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Clemson UniversityTigerPrints
All Theses Theses
12-2008
Transformation of Sulfonamide Antibiotics on SoilMineral OxidesBaishu GuoClemson University, [email protected]
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Recommended CitationGuo, Baishu, "Transformation of Sulfonamide Antibiotics on Soil Mineral Oxides" (2008). All Theses. 517.https://tigerprints.clemson.edu/all_theses/517
TRANSFORAMTION OF SULFONAMIDE ANTIBIOTICS ON
SOIL MINERAL OXIDES
A Thesis
Presented to
the Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Environmental Engineering and Science
by
Baishu Guo
December 2008
Accepted by:
Dr. Treavor A. Kendall, Committee Chair
Dr. Cindy M. Lee
Dr. Elizabeth R. Carraway
ii
ABSTRACT
Determining the occurrence and potential adverse effects of antibiotic
pharmaceuticals released into the environment is a critical step towards responsible
environmental stewardship. Under aqueous conditions typically found in soils and natural
waters, the antibiotic agent sulfamethoxazole (SMX) was transformed in the presence of
pyrolusite MnO2 via oxidative pathways. At least 50% loss of SMX was observed after
269 hr in acidic and basic solutions (pH 3-9). A maximum of 100% loss was recorded at
pH 3 and 66% loss was recorded at circumneutral pH. Concomitantly, aqueous
manganese concentrations increased to around 10 µM at pH 3 and 2 µM at pH 6 over the
same time period which suggests an oxidative transformation associated with the mineral
surface. Initial pseudo-first order reaction rate constants (kinit) increase with decreasing
pH from 0.0039 hr-1
at pH 9 to 0.019 hr-1
at pH 3. HPLC results showed similar
transformation products at different pH values. Mass spectrometry of the reaction
products collected at pH 3 and 4 confirm an oxidative pathway by which oxidation and
hydroxylation occurs at the aniline moiety and isoxazole ring of SMX, and hydrolysis
occurs at the sulfonamide moiety. Transformation kinetics of SMX on the surface of
manganite was also studied. The initial transformation of SMX on manganite was faster
than on pyrolusite at pH 4 to 9; however, at pH 3, the transformation rate was much
slower. The observed transformation of SMX by manganese oxides suggests that this
iii
ubiquitous component of soils and sediments likely plays an important role in the fate of
this antibiotic agent in the environment.
iv
DEDICATION
This Thesis is dedicated to my dear husband Jianghong Chen and my son Shawn
Chen who have supported me all the way since the beginning of my studies.
Also, this thesis is dedicated to all mothers who redoubled their efforts on
children and school lives.
v
ACKNOWLEDGEMENTS
With my deep sense of gratitude, I wish to express my sincere thanks to Dr.
Treavor A. Kendall for his immense help in selecting the research topic and executing the
work in time. Profound knowledge and timely wit came as boon under his guidance, his
valuable suggestions as final words during the process of research are greatly
acknowledged.
My sincere Thanks are due to my committee members Dr. Cindy M. Lee and Dr.
Elizabeth R. Carraway, for their providing me constant encouragement and the valuable
discussion that I had with them during the course of research and their helps in arranging
the LC/MS and ICP analysis which are very important to this research.
Words fails me to express my appreciation to my husband Jianghong Chen whose
sharing the load of family trivial and taking care of the child, makes it possible for me to
start this thesis. Furthermore, his scientific advices and knowledgeable in Chemistry
helps a lot to get this thesis deeper and further. My son Shawn deserves special mention
for his cooperative support and encouragement, his love and growth are always the
motivations for my achievement.
The appreciations are due to Drs. Colleen Rostad, Shujuan Zhang and Kathy
Moore for LC/MS, BET surface area, and ICP analysis, respectively.
Finally, I would like to thank everybody who was important to the successful
realization of these, as well as expressing my apology that I could not mention personally
one by one.
vi
TABLE OF CONTENTS
Page
TITLE PAGE................................................................................................................ i
ABSTRACT.................................................................................................................. ii
DEDICATION.............................................................................................................. iv
ACKNOWLEDGEMENTS.......................................................................................... v
LIST OF TABLES........................................................................................................ viii
LIST OF FIGURES ...................................................................................................... ix
CHAPTER
1 INTRODUCTION ......................................................................................... 1
2 BACKGROUND AND MOTIVATION ....................................................... 5
Properties and sorption of SMX................................................................ 5
Degradation of SMX in engineered systems............................................. 6
Properties and reactivities of goethite, pyrolusite and manganite............. 7
3 RESEARCH OBJECTIVES .......................................................................... 10
4 TRANSFORAMTION OF SMX ON MANGANESE DIOXIDES .............. 11
Abstract ..................................................................................................... 11
Introduction ............................................................................................... 12
Materials and methods............................................................................... 14
Results ....................................................................................................... 19
Discussion ................................................................................................. 24
5 TRANSFORMATION AND ADSORPTION OF SMX ON
GOETHITE.................................................................................................... 31
Introduction ............................................................................................... 31
Materials and methods............................................................................... 32
Results and Discussion.............................................................................. 33
Table of Contents (Continued)
Page
vii
6 TRANSFORMATION OF SMX ON MANGANITE................................... 36
Introduction ............................................................................................... 36
Method....................................................................................................... 36
Results and Discussion.............................................................................. 37
7 CONCLUSIONS............................................................................................ 39
8 FUTURE WORK........................................................................................... 41
REFERENCES ............................................................................................................. 72
viii
LIST OF TABLES
Table Page
1 Properties of Goethite, Pyrolusite and Manganite ................................................ 42
2 List of products identified from LC/MS ............................................................... 43
3 List of products identified from GC/MS............................................................... 44
4 Initial reaction constant (Kinit) at different pH and SMX/MnO2 ratio .................. 45
5 UV-Vis absorption of SMX (24.88 µM) in DDI, HCl, and Fe (III) solutions...... 46
ix
LIST OF FIGURES
Figure Page
1 a. General structure of sulfonamide antibiotic; b. General structure of N4-
acetyl sulfonamide antibiotic; c. Structure of SMX (pKa1=1.6, pKa2=5.7 (30)
; Kow=8.222; Ciw(25°C) =0.37 g L-1
(pH=4) (31)
);......................................... 47
2 Eh-pH diagram of goethite, pyrolusite, and manganite (Mn (II) and Fe (II)
= 10-5
M). .......................................................................................................... 48
3 a.Comparison of SMX transformation by pyrolusite at pH between pH 3
and 9 ([SMX]0=4.4 µM, [pyrolusite]0=5 g/l ) ................................................... 49
4 SMX loss after 6 hr and after 269 hr at different pH in the presence of
pyrolusite........................................................................................................... 50
5 Aseptic transformation of SMX on pyrolusite after 3 days (Error bars,
which represent 95% confidence intervals, are smaller than the symbols
used) .................................................................................................................. 51
6 Time-dependent Mn (II) concentrations increase as SMX reacts with
pyrolusite........................................................................................................... 52
7 a. Overall kinetics of transformation of SMX by pyrolusite at the pH 3-9
([SMX]0=4.4 µM, [pyrolusite]0=5 g/l ); (b) Initial reaction kinetics of
SMX with pyrolusite at different pH ................................................................ 53
8 Effect of pH on the initial rate constant of SMX oxidation by pyrolusite.
The initial reaction rate constant kinit (hr-1
) was calculated based on the
pseudo-first-order reaction kinetics ([SMX]0=4.4 µM, [pyrolusite]0=5 g/l ).
Duplicates of experiments are reported with 95% confidence intervals........... 54
9 HPLC profile of products of SMX by pyrolusite after 269 hr. a. pH=3; b.
pH=4.................................................................................................................. 55
10 Mass balance of SMX transformation on pyrolusite after 3 days..................... 56
12 Proposed transformation pathway of SMX by pyrolusite................................. 62
13 Plausible scheme of oxidation on aniline and benzene ring of SMX by
pyrolusite........................................................................................................... 63
List of Figures (Continued)
Figure Page
x
14 Redox scheme on the isoxazole ring on the surface of MnO2 .......................... 64
15 Solubility of goethite depending on pH............................................................ 65
16 Transformation of 4-chloroaniline by goethite ................................................. 66
17 a. SMX concentration of control and reaction depending on pH. Symbols
represent the averages of triplicate measurements made on duplicate vial
sets.; b. absorbed SMX to goethite and Kd value. Samples were collected
after 7 days. Error bars indicate 95% confidence. ........................................... 67
18 UV absorption of SMX within different matrices ............................................ 68
19 X-Ray Diffraction (XRD) of synthesized manganite MnOOH (BG021408,
BG021208) and naturally formed manganite MnOOH (VT sample Ma5)
and pyrolusite MnO2 (BG021308) .................................................................... 69
20 a. Comparison of SMX transformation by MnOOH at pH 3 -9
([SMX]0=4.6 µM, [MnO2]0=5 g/l ); b. Time dependent SMX concentration
between pH 3 and 9 in the absence of MnOOH ([SMX]0 = 4.6 µM) ............... 70
21 Time-dependent Mn (II) concentrations increase as SMX reacts with
MnOOH. ........................................................................................................... 71
1
CHAPTER 1
INTRODUCTION
Pharmaceuticals received little attention as potential environmental pollutants
until the latest decade1. Since, a wide range of pharmaceuticals have been detected in
fresh and marine waters, stimulating recent research on their fate and transport within
natural aqueous systems and their ecological impact. Pharmaceuticals are produced and
used in increasingly larger volumes every year, therefore raising concerns that the
occurrence and potential adverse effects will increase with time2. There are a wide variety
of pathways by which pharmaceuticals and their bioactive metabolites enter natural
waters, including direct introduction to soils and groundwater during animal husbandry
operations or waste disposal, or indirect release as effluent from wastewater treatment
processes that are commonly not designed to remove pharmaceuticals from the waste
stream3.
