SORPTION OF TETRACYCLINES IN DESERT-SOIL ENVIRONMENTS
By
Mohammed Ahmed Alsanad

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Crop and Soil Sciences - Doctor of Philosophy
2018

ABSTRACT
SORPTION OF TETRACYCLINES IN DESERT-SOIL ENVIRONMENTS
By
Mohammed Ahmed Alsanad
The broad-spectrum antibiotic tetracycline is used extensively for human and animal
health, but causes unintended environmental consequences. Significant amounts of tetracycline are excreted with animal manures and can pollute soil, surface water, and groundwater.
The overall objective of this dissertation is to combine experimental and modeling work to
determine the environmental chemistry of tetracyclines in desert soils.
Some previous studied observed that a) when Ca2+ -to-tetracycline ratios were large,
then Ca2 H(T ec)2+ species dominate in the solution above pH 5, and b) at low concentration of cation and high pH, Ca2+ - and M g 2+ -clays sorbed significantly large amounts of
oxytetracycline. Since Saudi Arabian topsoils can have pH near 7.5 along with large Ca2+
concentrations, these conditions together may enable relatively strong sorption of cationic
Ca2+ -tetracycline complexes by cation exchange capacities (CEC) of clay minerals. Thus,
the objective of current study was to test this hypothesis. To do so, soil samples were collected from the Agricultural and Veterinary Training and Research Station at King Faisal
University, Al-Ahsa, Saudi Arabia. Three soils with relatively high clay contents were selected, and sorption isotherms were measured by using liquid chromatography/tandem mass
spectrometry (LC-MS/MS) instrument to quantify oxytetracycline (OTC) concentrations.
The results of this study showed that oxytetracycline sorption was significant at pH 7.5 for
all three soils, and the hypothesis of this study - that desert soils at pH 7.5 may adsorb
reasonably large amounts of oxytetracycline - was supported.
The speciation of tetracycline is complicated by several ionic species that form complexes

with aqueous cations and also with mineral surfaces, so computational tools are needed to
understand and predict partitioning of tetracycline into its various species. The objective of
this study was to use many experimental data sets to create new thermodynamic parameters
using Phreeqc for modeling the sorption speciation of tetracyclines. Since clay minerals are
important sorbents, cation exchange parameters were developed for tetracycline and its
K + - and Ca2+ -complexes for better understanding of that very complicated system. A
self-consistent set of parameters was derived that enabled tetracycline cation-exchange to be
modeled in both K + - and Ca2+ -systems over a range in pH from 4 to 8.

ACKNOWLEDGMENTS

I would like to take this chance to thank my major advisor Dr. Brian J. Teppen for his
uninterrupted assistance, longanimity, prompting, and guidance during my Ph.D. study.
Also, I would like to thank my committee members: Dr. Stephen A. Boyd, Dr. Hui Li
from the Department of Plant, Soil and Microbial Sciences, and Dr. David T. Long from the
Department of Earth and Environmental Sciences for the assistance they gave. Also, I would
like to thank Dr. Yingjie Zhang who is a research associate in the Department of Plant, Soil,
and Microbial Sciences for his assistance that he gave me in MSU laboratory. Moreover,
I would like to thank Agricultural and Veterinary Training and Research Station at King
Faisal University, Dr. Abdullah M. Algosaibi, and Dr. Abdulrahman M. Almadini from the
Department of Environment and Natural Agricultural Resources at King Faisal University
for allowing and helping me to collect and analysis soil samples of this study at that research
station. Also, I like to thank each one in my family, especially my parents and my wife for
encouraging me morally throughout my higher studies. Lastly, I like to thank King Faisal
University and Saudi Arabian Cultural Mission to the U.S. for the financial support they
gave me during studying my Ph.D. degree.

iv

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Chapter 1 Tetracycline Sorption in Saudi Arabian Soils . . . . . . . . . . .
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1 Study Area and Soil Characterization . . . . . . . . . . . . . . . . . .
1.2.2 Facts about the Agricultural and Veterinary Training and Research
Station at KFU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.3 Oxytetracycline Sorption to Soils 23 and 4 . . . . . . . . . . . . . . .
1.2.4 Oxytetracycline Sorption to Soil 19 . . . . . . . . . . . . . . . . . . .
1.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.1 Effects of Background Cations on Oxytetracycline Sorption . . . . . .
1.3.2 Sorption at Higher Concentrations . . . . . . . . . . . . . . . . . . .
1.3.3 Sorption Coefficient (Kd ) . . . . . . . . . . . . . . . . . . . . . . . . .
1.4 Conclusions from Experimental Work . . . . . . . . . . . . . . . . . . . . . .

