added to aqueous growth media reduced
this toxicity substantially, presumably by reducing 2,4-DNT bioavailability via
sorption. There is a need to optimize bioavailability of NACS during bioremediation
so that they are sufficient available for effective plant uptake or microbial degradation
without being present at levels that are toxic to organisms used for bioremediation.
Release of clay-bound NACs can be achieved by simple ion exchange reactions on

the clays.

32

Introduction

Smectites are layered 2:1 aluminosilicates with structural negative charges
arising from isomorphous substitution in octahedral and/or tetrahedral sheets. In
nature, inorganic cations (e.g., Ca“, Mg“, Al”, K+, and Na+) are associated with
smectite clay surfaces to balance these negative charges. When smectites are wetted,
these cations hydrate causing greater separation of the clay sheets (interlayer
expansion). Among the clay minerals commonly found in soils, smectites are
especially important as potential adsorbents of organic contaminants and pesticides
because of their widespread occurrence (Allen and Hajek, 1989), high surface areas,
large cation exchange capacities, and reversible interlayer expansibility.

Sorption to soils and sediments is a major determinant of environmental fate
and impact of contaminants, such as potential for leaching and bioavailability to
microorganisms and plants (Koskinen and Harper, 1990). Sorption often renders
contaminants less available to receptor organisms (Hatzinger and Alexander, 1995;
Luthy et al., 1997; Ogram etal., 1985; Pignatello and Xing, 1996). Aqueous
contaminant concentrations are lowered by sorption, thereby inhibiting root uptake by
plants. Similarly, soil microorganisms may ineffectually degrade organic
contaminants if they cannot readily access the sorbed contaminant pool. Harris
(1964) showed that insecticide adsorption by soil minerals under dry conditions
substantially raised the lethal dose (LDso) towards crickets, whereas hydration caused
release of sorbed contaminants hence increasing bioavailability of the insecticides. As
a consequence, lowered LDso values were observed for insecticides added to wet soils

compared to the same soils in a dry state. Steinberg et a1. (1987) and Scribner et a1.

33

(1992) showed that sorption and sequestration of ethylene dibromide and sirnazine
pesticides in field-weathered soils reduced bioavailability to contarninant-
biodegrading microorganisms thereby prolonging pesticide residence times. Reduced
bioavailability in these instances was associated with prolonged pesticide-soil contact
times. In contrast, there are examples where bacteria are able to access directly the
pool of sorbed substrate (Feng et al., 2000; Guerin and Boyd, 1992; Park et al., 2004).
So, it is unclear, a priori, whether or not sorbed contaminants are available to
biological organisms. At this point, no generalizations regarding the bioavailability
of sorbed organic contaminants are apropos.

Nitroaromatic compounds (NACS) are a class of prominent soil contaminants
that are toxic to plants and microorganisms (Gong et al., 1999; Sunahara et al., 1998).
Nitroaromatic compounds demonstrate high affinity for certain clays, especially
smectites, which is strongly influenced by the type of cation occupying cation
exchange sites of the clay. For example, smectites exchanged with Cs+, K+ and NH:
have high affinities for NACs whereas smectites manifest much lower NAC
adsorption when exchanged with Na+, Ca2+ and Mg2+ (Boyd et al., 2001; Haderlein
and Schwarzenbach, 1993; Haderlein et al., 1996; Johnston et al., 2001; Johnston et
al., 2002; Li et al., 2004; Sheng et al., 2002). The effect of exchangeable cation type
on NAC sorption appears to be related to their hydration energies. The presence of
strongly hydrated exchangeable cations creates an unfavorable clay interlayer
environment for NAC adsorption, whereas the comparatively weak hydration of other
exchangeable cations creates a much more favorable interlayer environment for NAC

adsorption. Adsorption of NACs occurs primarily in the clay interlayer (Boyd et al.,

34

2001; Sheng et al., 2002), and cation hydration determines the interlayer distance,
size of adsorptive domains, and ability to form NAC-cation complexes.

Boyd and co-workers (Boyd et al., 2001; Johnston et al., 2001; Li et al.,
2004a; Sheng et al., 2002) conducted systematic studies on the adsorption
mechanisms of NACs by smectite clays using a series of complimentary investigatory
techniques including detailed macroscopic sorption measurements, Fourier transform
infrared spectroscopy, X-ray diffraction, quantum calculations, molecular dynamics
simulations, and thermodynamic measurements. Their results revealed that weaker
hydration of exchangeable cations (e. g., K“) on smectite surfaces promotes favorable
interactions between cations and the -N02 groups of NACs via formation of inner-
and/or outer-sphere complexes. In addition to the formation of such complexes, an
optimal basal distance of ~ 12.5 A was observed for K+-smectite clays (Li et al.,
2004a; Sheng et al., 2002). This allowed simultaneous interactions of the planar
aromatic rings of intercalated NACS with the opposing clay siloxane surfaces, as well
as partial solute dehydration; both processes are energetically favorable (Li et al.,
2004a). Furthermore, compared with cations having higher hydration enthalpies, e. g.
Ca2+, Mg2+ or Na+, the smaller hydration spheres surrounding Cs+, K+ or NH4+
provide larger regions of siloxane surface between exchange cations that are
unobscured by water. These siloxane surface regions are the primary adsorptive
domains where NACS align in an orientation parallel to the clay sheets.

Nitroaromatic compounds are common components of munitions and are
commonly found as soil and ground water contaminants in military training facilities

and manufacturing sites. Important classes of pesticides such as the dinitrophenol

35

herbicides may persist in soils where they are used in crop protection (Broholm,
2001; Hawley, 1981), leaching to contamination of surface and ground waters. For
these reasons, NACS pose the threat of adverse effects on human and ecosystem
health (Berthe-Corti et al., 1998; Gong et al., 1999; Hawley, 1981; Neumann et al.,
1995). The —N02 groups of absorbed NACS can also be reduced to amino groups that
can conjugate with the phenolic sites in biota (Morgan, 1989; Ware, 1997).

Understanding the sorption mechanism(s), and the molecular scale forces and
properties of smectite clays that influence sorption, provides the scientific basis for
developing protocols to control the mobility and bioavailability of NACs to target
organisms such as plants and microorganisms utilized in bioremediation technologies.
Li et al., (2004a) measured adsorption of the pesticide dichlobenil by K+- and Ca2+-
smectites fiom binary aqueous solutions of KCl and CaClz, and found that the
adsorption increased with increasing molar ratio of K+ to Ca“. At lower fractions of
K+ occupation of cation exchange sites, 1C ions were randomly distributed on the
external and interlayer clay surfaces and did not substantially enhance pesticide
sorption. At higher populations of K+ (vs. Ca“) demixing occurred causing some
clay interlayers, interlayer regions or tactoids to become fully saturated with K+
thereby manifesting greatly enhanced pesticide sorption. These results demonstrated
that the degree of pesticide adsorption by smectite clays could be controlled by
adjusting the amount of K“ and Ca2+ associated with clay through simple geochemical
cation exchange processes.