Antimicrobial agents represent one of the largest classes of pharmaceuticals with
widespread application in human and veterinary medicine. These compounds are of
particular environmental importance because of their ubiquity and their potential to
disrupt bacterial populations in natural systems. An estimated 50,000,000 lbs of
antimicrobials are produced in the US4 alone; the percentage of this mass that reaches
natural systems in an unaltered form is unknown.
Sulfonamides (Fig.1a) are a class of widely used human and veterinary antibiotics
that consist of synthetic bacteriostatic, sulfanilamide derivatives. Sulfonamides are active
2
against a broad range of Gram-positive and Gram-negative bacteria and function as
competitive antagonists to bacterial folate synthesis5, 6
. The sulfonamides are the fifth
most widely used group of veterinary antibiotic within the European Union (EU) and the
second most widely used within the UK and the Netherlands2. Sulfonamides typically
enter the environment through hospital waste disposal and runoff from animal feeding
operations3 as unaltered parent compounds, or in an acetyl form that readily hydrolyzes to
the parent compound. Sulfonamides, sulfomethoxazole (SMX) (Fig 1b) in particular,
were detected in 30% of the samples collected from the waters that were suspected to be
contaminated by antibiotics used in animal husbandry7. Sulfonamide antimicrobials have
been detected in groundwater8, 9
, landfill leachate, and runoff and soils associated with
effluent-irrigated lands. Sulfonamides, especially SMX, were also detected in drinking
water10
and surface waters11
. Sulfonamides are among the most frequently detected
pharmaceuticals in U.S. streams7, 12-17
.
Reported environmental sulfonamide concentrations vary from parts per trillion
(ppt) to parts per million (ppm). Maximum concentrations are found in manure and can
be as high as 91 mg/kg.18
Sulfonamides appear frequently in sewage treatment plant
effluents with concentrations reported to be 6 µg/l in Germany19
, 290 ng/l in
Switzerland20
, 395-575 ng/l in Georgia, USA21
, and 0.06-0.21 µg/l in Colorado, USA22
.
Some of these reported concentrations may be underestimated, however, because the N4-
acetyl sulfonamide form (Fig. 1c) may not have been included in the analyses. Yet, the
N4-acetyl form is found in significant concentrations and is readily retransformed to
sulfonamide20
. For example, Hilton and Thomas23
reported concentrations of 2235 ng/L
3
and 239 ng/L of N4-acetyl SMX in a treated grab sample and receiving surface waters,
respectively.
The full ecological and toxicological significance of ppt to ppm antibiotic
environmental concentrations is still far from known; however, it is generally
acknowledged that the greatest potential risks include the disruption of natural ecosystem
functions that are dependent on microbial activity such as bacterial nitrification and
denitrification24-26
; the toxicity associated with long-term exposures to low levels of
antibiotics27
; and the development of antibiotic resistance within microbial populations
found in soils and natural waters. In an example of the latter, resistance to sulfonamide
antibiotics was frequently identified in E. coli and salmonella isolates collected from a
natural river basin28-30
.
Critical to evaluating these risks is an understanding of the fate and transport of
sulfonamides that is based on accurate, quantitative descriptions of sulfonamide-mineral
surface reactions, including sorption and mineral surface-catalyzed degradation via redox
processes. At present, these are poorly understood phenomena, particularly for the
mineral oxide-sulfonamide model system. The overarching objective of this thesis is to
investigate the transformation kinetics and potential degradation pathways of SMX on
mineral oxide surfaces. This work should aid in evaluating the natural attenuation of
SMX by minerals contained in soils and sediments.
A Note on the Structure of this Thesis:
This thesis contains relevant background information (Chapter 2) in support of the
research objectives outlined in Chapter 3. Chapter 4 is a draft manuscript in preparation
4
for Environmental Science and Technology. The manuscript highlights a key finding of
the thesis: the transformation of SMX on the manganese oxide pyrolusite. Additional
results which are not included in the manuscript, but still deemed useful for future work,
are included in Chapters 5 and 6. Overall conclusions are contained in Chapter 7.
5
CHAPTER 2
BACKGROUND AND MOTIVATION
This chapter contains detailed background and additional overview of the
literature relevant to this research as well as motivation for the research objectives in
Chapter 3. Included are the physicochemical properties of both the sulfonamide and the
mineral oxides to be studied. Existing work on sulfonamide sorption to environmentally
relevant substrates, and potential degradation pathways of sulfonamide antibiotics in
natural and engineered systems is also evaluated.
Properties and sorption of SMX
Sulfomethoxazole (Fig. 1b) is considered the most predominant and toxic (lowest-
observed-effect concentration (LOEC) for SMX is 37µg/l) sulfonamide antibiotic in the
environment20, 31
. SMX undergoes two proton dissociations at pH 1.56, and 5.727
,
resulting in cationic, anionic, neutral, or zwitterionic forms (Fig.1d). SMX is hydrophobic
with an octanol-water coefficient (Kow) of 8.222 and a deionized water solubility of 0.37
g/L at 25oC; pH of 4
31, and, therefore, favorably interacts with surfaces. Sorption of SMX
to activated sludge was reported in waste water treatment plants20
, where N4-acetyl SMX
was hydrolyzed to SMX, and SMX was detected at an average concentration of 68 µg/kg
in the dried sludge. No SMX was detected in the digested sludge. The average sorption
constant of SMX to activated sludge was reported as 256 L/kg20
. Species-specific organic
carbon absorption coefficients (Koc) values decrease in the order of Koc (SMX cation) >
6
Koc (SMX neutral) > Koc (SMX anion) when SMX was adsorbed to manure and humic
acid20
.
A few studies on the sorption of sulfonamides to whole and size-fractionated
soils32-34
and to organic materials35
have been reported. It was found that SMX adsorption
was pH dependent and increased with decreasing grain size in the sequence of coarse silt
< whole soil < medium silt < sand < clay < fine silt. Gao and Pedersen36
studied
sulfonamide sorption to the clays montmorillonite and kaolinite, and found that sorption
was influenced by pH, the charge density on mineral surfaces, ionic strength, and the
nature of exchange cations in solution. In particular, sulfonamide sorption generally
decreased with increasing pH. Sorption of the cationic form of sulfonamide antibiotics to
montmorillonite was more than 1-2 times that of the neutral species, and sorption to
kaolinite was highly sensitive to ionic strength. To my knowledge, sulfonamide sorption
to common mineral oxides such as goethite and manganese oxide has not been
investigated, and thus will be one focus of this work.
Degradation of SMX in engineered systems
Sulfonamide degradation in natural and engineered systems has been reported.
Both biotic and abiotic processes in marine sediments37
, activated sludge38
, and
seawater39
are operative in degrading sulfonamides, although reaction and retention times
vary. In general, the stability of sulfonamides in environmental media is described by the
following sequence: activated sludge (least stable) < manure < marine sediment < water
(most stable). As one example, the half-life of sulfadiazine under 1-2 cm of marine
7
sediments is 50 days37
. Specific reactions which control the fate of sulfonamide in
sediment systems have not been identified, and are thus another focus of this thesis.
Biodegradation and oxidative pathways are suspected to be dominant. In engineered
systems, oxidation is a common sulfonamide treatment strategy. Free chlorine,
monochloramine, chlorine dioxide, ferrate, and ozone have all been proven effective in
water treatment systems40-53
. Oxidation of sulfonamides on naturally occurring mineral
surfaces has not been demonstrated; however, it is possible such oxidation reactions may
degrade and naturally attenuate sulfonamides in the environment. Thus, an understanding
of possible sulfonamide oxidation by minerals, particularly mineral oxides, will enable a
comprehensive evaluation of sulfonamide fate, transport, and toxicology.
Properties and reactivities of goethite, pyrolusite and manganite
Goethite (α-FeOOH), pyrolusite (MnO2), and manganite (γ-MnOOH) are selected
for this study as model mineral oxide systems. These three secondary minerals are
common in the environment, especially in soils and sediments. Goethite is an important
mineral component in soils and the most abundant source of iron in near-surface
environments. Goethite is often investigated as a sorbent to immobilize and carry out
redox transformations of inorganic and organic environmental contaminants. For
example, goethite has been studied as a sorbent for other antibiotics such as tetracycline
and fluoroquinolone54-56
. Specifically, 40-70%55
and 50-76%56
( / ) of
tetracycline and fluoroquinolone, respectively, was adsorbed to goethite at pH 5.
Tricarbonylamide and carbonyl functional groups in tetracycline and carboxylic group in
8
fluoroquionlone were identified as critical moieties to the goethite sorption reaction54-56
.
Under aqueous conditions, goethite (pHpzc of 7-9) has a positive surface charge at pH < 7-
9 and negative surface charge at pH > 7-957
, Electrostatic association between the iron
and electron sufficient functional groups of sorbates such as carbonyls were considered as
the main sorption mechanisms. Similarly, various cationic, anionic and neutral forms of
SMX (Fig. 1d-f) emphasize the potential importance of pH and electrostatic forces when
considering sulfonamide-goethite interaction. Under the circumneutral conditions of pH
6-7, electrostatic forces favor the sorption of the SMX anion to the positively charged
goethite surface. Moreover, additional functional groups such as the anilinic amine, the
amide moieties, and the aromatic ring contain electron-dense moieties capable of
interaction with charged goethite surface groups. While transformation of SMX by
goethite has not been demonstrated, goethite (E0 = 0.63 V)
57 is a proven oxidant in soils,
and has been reported to oxidize a range of pollutants such as phenol, fluoroquinone, and
hydroquinone56, 58-60
.