1
1
7
7

Chapter 2
2.1
2.2

2.3

2.4

Geochemical Speciation Modeling of Tetracycline Sorption to
K- and Ca-Smectites . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1 Treatment of Cation-Exchange in Phreeqc . . . . . . . . . . . . . . .
2.2.2 Experimental Data on Tetracycline Exchange with K + or Ca2+ on
Clay Minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1 Possible Impurities in the Clay or Deionized (DI) Water . . . . . . .
2.3.2 Inorganic Cations Released When Tetracyclines Sorbed to Clay . . .
2.3.3 Fraction of Tetracycline Apparently Sorbed as Uncomplexed Cations
Conclusions from Modeling of Tetracycline Sorption to K- and Ca-Smectites

8
11
12
13
13
18
22
23

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

25
25
27
28
31
33
33
51
54
59
60

LIST OF TABLES

Table 1.1 Mineralogies of the clay fractions found in eight topsoils of Al-Ahsa city
near the King Faisal University research station [1]. . . . . . . . . . . . . .

5

Table 1.2 Physical characterizations of all soil samples . . . . . . . . . . . . . . . . .

9

Table 1.3 Chemical characterizations or properties of all collected desert soil samples

10

Table 1.4 Sorption coefficient (Kd ) and sorption per unit mass of soil organic carbon
(Koc ) values for plain soils; and comparison between the three selected soils. 23
Table 2.1 Compilation of all chemical equations and equilibrium constants used in
the present study. All equations are written as they appear in the Phreeqc
model database. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vi

57

LIST OF FIGURES

Figure 1.1

Tetracycline chemical structure involves of four bonded cyclic rings, and
three different pKa values [2]. . . . . . . . . . . . . . . . . . . . . . . . .

5

Tetracycline species distribution in pure water as a function of solution
pH calculated using the Phreeqc model [3]. . . . . . . . . . . . . . . . . .

6

Dominant species of tetracycline as a function of pH and Ca2+ concentration (mol/L) in aqueous solution [4]; L = tetracycline. . . . . . . . . .

6

Map of the study area which created using the Google Maps, and the blue
balloon refers to research station at King Faisal University. . . . . . . .

8

Locations map of all soil samples in the Agricultural and Veterinary Training and research station at KFU which created using Google Maps. . . .

11

The oxytetracycline sorption to soil 23 at three different concentrations of
CaCl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15

The oxytetracycline sorption to the soil 23 at three different concentrations
of NaCl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

The oxytetracycline sorption to soil 4 at three different concentrations of
CaCl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17

The oxytetracycline sorption to the soil 4 at three different concentrations
of NaCl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

18

Figure 1.10 Oxytetracycline sorption to soil 4 at three different concentrations of
CaCl2 or N aCl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19

Figure 1.11 Oxytetracycline sorption to soil 19 when soil mass about 0.5 g with no
added CaCl2 and N aCl. . . . . . . . . . . . . . . . . . . . . . . . . . . .

20

Figure 1.12 Oxytetracycline sorption to soil 19 when soil mass about 0.2 g with no
added CaCl2 and N aCl. . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Figure 1.13 Oxytetracycline sorption to soil 19 at two different soil masses (0.5 g and
0.2 g). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 1.9

vii

Figure 2.1

Calculated equilibria for K- Ca-exchange using Phreeqc and the cation
exchange coefficients of Appelo and Postma [5] and Table 2.1 at three
different ionic strengths. IS= ionic strength, and CEC= cation exchange
capacity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30

Comparison of modeled and experimental [6] pH values of initial tetracycline solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34

Sorption of tetracycline to the K-smectite clay in dilute solution (no added
KCl). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38

The sorption of tetracycline to the K-smectite clay at 0.01 M KCl ionic
strength. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39

Sorption of the tetracycline to the K-smectite clay at 0.10 M KCl as ionic
strength. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39

Sorption of tetracycline to the K-smectite clay at 0.80 molal KCl ionic
strength. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40

Ca2+ and tetracycline speciation and complication in aqueous phase as a
function of pH solution which calculated using Phreeqc model [7]. These
calculations were done with 0.00076 mol/L tetracycline in calcium chloride
with ionic strength of 0.004 M . . . . . . . . . . . . . . . . . . . . . . . .