The objective of this study was to demonstrate the ability smectite clay to

attenuate the bioavailability and hence toxicity of NACS to target organisms. There is

36

a need to optimize bioavailability of contaminants during bioremediation so that they
are sufficiently available for effective plant uptake or bioremediation without being
present at levels that are toxic to the organisms used for biodegradation. The ability
to modulate the binding and release, i.e. the bioavailability, of NACS via ion
exchange processes on smectite clays is the focus of a subsequent study. The target
organisms used in this study are two species of duckweed (Lemna gibba and Lemna
minor) which are a group of free-floating aquatic plants often found growing on
nutrient-rich water in ponds and lakes. Toxicity testing using duckweeds is an
accepted and common protocol to evaluate aquatic toxicity of organic contaminants
and heavy metals present in natural bodies of water (ASTM and Materials, 1991;
Hilhnans, 1961; Holst and Ellwanjer, 1982; Smith and Kwan, 1989; Taraldsen and
Norberg-King, 1990; Wang and F reemark, 1995; Wu, 1980). The toxic effects of
NACs to duckweed were evaluated to assess the bioavailability and toxicity of NACs

in aqueous systems containing, and devoid of, K- smectite.

37

Materials and Methods
Chemicals

2,4-Dinitrotoluene (2,4-DNT) was purchased from Aldrich Chemical
Company Inc. (Milwaukee, WI) with a reported purity > 97%. Potassium chloride
(KCl), calcium chloride (CaClz), magnesium sulfate (MgSO4), potassium nitrate
(KNO3), potassium phosphate dibasic (KHzPO4), boric acid (H3B03), sodium
molybdate (N azMoO4), sodium acetate (CH3COONa) and cupric sulfate (CuSO4)
were purchased fi'om Millinckrodt Baker Inc. (Phillipsburg, New Jersey); ferric
chloride (FeCl3), manganese chloride (MnClz), and zinc sulfate (ZnSO4) were
obtained from Sigma (St. Louis, MO); ethylenediaminetetracetic acid (EDTA) was
obtained from Invitrogen Corporation (Carlsbad, CA.). All chemicals were used as
received.
Clay preparation

Reference smectite clay (Wyoming montmorillonite, SWy-2) was obtained
from the Source Clays Repository of the Clay Minerals Society (Purdue University,
West Lafayette, Indiana) and used throughout this study. The SWy-2 clay has a CBC
of 82cmolc/kg and a theoretical surface area of 750m2/g (van Olphen and Fripiat,
1979). The < 2pm clay particle-sized fraction was obtained by centrifirging a dilute
clay suspension for 6 min at 60 g (~600rpm) using a Sorvall GSA rotor and RC-5C
centrifuge (DuPont Co., Wilmington, Delaware) (Costanzo and Guggenheim, 2001).
Prior to separating the clay-sized particles, the SWy-2 clay sample was treated with
0.5 M sodium acetate to remove carbonate impurities (Arroyo et al., 2005). The K-

saturated homoionic clay was prepared using cation exchange reactions. Specifically,

38

the naturally occurring inorganic exchangeable cations (predominately Na+) on SWy-
2 were replaced with K+ by washing the clay-sized fiaction of SWy-2 three times
with 0.5M KCl solution. K-smectite clay suspensions were then dialyzed against
Milli-Q water to remove excess electrolytes; the absence of white AgCl precipitate
upon addition of AgNO; indicated that the clay suspensions were free of chloride.
The prepared clay suspensions were quick-frozen and freeze-dried. Prior to use in
phytotoxicity assays, K-SWy-2 clay was sterilized by heating at 120°C for 3-5 days.
Duckweed culture

Duckweeds were used as the phytotoxic indicator species for evaluating NAC
exposure. Lemna minor and Lemna gibba duckweed cultures were obtained from the
Culture Collection of Algae and Cyanobacteria (CCAC, University of Toronto;
Toronto, Ontario, Canada). Axenic duckweed cultures were maintained in 300-mL
Erlenmeyer flasks containing 150 mL of full-strength (1X) Hoagland’s nutrient
solution (Cowgill and Milazzo, 1989) at pH 5.8, supplemented with 10 g of sucrose
per liter as recommended by the CCAC (Judy Acreman, personal communication).
After autoclave sterilization of the sucrose-arnended Hoagland’s solution, 20 mL of a
FeEDTA solution (prepared by dissolving 0.121 g F eCl3 and 0.375g EDTA into 250-
mL Milli-Q water and passed through a 0.22-um filter) were added to complete the
1X duckweed medium (1X DWM). Culture flasks inoculated with duckweed were
closed with sterilized foam plugs (~ 27 to 34 mm), the plugs covered with aluminum
foil, and placed in a plant growth chamber (Boone, Iowa) at 25°C with a 16 hr day

length and a photosynthetically active radiation (PAR) intensity of ~ 43 mol s’l m'z.

39

Plants were subcultured every 3 weeks by transferring approximately 16 fronds into a
flask containing fresh sterile culture medium.
NAC phytotoxicity

Phytotoxic assays for 2,4-DNT were carried out using Lemna minor and
Lemna gibba duckweed cultures in in-vitro bioreactors (described above). Using
aseptic techniques in a laminar flow hood, 300 mg sterile K-SWy-2 clay were
weighed into the 300 mL Erlenmeyer flasks, 150 mL of sterilized l/SX DWM
containing 7.5 mg/L 2,4-DNT and 0.01M KC] were added, and sterilized foam plugs
partially inserted into the mouth of the flasks. Foam-plugged flasks were covered
with aluminum foil to reduce the evaporative loss of water. The 1/5X DWM
supplemented with 7.5 mg/L DNT and 0.01M KC] was sterilized by passing through
a 0.2-um membrane. Our preliminary study indicated that 150 mL of 1/5X DWM
provided sufficient nutrients for duckweed growth and frond multiplication during the
two week experimental period.

The effect clay sorption of 2,4-DNT on duckweed growth was evaluated as
follows. Prior to inoculation with duckweeds, 150 mL of 1/5 X DWM containing
7.5mg/L 2,4-DNT and 0.01M KC] was mixed with 300 mg of K-SWy-2 clay in a
300-mL Erlenmeyer flask for four hours at room temperature to establish sorption
equilibrium of 2,4-DNT with K-SWy-2 clay. To ascertain sorption equilibrium, the
aqueous phase was periodically sampled and analyzed for 2,4-DNT concentrations by
a high-performance liquid chromatography (HPLC). This study and a previous study
(Sheng et al., 2002) have both indicated that sorption of NACS by K-smectites

reached equilibrium within four hours.

40

To initiate the phytotoxicity assay, 8-10 green duckweed fronds were
aseptically transferred to each flask. Foam plugs and foil were replaced and the
flasks incubated without shaking in a growth chamber under the conditions described
above. Controls consisted of the same 1/5X DWM without clay and/or without 2,4-
DNT. Duckweed frond numbers were counted manually at selected sampling times
as an index of NAC phytotoxicity. At pre-selected sampling times, 2 mL of solution
were removed and analyzed for 2,4-DNT aqueous-phase concentrations using HPLC.
In similar experiments, but in the absence of clay, 2,4-DNT toxicity to duckweed
growth was measured over a range of 2,4-DNT concentrations from 0 to 20 mg/L in
order to establish a dose-response relationship.

The HPLC system used to measure aqueous phase 2,4-DNT concentrations
consisted of a Perkin-Ehner (Shelton, Connecticut) Binary 250 LC pump, a Series
200 autosarnpler and a Series 200 UV-visible detector. The mobile phase was an
isocratic mixture of methanol and water (50:50) with the flow rate at 1.0 ml/min. The

wavelength of the UV-visible detector was set at 250 nm.