Manganese oxides are among the most important, naturally occurring catalysts for
organic pollutant transformation58, 59, 61
. These minerals are critical to inorganic, organic,
and biological redox processes in the environment62-64
. Manganese dioxide and
manganite are generally regarded as superior oxidants because of large standard
reduction potentials of +1.23V and +1.50V, respectively57
. Manganite is considered to be
a much stronger oxidant than manganese dioxide at pH <765
.
Again oxidation of SMX
has not been demonstrated; however, manganese oxide in combination with goethite and
hydrated FeOOH in soils and sediments oxidize potential SMX analogs, intermediates, or
9
rough equivalents such as amines66
, and 4-chloroaniline [E0 (PhNH2
·+/PhNH2) =1.01
V]15
. The structure of SMX is similar to a 4-sulfonamide substituted aniline (E0=1.1 V)
67
and, therefore, SMX transformation on the surface of iron and mineral oxides is
hypothesized. For pyrolusite and manganite, thermodynamic evidence supports this
hypothesis at least for pyrolusite and manganite, as both minerals have reduction
potentials that are above that of 4-sulfonamide substituted aniline (Fig. 2) at low pH.
10
CHAPTER 3
RESEARCH OBJECTIVES
This thesis examines the sorption and possible transformation of sulfonamide on
iron and manganese oxide surfaces. In doing so, relationships between sulfonamide
structure-reactivity and mineral surface chemistry are developed. An end goal is to assess
the role of mineral oxides in sulfonamide fate and transport within sediment-water
environments.
Specific objectives of the research include:
1. Measure the sorption of SMX to oxides over a wide pH range and at
environmentally relevant sulfonamide concentrations.
2. Investigate potential redox transformation of SMX on the surface of goethite,
pyrolusite, and manganite over a range of pH values.
3. Investigate the transformation kinetics of SMX.
4. Identify the intermediates and products of possible surface mediated reactions
with a goal of resolving potential degradative reaction pathways for SMX.
11
CHAPTER 4
TRANSFORAMTION OF SMX ON MANGANESE DIOXIDES
Manuscript in preparation to be submitted to Environmental Science and Technology
Abstract
Determining the occurrence and potential adverse effects of antibiotic
pharmaceuticals released into the environment is a critical step towards responsible
environmental stewardship. Under aqueous conditions typically found in soils and natural
waters, the antibiotic agent sulfamethoxazole (SMX) was transformed in the presence of
pyrolusite MnO2 via oxidative pathways. At least 50% loss of SMX was observed after
269 hr, in both acidic and basic solutions (pH 3-9). A maximum of 100% loss was
recorded at pH 3 and 65.6% loss was recorded at circumneutral pH. Concomitantly,
aqueous manganese concentrations increased to around 10 µM at pH 3 and 2 µM at pH 6
over the same time period suggesting an oxidative transformation associated with the
mineral surface. Initial pseudo-first order reaction rate constants (kinit) increased with
decreasing pH from 0.0039 hr-1
at pH 9 to 0.019 hr-1
at pH 3. HPLC results showed
transformation products were similar at different pH. Mass spectrometry of the reaction
products collected at pH 3 and 4 confirm a transformative pathway by which oxidation
and hydroxylation occurs at the aniline moiety and isoxazole ring of SMX, and
hydrolysis occurs at the sulfonamide moiety. The observed transformation of SMX by
manganese oxides suggested that this ubiquitous component of soils and sediments likely
plays an important role in the fate of this antibiotic agent in the environment.
12
Introduction
The occurrence and potential adverse effects of pharmaceuticals in the
environment have been the subject of increasing interest in recent years2. Antimicrobial
agents are of particular importance because of their widespread use in human and
veterinary medicine. Sulfonamides including sulfamethoxazole (SMX) are one of the
most commonly used antibiotics within the European Union (EU)2; they are ranked
among the most frequently detected pharmaceuticals in U.S. streams7, 12-17
; and were
detected repeatedly in surface water11
, ground water8, 9
, and drinking water10
. While
typical environmental concentrations are ppt to ppm, values as high as 91mg/kg have
been reported in livestock manure18
. Quantitative information on the risks associated with
these concentrations is lacking; however, the following are regarded as potentially
problematic: a) the development of antibiotic resistance within microbial populations
found in soils and natural waters, b) the disruption of natural ecosystem functions that are
dependent on soil microbial activity such as bacterial nitrification and denitrification24, 25
;
and c) the toxicity associated with long-term exposures to low levels of antibiotics27
.
Critical to evaluating these risks is an understanding of the fate and transport of
sulfonamides in earth materials that is based on accurate, quantitative descriptions of
sulfonamide degradation, including mineral surface-catalyzed degradation via redox
processes. At present, these are poorly understood phenomena, particularly for the
mineral oxide-sulfonamide model system.
Sulfonamide degradation in engineered systems via inorganic oxidation is a
common sulfonamide treatment strategy. Accordingly, free chlorine, monochloramine,
13
chlorine dioxide, ferrate, ozone UV/ozone, UV/H2O2 and UV/TiO2 have been proven
effective in oxidizing SMX in water treatment systems40-53
. Photolytic degradation of
SMX by UV with other catalysts has been shown; more than 10 products may be
generated via various pathways including hydroxylation, oxidation, and ring cleavage
reaction68, 69
. In natural systems, specifically marine sediments37
and seawater39
,
sulfonamide degradation was also reported. Here, both biotic and abiotic processes
resulting in sulfonamide oxidation appear to be operative; however, specific reaction
pathways were not identified. Moreover, sulfonamide sorption to soils32-34
, organic
materials35
and clays36
have been proposed as important environmental sinks sulfonamide
antibiotics. For example, Gao and Pederson36
found that the adsorption of several
sulfonamide antibiotics include SMX particularly the charged speciesto clays such as
montmorillonite and kaolinite were pH influenced . Specifically, the adsorption
coefficient of SMX to clay decreases as pH increases from 3 to 6 and then remains steady
above pH 6.
In addition to examining the pH influence on SMX sorption, another critical task
is to identify potential pathways for the transformation of sulfonamide on naturally
occurring minerals which, to our knowledge, has not been demonstrated, but could be
important in quantifying sulfonamide fate, transport, and toxicology. As our model
sulfonamide-mineral model system, we selected sulfomethoxazole (SMX) and pyrolusite
MnO2. Sulfomethoxazole (Fig. 1b) is the most predominant sulfonamide antibiotic in the
environment20, 31
. MnO2 is the most common manganese mineral and is among the most
important, naturally occurring catalysts for organic pollutant transformation58, 59, 61
. For
14
example, manganese oxides are reported to play a significant role in the irreversible
oxidation and transformation of aromatic amines in soil66
.
Specific aims of this study are to 1) examine the transformation of SMX by MnO2
using bulk aqueous methods 2) investigate the influence of pH on the reaction kinetics of
SMX with MnO2; 3) characterize stable reaction intermediates and products with mass
spectrometry; and 4) elucidate the possible transformation pathways.
Materials and methods
Chemicals. Sulfamethoxazole (SMX) and manganese dioxide were purchased
from Aldrich-Sigma with purity of more than 98.5%. SMX and manganese dioxide purity
and phase were confirmed using HPLC and XRD, respectively (data not shown). The
Brunauer-Emmett-Teller (BET) surface area of the manganese dioxide was ~82.44m2/g,
the phase of manganese dioxide was confirmed to be pyrolusite. Additional chemical
reagents described below were obtained from VWR or Sigma-Aldrich-Fluka with purity
of more than 98% (for solids) or of UV grade (for the solvent used in LC/MS) and HPLC
grade (for the solvent used in HPLC). All dry chemicals were used directly without
further purification. All solutions were prepared from reagent grade chemicals without
further purifications. Reagent grade water was supplied by a Millipore system (18
MΩ·cm). Stock solutions of 250 µM SMX solution were prepared in deionized water. All
stocks were protected from light, stored at 4ºC, and used within one week of preparation.
Batch Reaction Setup:
15
Solution experiments were carried out in 40 mL VWR amber borosilicate glass
vials with Teflon-lined solid-top screw caps. The vials were presoaked in 5 M HNO3
solution for 24 hr and thoroughly rinsed by distilled water then dried prior to use. Stock
solution pH was adjusted by using 0.1 M and 0.01 M HCl and NaOH. The ionic strength
of each buffer was maintained at 0.01 M using NaCl. SMX stock solution was added to
the buffer solution for a final, environmentally-relevant SMX concentration of 4.5 µM or
1.2 mg/kg (ppm). The pH of the diluted SMX solution was determined after thoroughly
stirring. Each vial was loaded with 30 mL of 4.5 µM SMX solution and 150 mg of
mineral oxides for a solids concentration of 5g/L and an SMX/MnO2 molar ratio of
1/12771. To assess the impact of solid mass loading on the reaction, additional vials with
4.5 µM SMX, a solids concentration of 0.0125 g/L MnO2 , and an SMX/MnO2 molar
ratio of 1/32 were set up at pH 3, 5, and 7. The reaction vials were tightly closed with
caps and continually shaken on a New Brunswick Scientific C10 Platform Shaker at 100
rpm at room temperature. Aliquots were periodically collected over 269 hr. Any mineral-
SMX reactions in the aliquots were quenched upon collection by centrifuging at 11,000
rpm for 25 min. The supernatant was separated and filtered using 0.2 µm VWR syringe
PTFE filter, then transferred to two vials, one 2 mL glass vial for later SMX analysis, and
one 1 mL plastic vial for later manganese ion analysis. Two control vials and duplicates
were prepared at each pH value examined. One control contained SMX and buffer, but
lacked mineral oxides. The other control contained the mineral oxides in buffer, but no
SMX. Each control was designed to assess the effect of the buffer on SMX degradation
and mineral oxide dissolution, respectively. In addition, an explicitly aseptic experiment
16
was conducted similar to above by filtering the SMX-buffer solution through 0.2 µm
VWR filter, and MnO2 was sterilized at 121oC for 20 min. All the samples were analyzed
after being shaken for 3 days.