40

Sorption of tetracycline to Ca-smectite in dilute solution (no added salt
other than tetracycline HCl and sodium carbonate) when log k value for
CaH2 (T ec)+ was set to 7. . . . . . . . . . . . . . . . . . . . . . . . . . .

41

Sorption of tetracycline to Ca-smectite at 0.01 M ionic strength when
log k value for CaH2 (T ec)+ was set to 7. . . . . . . . . . . . . . . . . . .

42

Figure 2.10 Sorption of tetracycline to Ca-smectite in dilute solution (no added salt
other than tetracycline HCl and sodium carbonate) when log k value for
Ca2 H(T ec)2+ was set to 6.6. . . . . . . . . . . . . . . . . . . . . . . . .

43

Figure 2.11 Sorption of tetracycline to Ca-smectite at 0.01 M ionic strength when
log k value for Ca2 H(T ec)2+ was set to 6.6. . . . . . . . . . . . . . . . .

44

Figure 2.12 Sorption of tetracycline to Ca-smectite in dilute solution (no added salt
other than tetracycline HCl and sodium carbonate) when log k values for
both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set to 4. . . . . . . .

45

Figure 2.13 Sorption of tetracycline to Ca-smectite clay in dilute solution (no added
salt other than tetracycline HCl and sodium carbonate). . . . . . . . . .

46

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

viii

Figure 2.14 Sorption of the tetracycline to Ca-smectite clay at 0.01 M CaCl2 ionic
strength. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47

Figure 2.15 Sorption of tetracycline to Ca-smectite at 0.10 M ionic strength strength
when the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species
were set to 6.6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

Figure 2.16 Sorption of tetracycline to Ca-smectite at 0.10 M ionic strength strength
when the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species
were set to 5.6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49

Figure 2.17 Sorption of tetracycline to Ca-smectite at 0.82 molal ionic strength when
the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set
to 6.6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50

Figure 2.18 Sorption of tetracycline to Ca-smectite at 0.82 molal ionic strength when
the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set
to 4.4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50

Figure 2.19 Contributions of three tetracycline species to the total sorption calculated
using Phreeqc model at an ionic strength of 0.01 M . . . . . . . . . . . .

51

Figure 2.20 Tetracycline sorption to K-smectite clay and K + released calculated using
Phreeqc in dilute solution (no added salt other than tetracycline HCl and
sodium carbonate). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52

Figure 2.21 Tetracycline sorption to Ca-smectite clay and Ca2+ released calculated
using Phreeqc in dilute solution (no added salt other than tetracycline
HCl and sodium carbonate) when log k values of both CaH2 (T ec)+ and
Ca2 H(T ec)2+ species were set to 6.6. . . . . . . . . . . . . . . . . . . . .

53

Figure 2.22 Tetracycline sorption to Ca-smectite clay and Ca2+ released calculated
using Phreeqc in dilute solution (no added salt other than tetracycline
HCl and sodium carbonate) when log k for CaH2 (T ec)+ species was set
to 5.6 while log k for Ca2 H(T ec)2+ species was set to 10.2. . . . . . . . .

54

Figure 2.23 Percentage of tetracyclines sorbed as cations to K-smectite clay in dilute
solution (no added salt other than tetracycline HCl and sodium carbonate). 55
Figure 2.24 Percentage of tetracyclines sorbed as cations to Ca-smectite clay in dilute
solution (no added salt other than tetracycline HCl and sodium carbonate). 56

ix

Chapter 1
Tetracycline Sorption in Saudi
Arabian Soils

1.1

Introduction

Tetracycline antibiotics are supplied to animal feeds for multiple purposes such as protection
and treatment from many diseases and to enhance the growth as well. Up to the present time,
antibiotic resistance is still to form fears into our environment system which requires further
particular research [8]. In 1950s, tetracycline antibiotics were discovered [9]. A recent report
showed that 51 tons of tetracyclines are consumed every day in the U.S. only, and around
80% of tetracyclines antibiotics are utilized in the livestocks while a small amount of about
20% is utilized for people and another purposes [10]. And the tetracycline antibiotic chemical
structure involves from four bonded cyclic rings substituted with functional groups, containing dimethylammonium, tricarbonylmethane, and diketone (Figure 1.1). Several studies and
research were notified that antibiotics uses in agricultural practices have a danger impact to
the human health [11–13].
Since great portions of tetracycline antibiotic are not digested entirely within the animals’
bodies, thus quantities of tetracyclines antibiotics are discharged with animal manures to
the environment, such as a parent compound or like their bioactive metabolites. Hence,