4]

Results and Discussion

The dose-response relationship of Lemna gibba and Lemna minor to aqueous-
phase 2,4-DNT at different intervals of exposure is shown in Figure l. The response
of the two duckweed species, in terms of number of fionds produced, to 2,4-DNT
solutions ranging from 0.5 to 20 mg/L (without clay) was evaluated to identify the
level at which 2,4-DNT inhibits duckweed growth. The results indicated that Lemna
gibba growth was negatively impacted to a measurable degree at 2,4-DNT
concentrations 2 1.0 mg/L. Lemna minor was somewhat more susceptible to the
toxicity of 2,4-DNT. For example, the observed difference in growth after 12 days
exposure to 0.5 mg/L of 2,4-DNT compared with the 2,4-DNT-free controls was

greater for Lemna minor compared to Lemna gibba (Figure 1).

42

 

160
140 Lemna gibba
‘ 0.0 (mg/L)

120 ~
0 0.5 (mg/L)-

 

100 -

.. 1.0 (mg/L)
. 2.0 (mg/L)

fl.200 (mg/L)

 

 

 

 

 

 

3
g .
r: 0 -
*5 180
E .
S 160 Lemna minor 0.0 (mg/L)
2 140 4
120 ~
100 i
0.5 (mg/L)
80 a 6
§ 1.0 (mg/L)
60 4
40 .
/? 2.0 (mg/L)
20 - ,_ I
o ._ 3__«.'__..——_-:;:_-:.;' 4 a20.0 (mg/L)
0 2 4 6 8 10 12 14

Time (Days)

Figure 1. Growth of two species of duckweed over a 12 day incubation time as
function of different solution concentrations (0 to 20 mg/L) of 2,4-dinitrotoluene.

43

To quantitate duckweed growth rate, the progress curves of Lemna minor and
Lemna gibba plotted as fiond number vs exposure period were fitted to a first-order
kinetic process equation:

ln F, = In F}, + kt [l]

where F, and F o are the frond numbers at time t (days) and initial time, respectively,
and k (day") is the growth rate constant. The results of fittings are shown as solid
lines in Figure l. The growth data were well described by the equation [1]; fitting
these data to the linear form of equation [1] gave 1'2 values 2 0.95. Doubling time, T2
(days ’1), of duckweed growth in terms of fi‘ond numbers was calculated using the

equation:

T. = 111% [2]

The growth rate constants and doubling times are listed in Table 1. For Lemna
minor exposed to 2,4-DNT concentrations 2 0.5 mg/L, growth was inhibited as
indicated by decreased grth rate constants and increased doubling time compared
with the 2,4-DNT-free control. The doubling time increased approximately 10 and 20
% at 2,4-DNT concentrations of 0.5 and 1.0 mg/L, respectively, relative to that of the
control when no 2,4-DNT is present. This corresponds to a > 50 % reduction in the
number of fronds afler 12 days of exposure to 2,4-DNT (Figure 1). As 2,4-DNT
concentration increased to > 1.0 mg/L, toxic symptoms were usually apparent as the
duckweed fronds changed fi'om green to yellow-brownish in color. After 7 d
exposure to 2,4-DNT 2 2.0 mg/L, duckweed fronds were white indicating the
occurrence of severe chlorosis and death of the plants. No apparent grth was

observed at 2,4-DNT concentration of 20 mg/L. The higher sensitivity of Lemna

minor to 2,4-DNT exposure made it more suitable for use in our bioassays designed to test

the effects of K-SWy-2 on 2,4-DNT toxicity and bioavailability (Figure 1 and Tablel).

45

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.E

 

 

 

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46

Among the factors influencing the bioavailability of organic contaminants in
the soil environment, sorption is a major process which can reduce the bioavailability
of organic compounds to target organisms. Nitroaromatic compounds have been
reported to demonstrate strong affinities for K+-saturated smectites (Boyd et al., 2001;
Haderlein and Schwarzenbach, 1993; Haderlein et al., 1996; Johnston et al., 2001;
Johnston et al., 2002; Li et al., 2004a; Sheng et al., 2002). As NACS are sorbed by K-
smectites, their availability to aquatic plants may be reduced. The growth responses
of Lemna minor to 7.5 mg/L of 2,4-DNT solutions in the presence and absence of K-
SWy-2 clay afier 11 (1 exposure are shown visually in Figure 2. In the system absent
of K-SWy-2, the 7.5 mg/L 2,4-DNT solution completely inhibited Lemna minor
growth as evidenced by failure to reproduce more fronds over the 1] (1 exposure
period. The toxicity of 2,4-DNT at this concentration was apparent afler 4 (1 exposure
as duckweeds showed signs of chlorosis consisting of pale yellow and white spots on
tissues. Death of Lemna minor occurred at ~ 9 d exposure. The addition of K-SWy-2
substantially reduced the inhibitory effects on frond production and toxicity arising
from the presence of 7.5 mg/L of 2,4-DNT in the absence of clay. Addition of K-
SWy-2 reduced 2,4-DNT concentration in water from 7.5 mg/L to 0.4 mg/L, an
aqueous phase concentration which had minimal impact on duckweed growth in the
absence of clay (Fig.1). In Figure 2, it is noted that duckweeds grew somewhat better
in the system containing K-SWy-2 clay alone (without 2,4-DNT). Thus, the clay
itself, independent of its ability to sequester 2,4-DNT, stimulated growth of Lemna

minor to some extent.

47

 

Figure 2. Photograph showing reduced bioavailability and toxicity of 2,4-
dinitrotolune (2,4-DNT) to Lemna minor in the presence of homoionic K-
montmorillonite (K-SWy-Z) clay (300 mg clay in 150 mL solution). The
photograph was taken after an incubation period of lldays. Treatments
consisted of various combinations of 2,4-DNT and/or K-SWy-Z.

The growth of Lemna minor in 1/5X DWM containing 7.5 mg/L 2,4-DNT in
the presence and absence of K-SWy-2 as a function of exposure period is shown in
Figure 3. The solid lines are fittings of the data to a first-order growth process
described by equation [1], and the corresponding growth rate constants and doubling

48

time are listed in Table 2. No apparent growth was noted in solutions containing 7.5
mg/L of 2,4-DNT without K-SWy-2 present. The amendment of K-SWy-2 to l/SX
DWM containing an initial 2,4-DNT aqueous concentration of 7.5 mg/L resulted in
the doubling time of Lemna minor of 2.5 days, which is comparable to the treatments
free of 2,4-DNT in the presence of K-SWy-2 (T2 = 2.3 days) and the absence of K-
SWy-2 (T2 = 2.8 days). Adsorption of 2,4-DNT by K-SWy-2 significantly reduced
the aqueous 2,4-DNT concentration (from 7.5 mg/L to 0.4 mg/L) and it is apparent
that this reduction caused decreased bioavailability and hence toxicity of 2,4-DNT to

duckweeds (Figures 2 and 3).