Analysis of sulfamethoxazole and Mn (II) ion
Decreases of sulfamethoxazole were monitored using Dionex Ultima 3000 HPLC
system equipped with a Dionex Acclaim PA reverse phase C16, 4.6×150 mm, 5 µm
particle size analytical column and a diode-array UV detector set at a wavelength of 265
nm. The mobile phase consisted of 50% phase A and 50% phase B, where phase A is
90:10 (v/v) of 20 mM phosphate buffers (pH=2.7) / methanol (VWR) and phase B is
10:90 (v/v) 20 mM phosphate buffers (pH=2.7)/ methanol. The flow rate was set at 1
mL/min. All the samples for HPLC were filtered using a 0.2 µm VWR syringe PTFE
filter before injection. Each sample was run twice. Standards were prepared at 0, 0.5, 1.5,
3.0, 5.0 µM and the standard curve was linear with R2 above 0.9995.
Aqueous manganese ion concentrations from the SMX-mineral reaction were
detected using an inductively coupled plasma atomic emission spectroscopy (ICP-AES)
spectrometer (Spectro ARCOS). At predetermined time intervals, 0.5 mL aliquots of the
experimental solutions were quenched by centrifugation and the supernatant was filtered
through a 0.2 µm VWR syringe filter. The filtrate was diluted and acidified using 0.5%
HNO3 to 5 mL and the emission of Mn was measured at 257.61 nm using yttrium as the
internal standard. Controls with only manganese oxide and pH buffers but without SMX
revealed that dissolution of manganese oxides was moreat low pH of 3 than at that of pH
6 and 9 in the absence of SMX (Fig. 4.)
17
Mass Spectrometry of Reaction Products
Liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/
mass spectrometry (GC/MS) were used complementarily in analyzing products. LC/MS
was suitable for the non-volatile and water-soluble compounds especially SMX and its
analog products. Although GC/MS is not typically considered for SMX analysis, it
proved useful in cross-evaluating our reaction pathways deduced from the LC/MS results.
LC/MS analysis was conducted on an Agilent Series 1100 liquid chromatograph/
mass spectrometer (Wilmington, DE, USA) system with various columns. Samples were
analyzed by flow injection analysis electrospray ionization mass spectrometry
(FIA/ESI/MS), slow liquid chromatography electrospray ionization mass spectrometry
(LC/ESI/MS) in both positive and negative modes. Samples were initially analyzed by
direct injection using both positive and negative electrospray ionization/mass
spectrometry. Analytical conditions were optimized previously to form molecular ions
without dimers or multiply-charged species, fragmentation, or adduct ion formation70-72
.
Using direct or flow injection analysis, 2 microliters of sample were injected into an
isocratic stream of 90/10 water/methanol at 0.2 milliliters per minute, transferring the
sample directly into the ion source without chromatography. All solvents used for LC/MS
were UV grade (Honeywell Burdick & Jackson, VWR Scientific). Nitrogen drying gas
was introduced at 350oC at 12 L/min with 35 psi nebulizer pressure, at a capillary voltage
of 4000 V. The capillary exit voltage of 50V maximized formation of molecular ions and
minimized adduct formation (positive mode) or fragmentation (negative mode) while
ensuring effective ion transmission. The quadrupole mass spectrometer was scanned from
18
m/z (mass-to-charge ratio) 50 to 1000 per second. Samples were injected every minute,
with at least triplicate analyses for every sample. Vial septa were replaced immediately
after injection.
For slow LC, samples were analyzed by liquid chromatography/electrospray
ionization/mass spectrometry in positive and negative mode on a Zorbax Bonus-RP
narrow-bore 2.1 × 150 mm, 5 micron column using water and methanol as the mobile
phase at 0.2 mL/min, starting at 90% water for 5 minutes, increasing to 98% methanol at
25 minutes, holding for 5 minutes, and returning to initial conditions in 5 minutes, with 5
minute hold to equilibrate. Nitrogen drying gas was introduced at 350oC at 10 L/min
with35 psi nebulizer pressure, at a capillary voltage of 4000 V and capillary exit voltage
of 50V. The quadrupole mass spectrometer was scanned from m/z (mass-to-charge ratio)
100 to 600 per second.
At all experimental pHs, the spectra of transformation products of SMX on
mineral oxides are similar. For the case that transformation products may be under the
limit of detection of or have no response to HPLC-PDA, GC/MS was used as a
complementary method in this research. The samples were prepared for GC/MS as
follows: Reactions (pH=3 and 4, 269 hr, 20 mL) were quenched by filtering with 0.2 µM
VWR PTFE syringe filter, the filtration was extracted with dichloromethane (2×10 mL).
The dichloromethane was condensed under vacuum at room temperature to 5 mL after
being dried by sodium sulfate. A Shimadzu QP 2010 Capillary GC/MS instrument was
used for the GC/MS analysis. Chromatographic separation was carried out using a fused-
silica capillary column RTx-5MS (30m×0.25 mm i.d., 0.25 µm film thicknesses). Helium
19
(99.999% purity) was used as the carrier gas at a flow-rate of 1.13 mL/min. The GC was
operated in split mode with a split ratio of 50. The GC temperature program was initially
set at 60 ºC, and increased to 300 ºC at a rate of 15 ºC min-1
. Effluents from the GC were
directed into the MS via a transfer line held at 300 ºC. Mass spectrometric analysis was
performed in full-scan mode from m/z of 50 to 900 under electron ionization (EI)
operating at 70 eV electron impact modes.
Results
Variations in SMX concentration in the presence of MnO2
Significant loss of SMX in the presence of MnO2 was observed at all
experimental pH values between 3 and 9 after 6 hr (Fig. 3a). Conversely, in the absence
of MnO2, SMX concentrations remained stable and deviated no more than 2.5% at each
pH throughout the experimental time period (Fig. 3b). A clear trend of increased SMX
loss with decreasing pH is observed. After 6 hr reaction time with the MnO2, 10% of the
SMX was transformed at pH 3-4, and less than 10% was lost at pH above 5; after 269 hr,
more than half of SMX was lost for pH of 4 to 9 and nearly all of the SMX was lost at pH
3 (Fig 4). All reaction vials were regarded as unaffected by biological contamination.
This assertion was confirmed when similar results were observed for an experimental set
that was explicitly rendered aseptic (Fig. 5). SMX loss was coupled with an increase in
Mn2+
(aq) concentrations, which is indicative of MnO2 reduction coupled to SMX
oxidation (Fig. 6). Manganese concentrations increase quickly with time in the first 30 hr
period at pH 3, 6 and 9. The increase became steady at pH 3 and 6. Mn (II)
20
concentrations remained stable after the first 30 hr at pH 9 and started to decrease after
100 hr. The highest aqueous Mn concentrations corresponded to the largest SMX mass
loss, both of which occurred at pH 3. Furthermore, the dissolved Mn (II) concentration at
pH 3 of 20 µM is four times as much as the initial SMX concentration.
Pseudo-first-order reaction kinetics best describe the SMX loss for the time range
of 0 hr to 150 hr at all experimental pH, after 150 hr at pH 4-6, the reaction kinetics
deviates from first-order. At pH > 6, the kinetics is pseudo-first-order over the entire
experimental time range (Fig. 7). Therefore, comparisons at different pH are made using
the initial reaction rates (rinit) and rate constants kinit (hr-1
) calculated from data collected
between 0 and 30hr (Fig. 8). Rate constants kinit were pH dependent, particularly at low
pH. The kinit value increases from 0.00314 hr-1
to 0.0193 hr-1
when pH decreases from 6
to 3. Above pH 6, kinit fluctuated around 0.004 hr-1
. The initial rate constant at pH 3 is
five fold larger than at pH ≥ 7. The results also show that at pH of 4 and above, the SMX
concentrations appear to plateau at a steady state value (Fig. 3a).
Transformation products of SMX
There are several organic intermediates recorded in the HPLC-PDA
chromatograms during the SMX reaction with manganese dioxide (Fig. 9). A mass
balance was calculated based on the equations as follows:
Where A (mAU) is the peak area of all products, C (µM) is concentration of products,
and ε (mAU/µM) is the coefficient UV absorption in HPLC for the products. Here we
21
assumed that the UV absorbance of the products have an overall ε and that the products at
different pH are the same (ε are the same). We calculated ε based on the new product
peaks appearing at pH 3 representing transformed SMX. At pH 3, most of the SMX was
transformed, at least to a level at or below our detection limit. The total concentration
results after 3 days is shown in Fig. 10. At all experimental pHs the total mass equals the
control mass of SMX, within error.
LC analysis reveals six reaction products from the supernatants of SMX+MnO2
mixture from pH 3-9 (Fig. 11a-e; Table 2). An additional five products are revealed in
the GC/MS analysis (Fig. 11 f-j; Table 3). In the LC/MS analysis, the molecular ion of
SMX was m/z 252 in negative mode and m/z 254 in positive mode. Intermediates with
molecular weights of 288, 268, and 190 in electrospray (positive) mode ES (+), and 453,
286, 284a and 284b in electrospray (negative) mode ES (-) were identified. The products
may also be written as M + 34 (Table 2; EIC spectrum), M + 14 (Fig. 11a), M – 64 (Fig.