1

tetracyclines antibiotics and their secondary forms are transported to soils, surface waters,
and groundwaters [14–16]. Paul Ebner reported some antibiotics concentrations ranges which
found in the environment [17]. For example, concentrations of tetracycline antibiotic in
animal manure were ranging from 0.04 mg/kg to 24.0 mg/kg, and tetracycline concentrations
in soil were ranging from 86 µg/kg to 172 µg/kg while tetracycline concentration in surface
water was 0.11 µg/L [17].
As we know that the sorption dominates the fate of substances and compounds in clays
and soils, therefore this process must be considered. The accumulation and stability of any
substance in soil or clay surfaces relies on soil properties, such as soil pH, clay content,
clay minerals, and cation exchange capacity [18, 19]. Further, the study of Sarmah et al.
indicated that physical and chemical parameters such as chemical structure, solubility in
water, and compound species have great impacts on sorption mechanism [20]. Many years
ago, exploring the fate of tetracyclines antibiotics in the environment started and, it remains
until nowadays in wide parts of the world.
Since pH of solution controls speciation of ionable compounds, tetracycline functional
groups usually turn into ionized and lead tetracycline to form cationic, zwitterionic (neutral),
or anionic species in the solution phase, (Figure 1.2).
Generally, solution phase will also consist of many different cationic metals, such as
Ca2+ , M g 2+ , and K + which will combine with the tetracycline compound. Hence, this
will decrease the activity of uncomplexed tetracycline and making new varied species which
should be estimated (Figure 1.3).
For soil systems that contain swelling clay minerals, it has long been known that low pH
enhances the formation of cationic tetracyclines that sorb quite strongly to the negatively
charged clay surfaces [21–24].
2

Early work [25] indicated another mechanism for tetracycline sorption, because tetracycline was found intercalated into clay interlayers at both pH 5 and 8.7, and spectroscopic
evidence showed Ca-tetracycline complexation. These authors concluded that tetracycline
could sorb by either cation-exchange of the H4 (T ec)+ species, or through sorption of unknown Ca-tetracycline complexes. This complexation hypothesis was further supported [26],
in that Ca2+ smectite sorb more tetracycline than that N a+ smectite at given pH and extraction of all sorbed cations implied Ca-tetracycline complexes had been sorbed to the clay.
Further work [27] showed that Ca2+ smectites sorbed more tetracycline than N a+ smectites
over the entire pH range from 7 to 9.
At moderate to neutral pH, strong sorption is also sometimes observed [21, 24], although
such sorption is often attributed to tetracycline interactions with soil organic matter [24, 26]
or oxide mineral surfaces [28–30]. However, in 2013, Parolo et al. studied sorption of
tetracycline to montmorillonite in 0.01 M NaCl, and they discovered that at pH 7 to 9, Ca2+ clays sorbed large amounts of oxytetracycline (50 cmol/kg) [7]. The authors hypothesize that
Ca2+ -oxytetracycline complexes are involved. Also, Parolo et al. observed that when [Ca2+ ]
= 15.2 mM and [tetracycline] = 0.76 mM (300 ppm), then Ca2H(T ec)2+ species dominate
in the solution above pH 5 [7]. This is a plausible species to participate in cation exchange.
Further, in 2016, Aristilde et al. their study showed that at low concentration of divalent
cation and high pH, Ca2+ - and M g 2+ -clays sorbed significantly larger amounts of oxytetracycline than N a+ -clays [31]. This implies Ca2+ - and M g 2+ -oxytetracycline complexes are
involved.
In studying tetracycline sorption to montmorillonite Xu et al. [32] observed that there
were two adsorption maxima, one below pH 4 and another at pH 7.7. Since their bulk clay
contained a variety of cations including Ca2+ , a reasonable hypothesis for explaining their
3