49

‘é

 

     
      

—e— 2,4-DN'I‘ + K-SWy-Z

—A- 2,4-DNT + no K-SWy-2

+ Control: no 2,4-DNT + K-SWy-2
+ Control: no 2,4-DNT + no K-SWy-2

Number of Fronds
.5 N a
8 8 o

§

 

 

 

 

Exposure Time (Days)

Figure 3. Effects of 2,4-dinitrotoluene (2,4-DNT) and clay on the growth rate of
Lemna minor in 1/5X DWM over a incubation period of 1] days. Treatments
were: 7.5 mg/L 2,4-DNT; 7.5 mg/L 2,4-DNT (added) plus homoionic K-
montmorillonite (K-SWy-Z); K-SWy-Z; control containing neither 2,4-DNT nor
K-SWy-Z.

50

H: 523:3
85 538» 3030—05. 550.65 95 3 532m we mafia .305— 55 3506808 cote—oboe .3 25cm : "538» “game e: o

 

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.5 .m: .m: 3&8 m5 Essa

133. .o o2...

 

«mas o. as
t a. Q ox 8553:.

 

gauge Sum 5380

 

62.5.— 9525: .95 3 5. «5.5.. 3385?! me 3:03.— ..5. 358.:— 2: 5 P75 4.6
5539553.” 9553535: 8 63.2.5 .35! 32.: me A": o5.» new—5.3. 55 a: 5553 85 5.395 .N 03:.

51

The results presented clearly indicate that sorption of 2,4-DNT by K-SWy—Z
manifested a diminished bioavailability and toxicity to aquatic plants. This
information could be useful in developing strategies for control of NAC mobility and
bioavailability to target organisms such as plants and microorganisms utilized in in-
situ bioremediation technologies. As part of a watershed geostabilization strategy,
clay-modulated NAC sorption would allow sufficient phytoremediative plant biomass
accumulation at otherwise phytotoxic soil contamination levels (e.g. of NACS). This
would allow for effective phytostabilization and contaminated particulate erosion
control. Furthermore, subsequent ion exchange of K+ (on clays) with Ca2+ could be
used to release target NACS in a controlled fashion (Li et al., 2004b) into an active
rhizosphere for effective plant uptake or bioremediation. This is the subject of an

ongoing study in our laboratory.

52

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chemicals to natural particles. Environmental Science and T echnology, 30, l-
l 1.

Scribner, S.L., Benzing, T.R., Sun, SB. and Boyd, S.A. 1992. Desorption and
bioavailability of aged sirnazine residues in soil from a continuous corn field.
Journal of Environmental Quality, 21 , 1 15-120.

Sheng, G.Y., Johnston, C.T., Teppen, BJ. and Boyd, S.A. 2002. Adsorption of
dinitrophenol herbicides from water by montrnorillonites. Clays and Clay
Minerals, 50, 25-34.

Smith, S. and Kwan, M.K.H. 1989. Use of aquatic macrophytes as a bioassay method
to assess relative toxicity, uptake kinetics and accumulated forms of trace
metals. Hydrobiologia, 188/189, 345-351.

Steinberg, S.M., Pignatello, J .J . and Sawhney, BL. 1987. Persistence of 1,2-
dibromoethane in soils - entrapment in intraparticle micropores.
Environmental Science and T echnology, 21, 1201-1208.

Sunahara, G.I., Dodard, S., Sarrazin, M., Paquet, L., Ampleman, G., Thiboutot, S.,
Hawari, J. and Renoux, A.Y. 1998. Development of a soil extraction

55

procedure for ecotoxicity characterization of energetic compounds.
Ecotoxicology and Environmental Safety, 185-194.

Taraldsen, J .E. and Norberg-King, T.J. 1990. New method for determining effluent
toxicity using duckweed (Lemna minor). Environmental Toxicology and
Chemistry, 9, 761-767.

van Olphen, H. and Fripiat, J .J . 1979. Data handbook for clay minerals and other non-
metallic minerals. Pergamon Press, New York, NY.

Wang, W. and Freemark, K. 1995. The use of plants for environmental monitoring
and assessment. Ecotoxicology and Environmental Safety, 30, 289-301.

Ware, G.W. 1997. Introduction to Insecticides University of Arizona, Deptment of
Entomology, Tucson, Arizona.

Wu, T.L., Lambert, L., Hasting, D. and Banning, D. 1980. Enrichment of the
agricultural herbicide atrazine in the microsurface water of an estuary.
Bulletin Environmental Contaminant and Toxicology, 24, 411-414.

56

CHAPTER [11

Geochemical Modulation of Bioavailability and Toxicity of Nitroaromatic

Compounds to Aquatic Plants

57

Abstract

Nitroaromatic compounds (NACs) are a prominent class of environmental
toxic contaminants that pose a threat to human and ecosystem health. Nitroaromatic
compounds demonstrate a high affinity with smectite clays which are widely
distributed in nature as components of soils and sediments, and in geological deposits.
The magnitude of NAC sorption by smectites is highly dependent on the
exchangeable cation. For example, smectites saturated with K+ have a very high
affinity for NACs while those saturated with Ca2+ do not. In this study the ability of
K+-saturated smectite to attenuate the bioavailability and hence toxicity of 2,4-
dinitrotoluene (2,4-DNT) to the aquatic plant duckweeds (Lemna gibba and Lemna
minor) was evaluated. 2,4-DNT at the concentration of 7.5 mg/L was highly toxic to
duckweeds. However, the presence of small amounts of K-smectite in aqueous
growth media reduced this toxicity substantially because the sorption of 2,4-DNT by
the added smectite reduced bioavailability. This reduced bioavailability could be
modulated by simple K+/Ca2+ cation exchange reaction occurring on mineral surfaces.

In the subsequent experiments, CaC12 was added and the Ca2+ replaced K+ associated

with smectite surfaces. Such reaction released clay-sorbed NACS to aqueous
solution, which is readily available to target plants. This study proves the concept
that we can modulate the type and fraction of cations occupying the cation-exchange-
capacity sites of smectite clays to control the sorption/desorption hence
bioavailability of NACs in smectite-rich soils and sediments. This information is

needed in developing strategies for control of NAC mobility and bioavailability to

58

target organisms such as plants and microorganisms utilized in in-situ bioremediation
technologies.
Introduction

Smectites are expandable 2:1 layered alumina silicate clays that are widely
distributed in nature (Allen and Hajek, 1989). They have structural negative charges
arising from isomorphic substitution in the octahedral and/or tetrahedral layers. In
nature, common inorganic cations (e.g., Ca2+, Mg2+, Al”, K+, and Na+) exist on clay
surfaces to balance the negative charges. These exchangeable cations control clay
interlayer expansion via hydration, and may interact with environmental
contaminants/ pesticides, thereby leading to sorption from water and immobilization.
Among the clay minerals commonly found in soils, smectites may function as
particularly effective adsorbents for organic contaminants and pesticides because of
their high surface areas, surface acidity and reactivity, large cation exchange
capacities, and reversible interlayer expansibility.

Sorption by soils/sediments is a major determinant of environmental fate of
organic contaminants and pesticides, such as potential for leaching and bioavailability
to microorganisms and plants (Koskinen and Harper, 1990). Sorption ofien renders
contaminants less available to receptor organisms (Hatzinger and Alexander, 1995;
Luthy et al., 1997; Ogram et al., 1985; Pignatello and Xing, 1996). Scribner et al.,
(1992) and Steinberg et al., (1987) showed that ethylene dibromide and simazine
sorption and sequestration by field weathered soils reduced bioavailability to
contaminant-biodegrading microorganisms thereby prolonging residence times.