11b) for ES (+) mode products and M + 201(Fig. 11c), M + 34 (Table 2; EIC Spectrum),
M + 32a (Fig. 11d) and M + 32b (Fig. 11e) for ES (-) mode products, indicating the net
mass loss or gain of the products from the parent compound (M) of SMX. Molecular ions
288 ES (+) and 286 ES (-) appear to have originated from the same product M + 34.
Product m/z = 453 (M + 201; containing even nitrogen) showed a much shorter retention
time (1.5 min) than SMX (25 min) in the chromatogram and is interpreted as a
dimerization product of SMX. The M + 201 dimer exhibited a fragmentation pattern (m/z
307, 157, 141) that was similar to a 4-sulfonic aniline dimer. For example, the m/z 307 (-
) fragmentation is the molecular ion of the 4-sulfonic aniline dimer, m/z 157 (-) is the
22
molecular ion of 4-sulfonic aniline, and m/z of 141 is a common base structure of
sulfonic benzene. Other LC/MS fragmentations suggest that the M + 201 dimer exhibited
loss of two H2O and two NH2, suggesting two –OH and two –NH2 groups were present in
this product. Product M + 34 exhibited a strong extracted ion current (EIC) response at
288 (+) and 286 (-) with a retention time of 30 min. We suggest M+34 represents a
dihydroxylated SMX structure with –OH groups added to the isoxazole ring. Similar
intermediates were identified upon photooxidation of SMX in previous work68
. Product
M + 32a is identified as a two hydroxyl substituted SMX, with two hydroxyl substituting
on the benzene and isoxazole rings, respectively. This assertion is supported by the
prominent m/z 113 (-) fragment associated with M + 32a spectrum that is believed to
represent a hydroxylated 5-methyl isoxazolamine. Product M + 32b may represent the
two hydroxyl sbustituted SMX with both hydroxyl occurring on aniline benzene ring.
Product M + 14 (m/z 268 (+)) is identified as N4-oxylated SMX which could result from
hydroxyl attack on aniline. Product M - 64, which has a relatively short 5 min retention
time and, therefore, is considered more hydrophilic, was identified as N4-hydroxyl
sulfanilic acid, a hydrolysis product of intermediate M + 14. This identification is
supported by fragment m/z 141 in the M - 64 spectrum which is suggestive of –OH and –
NHOH loss from a N4-hydroxyl sulfanilic acid backbone with mass loss of 49.
GC/MS was also applied as a complementary method for analyzing the trace and
comparable volatile products. The analysis of SMX transformation mixtures indicates the
presence of five additional transformation products with molecular ions of m/z = 503
(Fig. 11f-g), 172 (Fig. 11h), 119 (Fig. 11i) and 99 (Fig. 11j). There are two different
23
compounds which have the same molecular ion of m/z 503; both are identified as
dimerized SMX. This is supported by the presence of fragment m/z 281, which could be
derived from the dimer after the loss of two 5-methyl-isoxazolamine ring of SMX); and
the symmetry structures which are derived from the same mass loss of 74 (Fig. 11f) and
88 (Fig. 11g), respectively. Moreover, the presence of a dimer as a reaction product is
supported by the molecular ion m/z = 502 observed in the negative mode of FIA/ESI/MS
analysis. The 503a (Fig. 11f) and 503b (Fig. 11g) dimers are isomers with different
conjugative positions, one being the diimide (m/z 503a) and the other a secondary amine
(m/z 503b). The spectrum of reaction product m/z 503b shows m/z 415 and 399
fragments. The difference between the two fragments represents a -NH2 with mass of 16
loss. The spectrum of m/z 503a contains no fragments with an m/z 16 interval, which
suggests that one NH2 group exist in m/z 503b but not 503a. Product m/z = 172 having a
similar fragmentation pattern to SMX, matches the mass spectrum of known compound-
sulfanilic acid which can be considered as the hydrolysis product of SMX. Similarly,
product m/z 99 is another hydrolysis product of SMX, in this case, 5-methyl-3-
isoxazolamine which is a known SMX oxidation product. Both sulfanilic acid and 5-
methyl-3-isoxazolamine were previously identified in the photodegradation of SMX68
.
Product m/z 119 fragments to m/z 91, which represents loss of carboxide (m/z 119-CO)
and the open ring of hydroxylated 5-methyl-3-isoxazolamine. Overall, our MS results
agree well with previous findings of oxidation of SMX by TiO2 photocatolysis68
. Based
on the identification of these major reaction products, a summary of the proposed
transformation pathways of SMX in the presence of MnO2 is provided in Fig. 12.
24
Discussion
SMX loss is coupled to increased dissolved manganese concentrations at all
experimental pHs. Therefore, abiotic oxidation is suggested as the primary transformation
mechanism of SMX on the surface of manganese dioxide. Products identified by
LC/MS and GC/MS support an oxidative pathway, but also hint that other reactions such
as hydrolysis are occurring. The aniline benzene and isoxazole rings are identified as
specific sites of oxidation in the SMX structure. The 2:1 ratio of soluble manganese ion
to reacted SMX suggests multi-site oxidation on the MnO2 or further oxidation of
secondary reaction products. This model is also supported by the LC/MS and GC/MS
intermediates and products characterization (Fig. 13, 14). For example, oxidation
products for both the aniline group and isoxazole group were identified. SMX oxidation
by manganese dioxide is hypothesized to consist of several steps including sorption,
surface complex formation, electron transfer, and release of a radical and a reduced
manganese ion61
.
Sorption Initiation
Sorption between sulfonamide antibiotics and soils, sediment, organic material32-
35often precedes or even limits the rate of reactivity with mineral components in aqueous-
mineral systems. Increasing the sorption of SMX to the MnO2 surface may, therefore,
increase the surface complexes and thus increase the transformation of SMX. We
observed that the initial reaction rate constant kinit (Fig. 6) increases with decreasing pH .
The sorption can be rationalized with electrostatic considerations. SMX has two pKa
25
values of 1.6 and 5.7, which means that at pH 3 to 5.7, SMX exists mainly as neutral
species in the solution. Above pH 5.7, the main species of SMX is negatively charged.
The surface of manganese dioxide was negatively charged (pHpzc of 1.6~2.373
) under all
pH conditions studied. Therefore, electrostatic repulsion is expected to impede SMX
sorption to the MnO2 surface at high pH, but not low pH. This is in agreement with the
decrease of kinit within increasing pH. At pH 3, 5, and 7, the initial rate constants are 60,
200, and 600 fold higher, respectively, when the molar ratio of SMX/MnO2 is 1/12777
compared to 1/32 (Table 4). The results indicate that the impact of surface area at lower
pH was less than that at higher pH. At the higher pH 7, the reaction is slow and the
increase in kinit is comparable to the increase in surface area, which indicates that the
sorption may be rate-limiting. Specifically, with a fixed amount of SMX, the initial rate
increase is attributed to the additional active mineral surface sites that are made available
for sorption complex formation by adding more MnO2. If the initial rate does not increase
or does not scale with the amount of MnO2 added, then the sorption complex formation is
unlikely the rate-limiting step74
. This appears to be the case at low pH. At pH 3, the
initial rate increase is ten fold less than the surface area increase, which indicates sorption
may affect the reaction, but is not the rate-limiting step.
Dissolved manganese increases with a decrease in SMX concentration. At pH 3,
6, and 9, Mn (II) concentrations were 10 µM, 2 µM and 1.5 µM, respectively at 130 hrs.
Values at higher pH, however, may not be entirely reflective of the amount of SMX
degraded by MnO2. Zhang and Huang74
report that during MnO2 dissolution, 90% of Mn
(II) will be back-sorbed to MnO2 at pH 5 to 7. As a result, the reduced Mn produced
26
during SMX transformation at high pH could be higher than measured in our
experiments. This also allows for a stoichiometry of MnO2 to SMX consumed in the
oxidation that is higher than 1:1, further supporting additional and multi-site oxidation of
SMX. Moreover, absorption of Mn (II) to the MnO2 surface may block the active site and
slow the reaction rate, which could be reflected in the steady state concentration of SMX
at pH 4 to 9 after 250 hr (Fig. 3.b). At pH 3, complex formation may not be affected
because adsorption of Mn (II) to the surface of MnO2 is not favorable, potentially due to
the increased amount of positively charged surface sites. However, sorption of netural
SMX to MnO2 at pH 3 may be less affected because of the lower pKa1 (1.6) of SMX.