sorption maximum at pH 7.7 is the sorption of cationic Ca-tetracycline complex by the clay.
At low pH values, Ding [6] observed tetracycline sorption to montmorillonite clay of up
to 19% tetracycline by weight. For palygorskite which has a smectite like composition but
does not swell, Chang et al. [33] measured a pH maximum in sorption at about pH 8.7, at
which the palygorskite sorbed 10% tetracycline by weight.
Relevant, strongly-sorbing clay minerals are common in arid environments. For example,
the general area surrounding Al-Ahsa, Saudi Arabia is rich in smectites and especially palygorskite [34] which has a high surface area [35] as smectite but it doesn’t swell as a smectite
clay. However, it can hold cations in the channels (Channels size ≈ 6.4 × 3.7 Å [36]; S.A.
≈ 150 m2 /g [37]; CEC of palygorskite ≈ 20 meq/100g [36]) and water [35]. Reasonably,
palygorskite can hold tetracycline.
Furthermore, Al-Hawas [1] extensively characterized the clay mineralogies of eight soils
near the King Faisal University Research Station in Al-Ahsa. His results are in agreement,
and indicate (Table 1.1) that the general clay mineralogy of the area is;
palygorskite > smectite > kaolinite ≈ calcite.
Thus, Saudi Arabian topsoils, including those in Al-Ahsa city, can have substantial clay
contents that include smectite and palygorskite [1, 34]. In addition, pH values in such soils
are generally alkaline, and the soils are often rich in divalent cations such as Ca2+ . Understanding the sorption of veterinary antibiotics to such soils may be important for protecting
environmental quality. For example, there is a large, commercial dairy farm immediately adjacent to the King Faisal University Research Station. The main hypothesis of this study is
that desert-soil conditions (clays, pH, and soluble cations) may combine to enable relatively
strong sorption of cationic Ca-oxytetracycline complexes by the CECs of clay minerals [7,31].
This study could locate no references to previous work that measured sorption of tetracy4

Table 1.1 Mineralogies of the clay fractions found in eight topsoils of Al-Ahsa city near the
King Faisal University research station [1].
Estimated mineral∗ content (%) of the clay fraction
S C P K I
Ca Q
Typic Torripsamment 27 8 21 16 13 14 1
Gypsic Haplosalid
10 0 27 10 3 39 11
Typic Torrifluvent
5 12 56 18 6 2
1
Lithic Haplogypsid
1 2 49 9 15 23 1
Leptic Haplogypsid
33 11 39 8 5 2
2
Typic Torriorthent
73 0 9 6 7 4
1
Anthropic Torrifluvent 0 5 55 11 28 0
1
Cambic Gypsiorthid
4 2 55 3 9 26 1
Soil Classification

∗ S=smectite,

C=chlorite, P=palygorskite, K=kaolinite, I=illite, Ca=calcite, Q=quartz.

clines to desert soils. Indeed, there may be tacit assumptions that sorption would be low
under such high-pH conditions [21–23].
Therefore, the objective of this study was to test this hypothesis by quantifying sorption
to some desert-region topsoils.

Figure 1.1 Tetracycline chemical structure involves of four bonded cyclic rings, and three
different pKa values [2].

5

1.20E-04

Speciation	Fraction	

1.00E-04
8.00E-05
6.00E-05
4.00E-05
2.00E-05
0.00E+00
-2.00E-05

0.0

2.0

4.0

6.0

8.0

10.0

12.0

pH
H4(Tec)+	

H3(Tec)0	

H2(Tec)-

H(Tec)2-

Figure 1.2 Tetracycline species distribution in pure water as a function of solution pH calculated using the Phreeqc model [3].

Figure 1.3 Dominant species of tetracycline as a function of pH and Ca2+ concentration
(mol/L) in aqueous solution [4]; L = tetracycline.