Reduced bioavailability in these instances was associated with prolonged pesticide-

59

soil contact times. A number of studies have examined the bioavailability of sorbed
contaminants to microorganisms (Boyd et al., 1992; Feng et al., 2000; Park et al.,
2004). There are examples where bacteria can and cannot access contaminants
sorbed to soils. At this point, no generalizations can be made regarding the
bioavailability of sorbed organic contaminants. Harris (1964) showed lowered
bioavailability of insecticides (to crickets) sorbed to dry soils as evidenced by higher
LDso values compared to the insecticides in the non-sorbed state. Wetting the soils
apparently caused desorption of the insecticides, as LDso values decreased by an order
of magnitude or more. Soil minerals in the dry state have been shown to sorb much
higher amounts of organic contaminants than occurs in the presence of water (Chiou
2002). It was hypothesized that water molecules preferentially occupied the mineral
surfaces rendering the ability of soil mineral to sorb and retain organic molecules in
the presence of water. Recent work on the bioavailability of 2,4-dinitrotoluene (2,4-
DNT) showed that in water it was highly toxic to the aquatic duckweed (Roberts et
al., 2006). However, sorption of 2,4-DNT by K-saturated smectite in aqueous media
reduced its bioavailability and hence toxicity to duckweed.

Nitroaromatic compounds (N ACs) are components of munitions that are
commonly found as soil and ground water contaminants in military training facilities
and manufacturing sites. For example, 10% of the NACs in many munitions may fall
to the soil as unaltered NAC after an ordinance explodes (Taylor et al., 2004).
Nitroaromatic compounds also include important pesticides such as the dinitrophenol
herbicides that may contaminate ground- and surface-waters or persist in soils where

they are used in crop protection (Brohohn, 2001; Hawley, 1981). As such, NACS

pose threats to human and ecosystem health (Berthe-Corti et al., 1998; Gong et al.,
1999; Hawley, 1981; Neumann et al., 1995). For instance, the —N02 of NACS in
organisms can be reduced to amino groups that conjugate with the phenolic sites in
biota (Morgan, 1989; Ware, 1997), with toxic effects (Gong et al., 1999; Sunahara et
al., 1998).

Many smectites demonstrate high affinity for aqueous phase NACs, and
sorption is strongly influenced by the type of cations on ion-exchange sites of the
clays. For example, smectites exchanged with Cs+, K+ and NH: have high affinities
for NACS whereas smectites display much lower NAC adsorption when exchanged
with Na+, Ca2+ or Mg” (Boyd et al., 2001; Haderlein and Schwarzenbach, 1993;
Haderlein et al., 1996; Johnston and Premachandra, 2001; Johnston et al., 2002; Li et
al., 2004a; Sheng et al., 2002). Strong hydration of Na+, Ca2+ and Mg“ cations
creates an unfavorable clay interlayer environment for NAC adsorption, whereas the
comparatively weak hydration of Cs+, K+ and NIrIa+ cations creates a much more
favorable interlayer environment for NAC adsorption. Adsorption of NACs occurs
primary in the clay interlayer and weaker cation hydration manifests a more optimal
interlayer distance, larger adsorptive domains, enhanced ability to form NAC-cation
complexes, and greater solute dehydration (Boyd et al., 2001; Johnston and
Premachandra, 2001; Johnston et al., 2002; Li et al., 2004a; Sheng et al., 2002).

Among the factors influencing the bioavailability of organic contaminants in
the soil environment, sorption is a major process that can reduce the bioavailability of
organic compounds to target organisms. In the case of NACs, smectite clay surface

chemistry can be used to control sorption and bioavailability of NACs by simple ion-

61

exchange reactions that change the interlayer ion composition of the clays. For
example, a K+-saturated system would maximize NAC retention and could be used to
minimize transport of the environmental contaminants. This was indicated in a recent
study where KC] solutions injected into an aquifer were shown to reduce transport of
nitrotoluene, and subsequent injections of CaClz were shown to remobilize
nitrotoluene (Weissmahr et al., 1999). In another study, K-smectite was used to
promote aqueous phase NAC adsorption, thereby reducing bioavailability and toxicity
of the NAC to aquatic plants (Roberts et al., 2006). Li et al., (2004a) measured
adsorption of the pesticide dichlobenil by mixed K+/Ca2+-smectites and found that the
adsorption increased with increasing molar ratio of K+ to Ca2+on the cation exchange
sites of montmorillonite. Their results demonstrated that the degree of pesticide
adsorption by smectite clays could be controlled by adjusting the amount of K+ and
Ca2+ associated with clay through simple geochemical cation exchange processes.
Thus, simple reactions such as Ca-for-K ion exchange might hold promise as a
method to modulate the bioavailabilities of NACS in soils and sediments.

The objective of this study was to demonstrate the ability to modulate the
bioavailability and toxicity of NACs by controlling their binding and release via
cation ion exchange reactions on smectite clays. There is a need to optimize
bioavailability of contaminants during bioremediation so that they are sufficiently
available for effective plant uptake or bioremediation without being present at levels
that are toxic to the organisms used for biodegradation. The target organism used in
this study was duckweed (Lemna minor), a group of free-floating aquatic plants

usually found growing on nutrient-rich water in ponds and lakes. Toxicity testing

62

using duckweeds is an accepted and common protocol for evaluating the aquatic
toxicities of organic and heavy metal contaminants (ASTM and Materials, 1991;
Hillmans, 1961; Holst and Ellwanjer, 1982; Smith and Kwan, 1989; Taraldsen and

Norberg-King, 1990; Wang and Freemark, 1995; Wu, 1980).

63

Materials and Methods
Chemicals

2,4-Dinitrotoluene (2,4-DNT) was purchased from Aldrich Chemical
Company Inc. (Milwaukee, WI) with a reported purity > 97%. Potassium chloride
(KCl), calcium chloride (CaClz), magnesium sulfate (Mg804), potassium nitrate
(KNO3), potassium phosphate dibasic (KH2P04), boric acid (I-I3B03), sodium
molybdate (N azMooa), sodium acetate (CH3COONa) and cupric sulfate (CuSOa)
were purchased from Mallinckrodt Baker Inc. (Phillipsburg, New Jersey). Ferric
chloride (FCC13), manganese chloride (MnClz), and zinc sulfate (ZnSOa) were
obtained from Sigma (St. Louis, MO), and ethylenediaminetetracetic acid (EDTA)
was obtained from Invitrogen Corporation (Carlsbad, CA.). All chemicals were used
as received.
Clay preparation

Reference smectite clay (Wyoming montmorillonite, SWy-2) was obtained
from the Source Clays Repository of the Clay Minerals Society (Purdue University,
West Lafayette, Indiana) and used throughout this study. The SWy-2 clay has a CEC
of 82 cmolc/kg and a theoretical surface area of 750 mz/g (van Olphen and Fripiat,
1979). The < 2pm clay particle-sized fi‘action was obtained by centrifuging a dilute
clay suspension for 6 min at 60 g (~600rpm) using a Sorvall GSA rotor and RC-SC
centrifirge (DuPont Co., Wihnington, Delaware) (Costanzo and Guggenheim, 2001).
Prior to separating the clay-sized particles, the SWy-2 clay sample was treated with
0.5 M sodium acetate to remove carbonate impurities (Arroyo et al., 2005). The K-

saturated homoionic clay was prepared using cation exchange reactions. Specifically,

the naturally occurring inorganic exchangeable cations (predominately Na+) on SWy-
2 were replaced with K’( by washing the clay-sized fraction of smectite three times
with 0.5 M KCl solution. K-smectite suspensions were then dialyzed against Milli-Q
water to remove excess electrolytes; the absence of white AgCl precipitate upon
addition of AgNO; indicated that the clay suspensions were free of chloride. The
prepared clay suspensions were quick-frozen and freeze-dried. Prior to use in
phytotoxicity assays, K-smectite clay was sterilized by heating at 120°C for 3-5 days.
Duckweed culture