Oxidation on the aniline benzene ring
Mass spectrometry suggests that the benzene ring in the aniline moiety is a target
oxidation site for coupling with manganese reduction. A scheme for the oxidation of the
benzene ring on the manganese dioxide surface is proposed (Fig. 13). Surface complex
formation and radical generation are key steps to the proposed process. SMX is adsorbed
to the MnO2 surface and one electron is transferred from SMX to the surface Mn (IV) to
yield an SMX radical intermediate. Mn (IV) is reduced via Mn (III) to Mn (II), which is
released from the surface of MnO2 to solution. The radical can be stabilized by the
benzene ring due to resonance. The formed radical SMX intermediate reacts further to
produce observed m/z products 503 (dimer), 268, 190, which are also released from the
MnO2 surface. Given the high surface area of manganese oxide, low concentration of
SMX, and sorption and kinetic data detailed above, electron transfer within the SMX-
MnIV
complex and product release are most likely rate-determining.75, 76
27
Electron transfer between the aniline group in SMX and MnO2 is believed to be
thermodynamically favorable, but only at low pH. Reduction potential data are
unavailable for SMX, however, may be estimated by using a value for a structural analog:
4-sulfonic group substituted aniline67
. The one electron half-reaction of SMX and MnO2
is hypothesized as follows:
1/2 Mn(IV)O2 (s) + 2H+ + e
-1/2 Mn2+ (aq) + H2O
R NH2 R NH2+ e R NH2 E1/2 = -1.10V0
E1/2 = +1.23V0
The total reaction equation is as follows:
R NH2 R NH++1/2Mn(IV)O2 (s) H++ 1/2Mn2+ (aq) + H2O
(1)
Following from equation 1 and omitting other affecting factors such as ionic strength and
a non-ideal solution, we could get the reaction driving force equation from the Nernst
Equation:
(2)
Equation 2 shows that the reduction potential of the redox reaction is determined by the
standard reduction potential (0.13V), pH, and species concentration. Equation 2 also
shows lower pH favors more positive reduction potentials and, therefore, predicts
reaction 1 will proceed as written. The opposite trend occurs for increasing pH.
Moreover, surface-sorbed Mn2+
increases and aniline concentrations decrease, further
moving reduction potentials to more negative values; therefore, unfavorable conditions
for oxidation to occur at high pH. However, SMX disappearance and an increase in
28
dissolved Mn(II) is observed at high pH (albeit at longer time periods) suggesting
additional, favorable oxidation involving moieties besides the aniline ring.
Oxidation on the isoxazole ring
LC/MS analysis indentified two intermediates directly related to the oxidation of
the isoxazole ring of SMX: 1) the dihydroxylation product of SMX (m/z = 288 (+) and
286 (-)), 2) the hydroxyl substituted products (m/z = 284a (-)), and 3) the dimer product
of isoxazole oxidized analog (m/z 453 (-)). By analyzing the mass fragmentations
patterns of these two intermediates, it was concluded that hydroxylation and oxidation
occurred on the isoxazole ring of SMX (Fig. 14). Oxidation at this location is considered
to be favorable according to the following rationalization. C1 in the isoxazole ring of
SMX is electrophilic due to its electron being drawn away by the more electron negative
oxygen, and the conjugation between the C1, C2 double bond and the C3 nitrogen double
bond ((a) in Fig. 14). This results in a favorable electrostatic interaction between C1 and
deprotonated oxygens on the MnO2 surface. Similarly, >Mn (IV) may also interact
electrostatically with the partially negative charged nitrogen on the isoxazole ring.
Collectively, formation of an SMX surface complex is favored, and stabilized by the
hexatomic ring formation ((b) in Fig. 14). Electron transfer then proceeds from the
nitrogen through resonance to the manganese atom in the complex, forming an epoxide
intermediate ((c) in Fig. 14), which can be easily oxidized by MnO2 to form a double
hydroxylated SMX then to carboxides; the epoxide can also be hydrolyzed to form a
mono-hydroxylation intermediate. Both dihydrolated SMX and secondary products of
monohydrolated SMX were detected. Furthermore, the proposed hydroxylation scheme
29
was supported by some other products such as the dimer with m/z of 453. The complex
formation and electron transfer proposed does not involve a proton, so the electron
transfer driving force is not pH dependent; this relationship predicts a constant initial
reaction constant at pH 6 to 9 where sorption is rate limiting. However, low pH may
impede the complex formation in other ways, for example, by forming a protoned MnO2
surface61
and altering the reactive sites. The observation that initial reaction constant
remains stable at pH 6 to 9 agrees very well with the above oxidative pathway.
Environmental Implication.
Although past studies have demonstrated the degradation of SMX in engineered
systems, this is the first report of SMX transformation on a common soil mineral oxide.
This work should be useful in evaluating natural attenuation of SMX in environmental
systems.
Specifically, we show that manganese dioxide is an effective and efficient mineral for
transformation of SMX in aqueous solutions. Reaction products include dimers and
hydroxylated and hydrolyzed forms. Dimerization shows the potential for further
polymerization which may immobilize and decrease the toxicity of SMX. Because of
drastic structural alteration, hydrolysis and oxidation products are believed to have
reduced antimicrobial activity relative to SMX. Confirmation of toxicity loss and an
assessment of SMX oxidation product biodegradability are objectives for future work.
Because they are relatively common components in soils and sediments, transformation
30
of SMX by manganese dioxides is likely to play an important role in the environmental
fate of this antibiotic agent.
Acknowledgments
We thank Dr. Colleen Rostad at USGS located in Denver, CO for LC/MS
analyses, Dr. Kathy Moore in Agriculture Service Center at Clemson University for
assistance in ICP-AES analyses, and Dr. Colin McMillen in Department of Chemistry at
Clemson University for his assistance with the XRD analysis.
31
CHAPTER 5
TRANSFORMATION AND ADSORPTION OF SMX ON
GOETHITE
Introduction
Goethite (α-FeOOH) is an efficient sorbent due to its large surface area. Goethite
often occurs in microcrystalline forms with surface areas of 60-200 m2/g or as coatings
on larger soil grains77
. Although goethite abundance in a soil may be between 1 and 5%,
goethite may account for 50 to 70% of the total surface area of the soil77
. Furthermore,
goethite has a high affinity for sorption of ions via electrostatic interaction, particularly
anions such as phosphate at circumneutral pH78
. Goethite has a strong tendency to form
complexes with negatively charged functional groups in organics, such as carboxylate79
.
Thus, ionized and carbonate organic compounds are likely to be adsorbed to goethite.
In general, goethite is very insoluble under circumneutral aqueous conditions
(Fig. 15), ligands such as EDTA can form chelates with dissolved iron to increase the
solubility of goethite. Goethite is a moderate oxidant in acidic environments, with an Eh
of around 0V at neutral pH (Fig. 2)
Transformation and adsorption of SMX to goethite in the pH range of 3 to 9 was
investigated. The distribution coefficient (Kd) was used to evaluate the impact of pH on
the adsorption of SMX. Furthermore, UV-Vis absorption of complex between SMX and
dissolute iron (III) were studied to validate the HPLC detection method used in the
sorption experiment.
32
Materials and methods
Batch Experiments with Mineral Oxide
Goethite was purchased from Sigma-Aldrich. The batch experiments were carried
out at eight pH values ranging from 3 to 9. At each pH, six vials (40 mL VWR amber
borosilicate glass vials with Teflon-lined solid-top screw caps, presoaked with 5 N
HNO3) were used: two duplicate SMX solution vials containing goethite, one SMX
solution control vial with no goethite, one goethite suspension with no SMX, and two 4-
chloroaniline control solutions with and without the mineral oxide.
Reagent water and SMX stock solution was prepared in the same manner as
described in Chapter 4. 4-chloroaniline (4-CA) oxidation on goethite has been previously
reported58
. We used a 4-CA goethite reaction setup as a control to evaluate our
methodologies and protocols. 4-CA was purchased from Aldrich-Sigma at greater than
98% purity and used without further purification. A 500 µM 4-chloroaniline stock
solution was prepared similarly to SMX stock preparation.
Experiments were carried out at room temperature, 1 atm pressure with target
concentrations of 5g/L for the mineral oxides and around 5µM/L for SMX and 4-CA. The
pH of all the vials were adjusted with 1M, 0.1M, 0.01M hydrochloric acid (VWR) and
sodium hydroxide (VWR) solution. All the vials were continually shaken on a New
Brunswick Scientific C10 Platform Shaker at a speed of 100 RPM. One (1 mL) sample
was periodically collected from each vial at 3, 7, and 14 days. The samples were
33
immediately quenched by centrifuging at 11000 rpm for 25 min. Supernatant
concentrations of SMX and 4-chloroaniline were detected by HPLC-UV/Vis.
UV-Vis analysis
UV-Vis analysis was conducted with a Varian Cary 50 Bio UV-Visible
spectrophotometer instrument with 1 cm quartz cuvvette. Data were collected between
200 nm-400 nm with 1nm interval and a scan rate of 24000 nm/min. DDI water was run
as a baseline. SMX samples were prepared with concentration of 24.88 µM in three
different matrices of DDI water (pH=6.76), hydrochloride acide (pH=3.40), and ferric
chloride (10-5
M, pH=3.29) solutions.
Results and Discussion
HPLC analysis showed that 4-chloroaniline was completely transformed in the
presence of goethite at pH 3 after 3 days, 4-CA transformation also occurred at pH 4 and
5 with around 20% and 10% loss, respectively, after 3 days. However, no transformation
occurred at pH of 6 and higher over the same time period (Fig. 16). This result agrees
very well with the literature58
, and demonstrates the oxidative properties and quality of
goethite we used. Moreover, the pH dependent reactivity of the aniline functional group
(which is found in the SMX structure) was confirmed.
HPLC results show that within error, there was no observed transformation of
SMX on surface of goethite; control SMX concentrations matched experimental SMX
concentrations at each pH (Fig. 17a). SMX sorption to goethite was quantified using a
standard apparent distribution coefficient ( ):
34
Where: : Distribution coefficient
: SMX concentration in solid phase after sorption
: SMX concentration in aqueous solution after sorption
: SMX concentration in aqueous solution in absence of goethite
: The volume of solution before sample
: Goethite weight
Despite the negative values of adsorption coefficient (this is addressed below), there is a
trend of decreasing Kd of SMX to goethite in the pH range of 3 to 5, then increasing in
the pH range of 5 to 7, and decreasing again in the range of 7 to 9 (Fig. 17b). The
maximum adsorption occurred in the pH range of 6 to 8. SMX has three species at
different pH, cation, neutral or zwitterionic with the anion as the dominant species at pH
below 1.6, 1.6 to 5.7, above 5.7, respectively. Goethite has pHpzc of 7-9, so the surface
was partially positively charged at pH below 7, and partially negatively charged at pH
above 9. At pH of 6 to 8, the interaction between SMX and the surface of goethite is
likely to be electrostatically favorable, therefore, explaining our observation (Fig. 17b).