6

1.2
1.2.1

Materials and Methods
Study Area and Soil Characterization

In this current research, in August of 2016, the soil samples were gathered from the Research
Station at King Faisal University which located in Al-Ahsa city at Al-hofuf district, in the
east of Saudi Arabia (Figure 1.4). Typically, the weather in the country is desert climate
which is cold and rainy in the winter while it is very hot in the summer time [38]. The
maximum temperature in August often reaches 44.5◦ C (112.1◦ F) while the relative humidity
(RH) often exceeds 90% at the same period [39]. Figure 1.5 shows soil samples locations of
this study in the Agricultural and Veterinary Training and Research Station, Al-Ahsa city.
The soil samples of this research were air dried at the room temperature for around 24 hours.
After that, the soil samples were sieved using 2 mm screen. Stones and coarse roots were
discarded, and comprised less than 5% of soil volume.
Saturated soil paste was prepared for each soil sample, 250 grams of soil sample was taken
and then added DI water to the sample until it reached the saturation point. Then, the soil
paste left for 24 hours [40]. After that, the soil paste was extracted using vacuum pump for
3 hours hence the liquid phase of soil sample was collected in plastic vial and stored in the
refrigerator. The physio-chemical characterizations of soil measured via standard methods
[41]. The particle size distribution (PSD) of soil was measured by hydrometer method [42].
The soil pH was measured by a pH meter while the electrical conductivity (EC) was measured
by EC meter [40]. Ca2+ and M g 2+ cations measured via titration (by volumetric analysis
using EDTA (p.44 of ref. [40]) while N a+ and K + measured via Photometer Flame [40].
Also, CO3 and HCO3 ions were measured via titration with H2 SO4 [43] while Cl− ion was
titrated with AgN O3 [40]. The calcimeter method was used to measure CaCO3 content [44].
7

Figure 1.4 Map of the study area which created using the Google Maps, and the blue balloon
refers to research station at King Faisal University.
All these measurements were done in King Faisal University Labs. Table 1.2 and Table 1.3
show the physical and chemical characterizations of the soil samples.

1.2.2

Facts about the Agricultural and Veterinary Training and
Research Station at KFU

It was established in 1977, and It is located away 15 km from King Faisal University campus.
Research Station land area is 6 km2 (600 hectares). Its soils are classified as Aridisols and
Entisols. Wind-blown erosion is very active in this area [45].

8

Table 1.2 Physical characterizations of all soil samples
Sample#

Depth (cm)

Clay (%)

Soil Texture

4
19
23
29
1-3
8-19
21
4-14
6-24
28
26
20
13
30
3
47
40
33
32
1

0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30
0-30

60
32
18
8
8
11
8
20
6
14
10
8
12
6
8
6
8
10
10
10

Clay
Sandy Clay Loam
Sandy Loam
Sand
Sandy Loam
Sand
Sand
Sandy Clay Loam
Sand
Loamy Sand
Sand
Loamy Sand
Sandy Loam
Loamy Sand
Sandy Loam
Sand
Loamy Sand
Sand
Sand
Sand

9

Table 1.3 Chemical characterizations or properties of all collected desert soil samples
Sample# Ca2+ ∗(meq/L) N a+ ∗(meq/L)

CaCO3 (%) pH

Total OC (%)

4
19
23
29
1-3
8-19
21
4-14
6-24
28
26
20
13
30
3
47
40
33
32
1

19.23
2.96
5.92
5.28
13.95
5.92
5.71
9.93
4.65
7.19
7.78
2.62
12.47
10.36
21.56
4.44
4.44
6.13
4.31
10.15

0.61
0.49
0.26
0.06
0.35
0.02
0.01
0.36
0.41
0.15
0.18
0.84
0.5
0.32
0.41
0.01
0.27
0.02
0.02
0.19

84.13
72.77
189.97
16.97
91.03
12.8
17.03
40.4
4.27
36.13
56.9
16.67
224.93
39.1
100.33
4.17
25.17
16.07
9.47
15.23

∗ Concentration

151.95
147.34
189.06
19.32
87.83
1.32
30.08
118.58
5.71
86.07
37.33
2.42
210.14
54.9
159.2
0.66
32.93
4.17
2.2
5.05

7.4
7.5
7.4
8
7.4
8.2
7.9
7.1
8.1
7.4
7.9
7.9
7.4
7.7
7.7
7
7.5
7.8
7.9
7.9

or amount as measured in the solution extracted from the saturated paste.

10

Figure 1.5 Locations map of all soil samples in the Agricultural and Veterinary Training and
research station at KFU which created using Google Maps.

1.2.3

Oxytetracycline Sorption to Soils 23 and 4

Two soils, designated as samples 23 and 4 (Tables 1.1 and 1.2), were chosen for initial study.
Given the study hypothesis that Ca2+ oxytetracycline complexes are strongly sorbed to
swelling clays [7, 31], the soils were chosen because of their relatively high concentrations of
both clay and extractible calcium. Approximately 0.5 g of soil was carefully weighed into
glass vials.
The study prepared stock solutions of 50 mg/L oxytetracycline (Sigma oxytetracycline
HCl, minimum 95% HPLC), 1.00 mol/L CaCl2, and 1.00 mol/L NaCl. The oxytetracycline
stock solution was made freshly each day. Stock solutions were then combined (salt solution
first and then oxytetracycline) to make working solutions (25 mL each) at concentrations of
11