Duckweeds were used as the phytotoxic indicator species for evaluating NAC
exposure. Lemna minor and Lemna gibba duckweed cultures were obtained fi'om the
Culture Collection of Algae and Cyanobacteria (CCAC, University of Toronto;
Toronto, Ontario, Canada). Axenic duckweed cultures were maintained in 300-mL
Erlenmeyer flasks containing 150 mL of full-strength (1X) Hoagland’s nutrient
solution (Cowgill and Milazzo, 1989) at pH 5.8, supplemented with 10 g of sucrose
per liter as recommended by the CCAC (Judy Acreman, personal communication).
After autoclave sterilization of the sucrose-amended Hoagland’s solution, 20 mL of a
FeEDTA solution (prepared by dissolving 0.121 g FeCl3 and 0.375g EDTA into 250-
mL Milli-Q water and passed through a 0.22-um filter) were added to complete the
1X duckweed medium (1X DWM). Culture flasks inoculated with duckweed were
closed with sterilized foam plugs (~ 27 to 34 mm), the plugs covered with aluminum
foil, and placed in a plant growth chamber (Boone, Iowa) at 25°C with a 16 hr day

length and a photosynthetically active radiation (PAR) intensity of ~43 umol s‘l m'2.

65

Plants were subcultured every 3 weeks by transferring approximately 16 fronds into a

flask containing fiesh sterile culture medium.

Sorption Experiments

Sorption of 2,4-DNT by K-SWy-2 fiom KCl or CaClz solutions was measured
using a batch equilibration technique. Clay was weighed into triplicate glass
centrifuge tubes, aqueous solutions over a range of initial 2,4-DNT concentrations
were added, and the tubes were closed immediately with Teflon-lined screw caps.
The tubes were shaken end-over-end at 40 rpm for 24 h at room temperature. The
tubes were centrifuged for 30 min to separate liquid and solid phases. Previous
studies demonstrated that sorptive equilibrium has been reached within 4 h (Sheng et
al., 2002). Concentrations of NACs in supematants were measured by a Perkin-
Ehner High-Performance Liquid Chromatography (HPLC) system that consisted of a
Binary 250 LC pump, a Series 200 autosarnpler and a Series 200 UV-visible detector.
The optimal wavelength was set at 250 nm for 2,4-DNT. The mobile phase
composition was a 1:1 mixture of methanol and water. Control samples included the
initial aqueous 2,4-DNT solutions in the absence of clay at each 2,4-DNT
concentration. No changes in solute concentrations were detected in the tubes
containing aqueous 2,4-DNT without clay; therefore, solute mass lost from the
supernatant in the presence of clay was assumed to be sorbed by clay. The sorbed
concentrations were calculated from the difference between control and final solute
mass in aqueous solution. Sorption isotherms were obtained by plotting the amount

of sorbed by clay against the equilibrium aqueous phase concentrations.

66

NA C phytotoxicity

Phytotoxic assays for 2,4-DNT were carried out using Lemna minor
duckweed cultures in in-vitro bioreactors containing duckweed culture medium with a
1/5-strength of Hoagland’s solution (1/5X DWM). Using aseptic techniques in a
laminar flow hood, 300-mg sterile K-SWy-2 clay were weighed into 300-mL
Erlenmeyer flasks, 75 mL of 1/5X DWM containing 7.5 mg/L 2,4-DNT added, and
sterilized foam plugs partially inserted into the mouth of the flasks. F oarn-plugged
flasks were covered with aluminum foil to reduce the evaporative loss of water.

The effects clay sorption of 2,4-DNT on duckweed growth was evaluated as
follows. Prior to inoculation with duckweed, 75 mL of 2,4-DNT solution was added
to flasks that contained 300 mg of sterilized K-SWy-2 clay and then the flasks were
shaken for four hours at room temperature to establish sorption equilibrium of 2,4-
DNT with K-smectite. This volume of solution (75 mL) provided sufficient surface
area and volume for duckweed growth and multiplication. To ascertain sorption
equilibrium, the aqueous phase was periodically sampled and analyzed for 2,4-DNT
concentrations using HPLC. This study and previous studies both indicated that
sorption equilibrium of NACs by K-smectites was reached within four hours (Sheng
et al., 2002).

To initiate the phytotoxicity assay, 8-10 green healthy fronds were aseptically
transferred to each flask. The foam plugs and foil were replaced and the flasks
incubated in a growth chamber under the conditions described previously. Thus, the
bioavailability bioassays consisted of 75 ml of 1/5-strength Hoagland solution

containing 0.01M KCl background electrolyte, 7.5 mg/L 2,4-DNT, 300 mg of K-

67

SWy-2 clay and 8-10 fi'onds. Controls consisted of the same l/SX DWM with clay
but without 2,4-DNT. Duckweed frond numbers were counted manually at selected
sampling times up to 408 hours of incubation as a measure of NAC phytotoxicity. At
pre-selected sampling time (at 0, 4, 9, 11 and 17 days) 2 mL of solution were
sampled, transferred to vials and then the 2,4-DNT aqueous-phase concentrations
were measured using HPLC.

Release of 2, 4-DNT into solution

Afier four days of incubation and upon the establishment of 20 duckweed
fi'onds, 75 mL of either 0.1 M CaClz or 0.01 M KCl (both in 1/5 DWM) were added
to the flasks to evaluate the release or retention of sorbed-2,4-DNT by the smectite
clay and the consequent effects on duckweed growth. These bioreactor flasks with a
final volume of 150 mL were shaken for four hours to mix the systems and then
incubated in a growth chamber under the same conditions described previously.
Controls consisted of the same solution treatments with duckweeds and clay, but
without 2,4-DNT.

A similar bioassay as was used to isolate the effects of cations themselves on
duckweed growth in the absence of clay and NAC. That is, after four days of
incubation, 75 mL of 0 to 0.2 mol/L C1 solutions of K+, NHX, or Ca2+ in 1/5 DWM
were added to the bioreactors, and subsequent duckweed growth was monitored.

Also, duckweed biomass, as an alternative index for duckweed growth was
measured using the following procedure. Duckweed fronds were removed fi'om
flasks, rinsed four times with distilled water, blotted with tissue paper, and the whole

plants (fronds and roots) immediately weighed to obtain the flesh weight. The tissues

68

were then dried in an oven at 70 °C until each sample achieved a constant weight
(dried biomass). The mean of four samples at each sampling interval was considered
one observation. Four replicate samples were sacrificed for measuring biomass at 0,
4, 9, 11 and 17 days of incubation.