The anomalous negative Kd values calculated above could be due to potential
matrix (e.g., dissolved iron) effects on our ability to quantify SMX concentrations. As
such, an additional experiment was run to measure SMX concentrations in the presence
of variable iron concentrations, and thus evaluate changes in the molar absorption
coefficient ε (L mol-1
cm-1
) as defined by the Beer-Lambert Law:
35
Where ε is the molar absorption coefficient or extinction coefficient (L mol-1
cm-1
), A is
absorbance, C is the molarity (mol L-1
), and L is the width of cuvette (cm). For our
experiment, C and L were kept constant, A was measured, and ε was calculated for the
following conditions: SMX with no added iron in DDI, soluble iron in acidic solution
with no SMX, and SMX in the presence of soluble iron and acidic solution. UV-Vis
spectra showed significant differences among the three SMX matrices (Fig. 18). SMX in
an acidic ferric matrix has a larger molar adsorption coefficient than SMX in HCl and
DDI water (Table 5). The maximum adsorption wavelength of SMX in DDI, HCl, ferric
are 259 nm, 267 nm, and 268 nm, respectively, and their adsorption at 265 nm are 0.367,
0.404, and 0.458, respectively. SMX may interact with ferric iron under acidic conditions
resulting in higher UV absorption and thus higher analytical concentration of SMX in a
ferric matrix. This could potentially lead to elevated SMX concentrations and thus
anomalously low Kd values for pH of 3 to 5. It is, therefore, possible that the maximum
distribution coefficient occurs at pH 3, where SMX is neutral. This would be in
agreement with reports of the neutral species of fluoroquinolone antibiotics being mostly
adsorbed to goethite at pH<756
.
36
CHAPTER 6
TRANSFORMATION OF SMX ON MANGANITE
Introduction
Manganite (γ-MnOOH) is a naturally occurring secondary manganese mineral
present in lakes and rivers in the temperate zone and in subarctic zones80-82
. Less studied
than other oxyhydroxide minerals such as goethite, manganite is potentially effective in
oxidizing inorganic elements and organic contaminants. For example, it was reported to
oxidize different organic materials such as oxalate83
, ascorbic acid84
, aromatic
compounds74
, and aminocarboxylates85
.
In this experiment, transformation experiments were conducted in the pH range of
3 to 9. The transformation kinetics was used to compare to those by pyrolusite.
Furthermore, as manganite is a well crystallized mineral with flat surfaces, it is well
suited to conduct surface transformation studies. The transformation will serve as the
preliminary experiment to provide the basis of conducting in-situ investigation of
manganite surface transformation by Atomic Force Microscopy (AFM).
Method
Synthesis of and characteristics of Manganite
Manganite was synthesized using a common hydrothermal protocol86, 87
. A
MnSO4.H2O aqueous solution (0.06 M) of 300 mL is mixed at room temperature with 6.2
mL H2O2 30% w/v solution in a ratio of 3:1 (moles/moles) with respect to the
37
stoichiometric Mn (II)/Mn (III) oxidation. The mixture was neutralized by adding 90 mL
NH4OH (0.2 N), rapidly producing a chocolate-brown precipitate. The suspension was
refluxed for 24 hr and the precipitate gradually turned yellow-brown. The precipitate was
filtered and washed with hot water, and the solid was dried at 95-105 °C for 12 hr and
stored in a desiccators. The synthesized manganite was compared to a naturally
crystallized manganite (Virgina Tech. Dr. Hochella’s Lab, Ma5) using X-Ray Diffraction
(XRD) (Fig. 19)
See Chapter 4 for details on the SMX batch reaction setup and HPLC protocols.
Results and Discussion
In the presence of manganite, SMX significantly decreased during the first 6.5 hr
at all experimental pHs above 3 (Fig. 20a). The transformation kinetics appeared to be
similar at pH of 4-9. In contrast, control SMX concentrations remained constant with no
decrease (Fig. 20b). Results of HPLC analysis showed that there is one polar intermediate
which appeared at lower pH (3-5). At pH of 6 and higher, the transformation products
have a longer retention time than SMX suggesting additional hydrophilic products.
ICP analysis (Fig. 21) showed that there is significant increase of manganese ion
after 6.5 hr at pH of 4, 6, and 9; the trend sequence is pH 6 > 4 > 9, which agrees well
with the decrease of SMX. After that, Mn (II) concentrations remainsalmost constant at
pH 9, but decrease from 6.5 hr to 30 hr at pH 4 and 6. At the time period of 30 hr to 50
hr, Mn (II) concentration stays constant at pH 4 but decreases at pH 6. At 50 hr, Mn (II)
38
has a concentration trend of pH 4 > 6=9. This results may be due to the oxidation with
oxygen and back-adsorption60, 74
of Mn (II) to the surface of manganite.
To better understand the transformation of SMX on manganite surface, LC/MS
analysis of the products and an evaluation of the kinetics at variable SMX concentrations
need to be carried out. Moreover, the observed SMX transformation on manganite in bulk
reaction, provides the foundation for examining concomitant mineral surface
transformations using in situ AFM on macroscopic, single crystals of manganite.
39
CHAPTER 7
CONCLUSIONS
Transformation of SMX on pyrolusite (manganese dioxide), goethite (iron
oxyhydroxide), manganite (manganese oxyhydroxide) were investigated in this thesis.
The results showed signficant SMX transformation on the surface of manganese oxides
including both pyrolusite and manganite, but no transformation was observed on the
surface of goethite. Though not observed as an effective oxidant for SMX under our
conditions, goethite is still a candidate mineral oxide for SMX immobilization in the
environment via sorption. Sorption of SMX on goethite was observed at neutral pHs.
Under acidic conditions sorption is likely operative, however, our reported distribution
coefficients (Kd) are underestimated due to the complexation of SMX and dissolved Fe.
Additional investigation of SMX sorption on goethite under acidic conditions is required.
SMX transformation kinetics and pathways are different for pyrolusite compared
to manganite. SMX can be completely transformed (approximately 100% SMX loss) on
the surface of pyrolusite and has highest initial reaction rate at pH 3 among all the
experimental pHs. Reaction rates with pyrolusite increase with decreasing pH. On the
surface of manganite, low rates are observed for low pH. An increase in Mn(II)
concentrations confirm SMX transformation consists of a redox process between SMX
(reductant) and MnO2 (oxidant). For pyrolusite, oxidation occurred on both the benzene
and isoxazole rings according to the MS evidence. At lower pHs, the benzene ring
associated with the aniline functional group is thought to be the focus of oxidation. At
40
higher pHs, the isoxazole ring is the favorable reaction site. Radical and inner complex
intermediates were proposed as the main mechanisms in the redox process between SMX
and pyrolusite. SMX hydrolysis products were also observed in the pyrolusite reaction.
Transformation pathways of SMX on the surface of manganite are still under
investigation and identified as an area of future investigation.
41
CHAPTER 8
FUTURE WORK
To further evaluate the effectiveness of manganese oxides as natural attenuators
of SMX, the toxicities of the SMX-Mn-oxide transformation products need to be
evaluated. Furthermore, the potential for metal reducing soil microbes to catalyze or
inhibit sulfonamide transformation should also be investigated. Finally, to better
understand the redox and dissolution processes of mineral oxides in the presence of
antibiotics, in-situ mineral surface transformation needs to be investigated using Atomic
Force Microscopy (AFM). Redox promoted etching and precipitation can be
characterized on the surface at nanometer scale using AFM. The preliminary experiment
(Chapter 6) provides evidence that oxidation occurs on manganite, which forms well-
formed flat crystal surfaces suitable for AFM imaging. As a result, manganite is
recommended as the model system for the AFM investigation.