200, 400, 600, 800, and 1000 µg/L oxytetracycline in salt solutions that had concentrations
of approximately pure water (no added salt), 0.01, or 0.1 mol/L. Each working solution was
made in duplicate for each soil; for a given soil, one set of 10 working solutions was prepared
at each concentration of each cation, with controls at each oxytetracycline concentration and
each concentration of added salt. Then, each working solution was added to the appropriate
vial of soil. The soil itself added both Ca2+ and N a+ to each solution (Table 1.3). Thus, 0.5
g of soil 23 added 2.5 mmol Ca2+ /L and 4 mmol N a+ /L to each working solution, while 0.5
g of soil 4 added 3.6 mmol Ca2+ /L and 12 mmol N a+ /L to each working solution. Then, the
study samples were covered with aluminum foil to exclude light and were put on a horizontal
shaker for around 24 hours. After that, the samples were centrifuged for 15 minutes at 3500
rpm. Then, about 1 mL of the solution phase was taken from each sample.
Finally, the samples were analyzed using liquid chromatography/tandem mass spectrometry (LC-MS/MS) with a SCIEX API 3200 instrument. An aliquot of the centrifuged supernatant was used to measure the pH; pH values were typically in the range of 7.4 to
8.

1.2.4

Oxytetracycline Sorption to Soil 19

A third soil, designated sample 19 (Tables 1.1 and 1.2), was also studied. The soil was
chosen because of its relatively high concentrations of both clay and extractible calcium.
Approximately 0.5 or 0.2 g of soil was carefully weighed into glass vials.
In this case, experiments were conducted only in the absence of added salt. The study
prepared stock solution of 50 mg/L oxytetracycline and working solutions (25 mL each);
concentrations of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and 5000 µg/L, with
controls at each concentration. Then, each working solution was added to the appropriate
12

vial of soil. The soil itself added both Ca2+ and N a+ to each solution (Table 1.3). Thus, 0.5
g of soil 19 added 1.3 mmol Ca2+ /L and 5 mmol N a+ /L to each working solution, while 0.2
g of soil 19 added 0.5 mmol Ca2+ /L and 2 mmol N a+ /L to each working solution. Then,
the study samples were covered with aluminum foil to exclude light and put on a horizontal
shaker for around 24 hours. After that, the samples were centrifuged for 15 minutes at 3500
rpm. Then about 1 mL of solution phase was taken from each sample and analyzed using a
SCIEX API 3200 LC-MS/MS. Again, pH was determined using an aliquot of the centrifuged
supernatant and pH values were typically in the range of 7.4 to 8.

1.3
1.3.1

Results and Discussion
Effects of Background Cations on Oxytetracycline Sorption

This research studied oxytetracycline sorption to two desert soils from background solutions
containing Ca2+ or N a+ at three different concentrations. For example, Figure 1.6 shows
oxytetracycline sorption to soil 23 after adding CaCl2 concentrations of zero, 0.01, and
0.1 mol/L and also several oxytetracycline concentrations (ranging from 200 µg/L to 1000
µg/L).
First, sorption was strong with no added Ca2+ , and then sorption increased somewhat
further in the presence of 0.01 mol/L added CaCl2 . These data seem to support the hypotheses that Ca2+ enhances oxytetracycline sorption to clayey soils at high pH. That is,
Table 1.3 shows that soil 23 contains 95 mmol/L water-extractable Ca2+ in its saturated
paste (250/65= 3.85 g soil/mL extract); under the experimental conditions with 0.5 g soil
suspended in 5 mL of aqueous solution (0.1 g/mL), the water-extracted Ca2+ would be approximately 0.0025 mol/L in the experimental suspension with no added Ca2+ . According
13