A completely randomized design was adopted. Before comparing treatment
differences, the natural log transformation was applied because we observed trends in
treatments of the original data which had larger variance at later time points
compared to the small variance of early time points. The analysis of variance
(ANOVA) was applied to determine the statistical significance (P<0.05) for
differences in frond numbers among treatments. Statistical analyses were performed
on data fiom exposure day 4 to day 17 using mixed procedures (PROC MD(ED; SAS

Inst, 2005) to analyze treatment differences in duckweed frond numbers.

69

Results and Discussion

Smectite clay is an effective sorbent for 2,4-DNT, with the extent of sorption
depending largely on the exchangeable cations occupying the CEC sites of the clay.
Sorption of 2,4-DNT fi'orn 0.01 M KCl and 0.005 M CaClz by K-smectite is
represented in Figure 1 as sorption isotherms, with amount sorbed per unit mass of
clay plotted against equilibrium aqueous concentration. Sorption of 2,4-DNT by K-
smectite was substantially reduced in the presence of 0.005 M CaClz compared to that
in KCl solution, For example, at aqueous 2,4-DNT concentration of 2.0 mg/L,
sorption of 2,4-DNT in 0.005 M CaClz was about 1/10 the sorption in 0.01 M KC1
solution. The lowered affinity of 2,4-DNT for K- smectite is attributed to ion
exchange of K+ on K-smectite by Ca2+. Specifically, in 0.005M CaClz, Ca2+replaces
some K+ on smectite eliminating some K+-saturated clay domains/interlayers where
NAC sorption is believed to occur (Li et al., 2004b).The favorable clay-NAC
interactions afforded by K- smectite such as the formation of complexes between the
-N02 groups and K+ associated with the clay and/or electron-donor-and-acceptor
interactions between clay surfaces and planar aromatic rings of NACs, are diminished
with Ca2+ as the exchangeable cation (Boyd et al., 2001; Haderlein and
Schwarzenbach, 1993; Haderlein et al., 1996; Li et al., 2004b). The relatively strong
hydration of Ca2+ results in a larger hydration sphere surrounding it thereby,
inhibiting complex formation with NACs and interactions with clay surfaces (Boyd et
al., 2001; Haderlein et al., 1996). Furthermore, Ca2+ on exchange sites of smectites
causes greater interlayer swelling compared to K-saturated smectites (Boyd et al.,

2001; Haderlein et al., 1996; Johnston and Premachandra, 2001; Johnston et al., 2002;

70

Sheng et al., 2002), which may also reduce hydrophobic contributions to NAC
sorption (Boyd et al., 2001; Li et al., 2004b). As a consequence, addition of Ca2+
caused 2,4-DNT sorption by K-smectite to be reduced. We propose that such a cation
exchange process can be utilized to control the retention/release of 2,4-DNT from

clay hence altering the bioavailability and toxicity of 2,4-DNT to target organisms.

 

5000

  

K-smectite + 0.01 M KCI

  
   

4000 -

3000 .

2000 -

1000 -

Amount of 2,4-DNT Sorbed, Q (mg/kg)

K-smectite + 0.005 M CaCl2

 

 

 

T I T

0 2 4 6 8
Pesticide Equilibrium Concentration, Ce (mg/L)

Figure 1. Adsorption of 2,4-DNT by K-smectite suspensions in presence of
0.01M KCI or 0.005 M CaClz.

Response of Lemna minor duckweed to added K+, NH4+ and Ca2+
Lemna duckweed is clearly sensitive to the toxic effect of 2,4-DNT, but for
subsequent assays in the presence of clay, it was important to distinguish between the

toxicity of the test compound and possible effects emanating from cations associated

71

with the smectite clay surfaces. The cations K+, NH4+ and Ca2+ at aqueous
concentrations between 0 and 0.2 M were tested for their effect on growth of Lemna
duckweeds (Fig.2). These cations are candidates for modulating the binding and
release of NACs by clays (Li et al., 2004b) and hence provide the basis for
controlling bioavailability and toxicity of NACS. Smectite clays saturated with K+ or
NHfare strong sorbents for NACs whereas Ca-smectite is much less effective in
removing NACS from aqueous solution (Boyd et al., 2001). Thus, by controlling the
type and distribution of exchangeable cations on smectite clays, NACs may be
selectively removed from water or released into solution (Li et al., 2004b). In Figure
2, the Lemna minor response to the cations at concentrations ranging between 0.01
and 0.2 M for K“, NHI, and Ca2+in solution are compared to controls without added
exchangeable cations. Evaluating the ability of plants to grow in the presence of
these cations (in absence of clay) is necessary to determine which cations, at what
concentrations, might cause toxicity thereby complicating our assay for evaluating the
effectiveness of smectites for controlling toxicity and availability of NACs
duckweeds. Even at the comparatively low aqueous concentration of 0.01M, NI-Lt+
substantially reduced the number of fronds. At 0.1 M, NI-If, K+ and Ca2+ all
substantially limited the number of fronds produced. Thus, NH; at all concentrations
tested, and higher concentrations of K+ and Ca2+ (Z 0.05 M) were found to inhibit
plant growth. Since 0.01 M of KCl had no toxic effect on plant growth, it was chosen
as a background electrolyte solution in our bioassays. K+-smectite clay was selected
as the model adsorbent in this study since it is known to be an effective adsorbent of

many NACs including 2,4-DNT. A background solution of 0.01 M K+ ensures that

72

small amounts of K+ in bulk solution, due to the presence of K-smectite, will not

contribute to the apparent toxicity of 2,4-DNT to duckweeds used in our bioassays.

73

 

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74

 

140

 
 
  
   

NH
120 q —a— +4
-A_' K

100 1 -e- Ca2+

m

U I I

E; 80 ‘ Controls wrthout cations

u.

3 so a

E

g 40 -

z

N
O
r

 

O
r

 

 

 

I

0.00 0.05 0.10 0.15 0.20 0.25
Cation Aqueous Concentration (M)

Figure 2. Growth of duckweed after 12 day bioassay in presence of different
electrolyte solutions at concentrations ranging from 0.01 and 0.20M. The
electrolytes chosen contain cations that might be used to control binding (NHI,
K") and release (Car) of nitroaromatic compounds by smectite clay.

To test the hypothesis that K+/Ca2+ exchange reactions can be used to
modulate the bioavailability of 2,4-DNT to a target organism (e. g. plants and
microbes), the duckweed Lemna minor was identified as an appropriate plant for this
study (Roberts et al., 2006). Figure 3 shows the responses of Lemna minor to the
presence of 2,4-DNT in K-SWy-2 suspensions in terms of frond numbers and

biomass production. The 2,4-DNT aqueous concentration was also monitored during

75

the l7-day incubation. At the initiation of the experiment K-smectite (300mg) was
added which substantially reduced the 2,4-DNT concentration in water from 7.5 mg/L
to < 0.31 mg/L, an aqueous phase concentration that has minimal impact on Lemna
minor growth (Roberts et al., 2006). This is seen during the first 4-d exposure, where
there were no differences in duckweed growth in the K-smectite suspensions with the
added 2,4-DNT and the 2,4-DNT-free controls (Figure 3). Figures 4a and 4b visually
show that there were no observable differences after 4 days of exposure.

It was necessary to determine growth conditions in our bioassays that would
allow us to distinguish the potential effects of Caz’L/K+ ion exchange on the release of
2,4-DNT into solution and subsequent effects on bioavailability and toxicity to
duckweed.