42
TABLES AND FIGURES
Table 1 Properties of Goethite, Pyrolusite and Manganite
Goethite Pyrolusite Manganite
BET Surface Area
(m2/g)
9.7 82.44 22.6
Single Point Pore
Volume (cm3/g)
0.039 0.181 0.094
pHpzc 7-9 1.8-2.2 8.273
43
Table 2 List of products identified from LC/MS
Molecular
weight
Electrospray
ionization
mode
Fragmentation
(base peak)
(m/z)
Retention
time (min) Structure
287 positive,
negative EIC 29.8 H2N S NH
O
O N O
OH
OH
267 positive 268, 190 (100) 20 N S NH
O
O N O
O
189 positive 190, 141, 121
(100) 4.9 HN S OH
O
O
HO
454 negative 453, 385, 317,
249(100), 181, 113 1.3
NN SO2NH
H2NOH
HNO2S
NH2OH
285 negative 284 (100), 113 32.7 HN S NH
O
O N O
HO
OH
285 negative 284 (100) 32.9 HN S NH
O
O N O
HO
HO
44
Table 3 List of products identified from GC/MS
Molecular
Weight
Main
Fragmentation
(m/z)
Retention
Time (min) Structure
504 503, 429, 355,
281, 207 (100) 10.9
HN S NH
O
O N O
HNS
O
O
HNN
O
504 503, 415, 399,
327, 281, 73 (100) 7.3
S
O
O
NHHN
N O
NH
SO
ONH
N
O
172 172 (100), 156,
168, 92 12.7 H2N S OH
O
O
119 119 (100), 91, 77
10.7
HNH2N
HO CH3
OH
98 98 (100), 55 4.1 NH2
N O
45
Table 4 Initial reaction constant (Kinit) at different pH and SMX/MnO2 ratio
pH Kinit (hr-1
) 95%Confidence
3 19.281×10-3
3.882×10-3
4 10.532×10-3
3.628×10-3
5 5.047×10-3
1.513×10-3
6 3.144×10-3
0.961×10-3
7 6.127×10-3
1.041×10-3
8 2.246×10-3
2.624×10-3
SMX/MnO2 = 1/12771
(molar ratio)
9 4.236×10-3
1.315×10-3
pH Kinit (hr-1
) 95% Confidence
3 32.041×10-5
26.634×10-5
5 2.280×10-5
0.687×10-5
SMX/MnO2 = 1/32
(molar ratio)
7 1.009×10-5
0.378×10-5
46
Table 5 UV-Vis absorption of SMX (24.88 µM) in DDI, HCl, and Fe (III) solutions
SMX + DDI SMX + HCl SMX + Fe (III) (10
-5
M)
pH 6.76 3.40 3.29
Maximum absorbance
Wavelength (nm) 259 267 268
Absorbance at 265nm
(HPLC detector) 0.367182 0.40428 0.458269
47
H2N
SNH
O O
N
O
SMX
NH
SNH
O O
R
H3C
O
H2N
SNH
O O
RH2N
SHN
O
OR
H3N
SN
O
OR
H3N
SHN
O
OR
H2N
SN
O
OR
Ka1 Ka2
Ka1'Ka2'
(a)
(b)
(c)
(d)
(e)
Figure 1 a. General structure of sulfonamide antibiotic; b. General structure of N4-acetyl
sulfonamide antibiotic; c. Structure of SMX (pKa1=1.6, pKa2=5.7 (30)
;
Kow=8.222; Ciw(25°C) =0.37 g L-1
(pH=4) (31)
);
48
d. Speciation of generic sulfonamides showing cationic, neutral, zwitterionic, and anionic
forms; e. Species fraction of SMX at different pH
Figure 2 Eh-pH diagram of goethite, pyrolusite, and manganite (Mn (II) and Fe (II) = 10
-5
M).
49
(a)
(b)
Figure 3 a.Comparison of SMX transformation by pyrolusite at pH between pH 3 and 9
([SMX]0=4.4 µM, [pyrolusite]0=5 g/l )
; b. Time dependent SMX concentration between pH 3 and 9 in the absence of pyrolusite
50
Figure 4 SMX loss after 6 hr and after 269 hr at different pH in the presence of
pyrolusite.
51
Figure 5 Aseptic transformation of SMX on pyrolusite after 3 days (Error bars, which
represent 95% confidence intervals, are smaller than the symbols used)
52
Figure 6 Time-dependent Mn (II) concentrations increase as SMX reacts with pyrolusite.
53
(a)
(b)
Figure 7 a. Overall kinetics of transformation of SMX by pyrolusite at the pH 3-9
([SMX]0=4.4 µM, [pyrolusite]0=5 g/l ); (b) Initial reaction kinetics of SMX
with pyrolusite at different pH
54
Figure 8 Effect of pH on the initial rate constant of SMX oxidation by pyrolusite. The
initial reaction rate constant kinit (hr-1
) was calculated based on the pseudo-first-order
reaction kinetics ([SMX]0=4.4 µM, [pyrolusite]0=5 g/l ). Duplicates of experiments are
reported with 95% confidence intervals.
55
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
-2.0
5.0
10.0
15.0
20.0
25.0SMX METHOD 04-22-2008 #35 [modified by CLEMSON UNIVERSITY] UV_VIS_1mAU
min
1: P
eak
6
2: P
eak
5
3: p
rodu
ct 1
4: p
aren
t com
poun
d
WVL:254 nm
(a)
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
-3.0
0.0
5.0
10.0
14.0SMX METHOD 04-22-2008 #36 [modified by CLEMSON UNIVERSITY] UV_VIS_1mAU
min
1: P
eak
6
2: P
eak
5 3: p
rodu
ct 2
4: p
rodu
ct 1
5: p
aren
t com
poun
d
WVL:254 nm
(b)
Figure 9 HPLC profile of products of SMX by pyrolusite after 269 hr. a. pH=3; b. pH=4
56
Figure 10 Mass balance of SMX transformation on pyrolusite after 3 days
57
SO2NHNO
N O
m/z 268 (+)(a)
HN S OH
O
O
HO
m/z 190 (+)(b)
NN SO2NH
H2NOH
HNO2S
NH2OH
(c) m/z 453 (-)
58
Figure 11a-c LC/Mass Spectra of transformation products of SMX by pyrolusite
HN S NH
O
O N O
HO
OH
(d) m/z 284 (-)
m/z100 200 300 400 500
0
20
40
60
80
100
*MSD1 SPC, time=32.876 of 080827\1707N01.D API-ES, Neg, Scan, Frag: 50, "neg scan 100-600"
Max: 77496
284
.4 2
85
.4
436
.4
550
.6
HNS
O
O
HN
(e) m/z 284 (-)
NOHO
HO
Figure 11 (cont’d) d-e LC/Mass Spectra of transformation products of SMX by pyrolusite
59
HN S NH
O
O N O
HNS
O
O
HNN
O
m/z 281
-74-74
(f) m/z 503a
S
O
O
NHHN
N O
NH
SO
ONH
N
O
NH2
-88-88
m/z 281
(g) m/z 503b
60
H2N S
O
O
OH
(h) m/z 172
Figure 11 (cont’d) f-h GC/Mass spectra of transformation products of SMX by pyrolusite
HNH2N
HO CH3
OH
-CO2
(i) m/z 119
61
NO
H2N
(j) m/z 98
Figure 11 (cont’d) i-j GC/Mass spectra of transformation products of SMX by pyrolusite
62
H2N S NH
O
N O
HN S NH
O
O N O
HNS
O
O
HNN
O
S
O
O
NHHN
N O
NH
SO
ONH
N
O
H2N S OH
O
O
NH2
N O
m/z 172
m/z 99
SMX: m/z 254(+) 253 (-)
m/z 503
m/z 503
H2N S NH
O
O N O
OH
H2N S OH
O
O m/z 172
HNH2N
HO CH3
OH
m/z 119
+
+
H2N S NH
O
O N O m/z 288 (+) 286 (-)
OH
OH
N S NH
O
O N O
O
HN S OH
O
O
m/z 190 (+)
HO
HN S NH
O
O N O
m/z 284 (-)
HO
OH
NN
m/z 453 (-)
SO2NH
H2NOH
HNO2S
NH2OH
m/z 284 (-)
O
HN S NH
O
O N O
HO
HO
m/z 268 (+)
Figure 11 Proposed transformation pathway of SMX by pyrolusite
63
SO2NHRH2N SO2NHRHN
Mn(IV) OH Mn(III)
SO2NHRHNHNRHNO2S
SO2NHRH2N
SO2NHRHN
NH2
RHNO2Sm/z 503m/z 503
SO2NHRNO
m/z 268 (+)
SO2OHHNHO
m/z 190 (+)
MnO2
Mn(II)
Mn(II)
Figure 12 Plausible scheme of oxidation on aniline and benzene ring of SMX by
pyrolusite
SH2N
O
O
NH
N O
SMX
Aniline
64
N ORHN
O
N ORHN
HOHO
H2O
Mn (III) Mn (II)m/z 288 (+); 286 (-)
Mn (III) Mn (II)MnO2+
N
O
O
MnRHN O
N
O
O
MnRHN O
12
3N O
RHN
HO HNH2N
HO CH3
OH
m/z 119 (+)
SH2N
O
O
OH
m/z 174 (+)
+
dimmerization
m/z 453 (-)
(a)
(b) (c)
Figure 13 Redox scheme on the isoxazole ring on the surface of MnO2
SH2N
O
O
NH
N O
SMX
Isoxazole
65
Figure 14 Solubility of goethite depending on pH
66
Figure 15 Transformation of 4-chloroaniline by goethite
67
(a)
(b)
Figure 16 a. SMX concentration of control and reaction depending on pH. Symbols
represent the averages of triplicate measurements made on duplicate vial
sets.; b. absorbed SMX to goethite and Kd value. Samples were collected
after 7 days. Error bars indicate 95% confidence.
68
Figure 17 UV absorption of SMX within different matrices
69
VT sample Ma5.rawBG021408.rawBG021208.rawBG 021308.raw
5.0 9.0 13.0 17.0 21.0 25.0 29.0 33.0 37.0 41.0 45.0 49.0 53.0 57.0 61.0
Deg.
166
500
833
1166
1500
1833
2166
2500
2833
3166
3500
3833
4166
4500
4833
5166
5500
CPS
Figure 18 X-Ray Diffraction (XRD) of synthesized manganite MnOOH (BG021408,
BG021208) and naturally formed manganite MnOOH (VT sample Ma5)
and pyrolusite MnO2 (BG021308)
70
(a)
(b)
Figure 19 a. Comparison of SMX transformation by MnOOH at pH 3 -9 ([SMX]0=4.6
µM, [MnO2]0=5 g/l ); b. Time dependent SMX concentration between pH 3
and 9 in the absence of MnOOH ([SMX]0 = 4.6 µM)
71
Figure 20 Time-dependent Mn (II) concentrations increase as SMX reacts with MnOOH.
72
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