At 4-d exposure, since there were a sufficient number of fronds present (but not too
many) and no apparent difference in the number of fronds produced between the two
systems (2,4-DNT and 2,4-DNT-fi‘ee controls), electrolyte solutions of KCl and
CaClz were added to initiate the ion exchange process. Such addition of electrolytes
at 4 d of exposure was appropriate for visualizing of the effects of toxicity of 2,4-
DNT. In our previous efforts developing the appropriate bioassays conditions there
was no apparent inhibition on growth ofLemna minor at the l/SX DMW
concentration used, and the 150 mL volume of solution provided sufficient surface
area of water for plants to grow and be distinguishable so that frond counts be made

during a two week incubation period.

76

1400
1200
1000
800
600
400
200

Frond Numbers

100

Dry wt. (mg)

3.0
2.5
2.0

1.5

Aqueous Concentration of 2,4-DNT, (mg/L)

 

 

(A)

On day 4,

added KCl or CaClz

  
      
 
 

+ 2,4-DNT + KCI
—A— No 2,4-DNT + KCI
+ 2,4-DNT + CaCI2

-V—- No 2.4-DNT + CaCI2

 

 

 

 

q

 

(C)

Ti—I-I—fl ——————— I—--— ———————— I

 

/

 

 

0

2

O
I I l I I I U

4 6 8 1O 12 14 16 18

Exposure time (Days)

Figure 3. The growth of Lemna minor in the presence of 2,4-dinitrotoluene (2,4-
DNT) and K-smectite as affected by addition (day 4) of KCl or CaClz solutions.
Growth and toxicity are indicated by frond numbers (A) and biomass (B).
Aqueous concentrations of 2,4-DNT during the 17 day growth experiment are
show in panel (C).

77

At day 4, 0.1M CaClz or 0.01M KCl were added into the clay-duckweed
systems with or without 2,4-DNT. Lemna minor growth curves were nearly identical
for KCl-added systems whether 2,4-DNT was present or not (Figure 3A). In the
absence of 2,4-DNT, the addition of CaClz slowed down frond production compared
to that with KCl addition. In the suspensions containing 2,4-DNT, the addition of
CaClz caused the aqueous concentration of 2,4-DNT to increase (Fig. 3C) resulting in
inhibition of duckweed growth. Therefore, in the case of adding 0.1M CaClz to 2,4-
DNT-clay system, itself apparently caused some inhibitory effects on duckweed
growth, consistent with results shown in Fig. 2, however the inhibitory effects if Ca2+
vs. 2,4-DNT could be easily discerned. In summary, the 2,4-DNT released into
solution as a result of CaClz addition (Fig. 3C) caused complete cessation of Lemna
minor growth, clearly demonstrating that 2,4-DNT became available hence toxic to
the duckweeds. We made comparisons of frond numbers (means of four replicates) at
different periods of exposure to determine at which point toxicity effects due to
release of 2,4-DNT associated with CaClz addition were most apparent (Table 3).
Between days 9 and 11, toxic symptoms were most obvious. This corresponded to
visual observation that by day 11 the Lemna minor fi'onds had changed from green to
yellow-brownish in color. After 17 days, in the systems with CaClz and 2,4-DNT,
fronds turned white indicating the occurrence of severe chlorosis and death of the
plants. The measured dried biomass (Figure 3B) also showed the same trend as that

revealed by frond numbers.

78

Exposure time (day 4)

Exposure time (day 17)

 

Figure 4. The effects of 2,4-dinitrotoluene (2,4-DNT) in K-smectite suspensions
on duckweed (Lemna minor) growth. Duckweed frond numbers were equal in
presence (A) or absence (B) of 2,4-DNT on day 4 when either KCI or CaCI;
solution were added. The effects of these treatments after 17 day incubations
are shown in panel (C). Addition of CaClz caused release of 2,4-DNT into
solution where it inhibited growth.

79

The addition of CaClz to K-smectite suspension containing (mostly sorbed)
2,4-DNT resulted in 2,4-DNT concentration in water phase increasing from 0.31
mg/L to 1.5 mg/L (Figure 3C), a concentration that inhibits the growth of duckweeds
(Roberts et al., 2006)). The addition of KCI did not cause an increase in the aqueous
2,4-DNT concentration. In fact, it resulted in a slight reduction of aqueous
concentration (from 0.31 to 0.23 mg/L).

To quantitate the effects of salt solutions and of 2,4-DNT toxicity on
duckweed growth, progress curves showing Lemna minor fi'ond numbers and biomass
as a function of exposure periods were fitted to the first-order kinetic processes.

In F; = in F}, + kt [1]
where F, and F0 are the frond number or biomass at the time t and initial time,
respectively, k (day'l) is growth rate constant. The results of fittings are shown as
solid lines in Figures 2, 3A and 3B. The effects of 2,4-DNT on growth data were
well described by the equation [1]; fitting the frond numbers data to the linear form of
equation [1] gave 1’2 values 2 0.95. Doubling times, T 2 (days) for duckweed growth in

terms of fiond number and biomass were calculated using the equation:

T. = 111% [2]

The growth rate constants and doubling times are listed in Tables 1 and 2.
The doubling times were lower (faster growth) for KCl treatments (with 2,4-DNT and
with no 2,4-DNT present) relative to those for both CaClz treatments, indicating the
different degrees of bioavailability and toxicity of 2,4—DNT to duckweeds over the
course of incubation. In the key experiment, i.e. the addition of CaClz to K-smectite

systems containing (mostly sorbed) 2,4-DNT, the effects of 2,4-DNT release (induced

80

by Ca2+ exchange for Ki) on growth was evidenced by substantially decreased growth
rate constants and increased doubling times compared with the effect of CaClz
addition when no 2,4-DNT is present, and to all other systems evaluated. The
doubling time (which expresses the level of 2,4-DNT toxicity) for the K-
smectite/CaClz system with 2,4-DNT present was 5 days compared to 1.9 days for the
K-smectite/KCI system with 2,4-DNT present (Table 2). In the K-smectite/CaCl;
system containing 2,4-DNT, the doubling times increased approximately 163 and 100
based on frond production and biomass, respectively, relative to the same system

amended with KCl rather than CaClz.

81

 

 

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82

Comparisons were made among the treatments with 2,4-DNT and controls
without 2,4-DNT at a given exposure time to determine which days 2,4-DNT toxicity
to duckweed became apparent. The treatment effects are easy to discern (Table 3).

At day 4, when systems were amended with KCI or CaClz, there were no significant
differences in frond numbers among treatments. At day 7, the KC] amended systems
frond numbers with and without 2,4-DNT, were not statistically different. Frond
numbers in the CaClz amended system with 2,4-DNT were significantly lower than in
the KC] plus 2,4-DNT system and in the CaClz system without 2,4-DNT. At
subsequent times differences between the CaClz plus 2,4-DNT and KCI plus 2,4-DNT
treatments remained significant, with the later having higher frond numbers. By day
17, the differences in these two treatments were very large; 982 vs. 76 fronds in the
KCl plus 2,4-DNT and CaClz plus 2,4-DNT systems, respectively. The results clearly
show the exchange of Ca2+ for K+ on the exchange sites of K-smectite in the CaClz
amended system induced the release of 2,4-DNT into solution. With the resultant
increased bioavailability of 2,4-DNT, its toxic effects (manifested as lower frond

numbers) were apparent by day 7 and thereafter.

83

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