to the working hypothesis of this study, this amount of solution-phase Ca2+ was apparently
enough to result in many cationic Ca-oxytetracycline complexes that sorbed to (clay) mineral
surfaces. The observation that sorption increased further when 0.01 mol/L Ca2+ was added
indicates that the increased Ca-oxytetracycline complexation enhanced sorption enough to
allow the complexes to out-compete the extra added Ca2+ for exchange sites.
However, at 0.1 mol/L added of CaCl2 , the oxytetracycline sorption to the soil sample
was strongly suppressed, with the slope of the sorption isotherm (Fig. 1.6) only about 1/3
that of the isotherm at 0.01 mol/L added CaCl2 . Presumably, the high Ca2+ concentration
results in much larger competition for sorption sites, resulting in sorption of fewer Caoxytetracycline complexes.
Figure 1.7 shows oxytetracycline sorption to soil 23 at three different concentrations of
added NaCl (zero, 0.01, and 0.1 mol/L) and several oxytetracycline concentrations (ranging
from 200 µg/L to 1000 ug/L). The study would expect that the isotherms described by blue
dots in both Figures 1.6 and 1.7 would be the same, since both are for soil 23 with no added
salts. The two differed somewhat, with isotherms slopes of 0.060 L/g in Fig.1.6 and 0.044
L/g in Fig. 1.7. In contrast to Fig. 1.6 when increasing the Ca2+ concentration by 0.01
m/L increased the oxytetracycline sorption, when the N a+ concentration was increased by
0.01 mol/L (Fig. 1.7), the sorption of oxytetracycline decreased. This can be rationalized
by noting that the amount of Ca2+ in the system stays the same in all three isotherms of
Fig. 1.7. If we assumed that the N a+ complexation with oxytetracycline was weak, then
the only effect of adding increasing N a+ concentration is to provide more competition by
N a+ that result in less sorption of Ca2+ oxytetracycline complexes. Since N a+ is a weaker
competitor than Ca2+ 0.1 mol/L, N a+ suppressed oxytetracycline sorption less than 0.1
mol/L Ca2+ did.
14

10

y	=	0.0665x	- 0.799
R²	=	0.94652

9
8

Q (µg/g)

7

y	=	0.0232x	- 2.3303
R²	=	0.91116

6

0 added CaCl2

5

0.01 M CaCl2
0.1 M CaCl2

4

Linear (0.01 M CaCl2)
3

Linear (0.1 M CaCl2)

2
1
0
0

100

200

300

400

Ce (µg/L)
Figure 1.6 The oxytetracycline sorption to soil 23 at three different concentrations of CaCl2 .
Figure 1.8 shows oxytetracycline sorption to soil 4 at three different concentrations of
added CaCl2 (zero, 0.01, and 0.1 mol/L) and several oxytetracycline concentrations (ranging
from 200 µg/L to 1000 µg/L). The results show a significant oxytetracycline sorption to soil
4 at zero added CaCl2 (pure water), but the isotherm slope was 0.023 L/g (Fig. 1.8),
about half the slope observed for soil 23 (Fig. 1.6). However, at 0.01 mol/L added of
CaCl2 , the oxytetracycline sorption to the soil sample decreased slightly while the sorption
of oxytetracycline decreased significantly at 0.1 mol/L added of CaCl2 .
Figure 1.9 shows oxytetracycline sorption to soil 4 at three different concentrations of
added NaCl (zero, 0.01, and 0.1 mol/L) and several oxytetracycline concentrations (ranging
15

10

y	=	0.0442x	- 0.9467
R²	=	0.9294

9
8

Q (µg/g)

7
6
0 added NaCl

5

0.01 M NaCl
4

0.1 M NaCl
Linear (0.01 M NaCl)

3
2
1
0
0

100

200

300

400

Ce (µg/L)
Figure 1.7 The oxytetracycline sorption to the soil 23 at three different concentrations of
NaCl.
from 200 µg/L to 1000 µg/L). The oxytetracycline sorption to soil 4 has an isotherm slope
of about 0.005 L/g for all three conditions, zero, 0.01, and 0.1 mol/L added NaCl. Again,
Figure 1.10 shows oxytetracycline sorption to soil 4 with no added salt and at N aCl or
CaCl2 concentrations of 0.1 mol/L, and it included several oxytetracycline concentrations
(ranging from 200 µg/L to 1000 µg/L). The results with no added salt show an isotherm
slope of 0.006 L/g, which compares with slopes at the same conditions of 0.023 L/g (Fig.
1.8) and 0.005 L/g (Fig. 1.9). Despite this variability, the trends agree in that the high

16

9

y	=	0.0233x	+	0.3296
R²	=	0.88599

8
7

Q (µg/g)

6
5

0 added CaCl2
0.01 M CaCl2

4

0.1 M CaCl2
3

Linear (0 added CaCl2)

2
1
0
0

200

400

600

800

1000

Ce (µg/L)
Figure 1.8 The oxytetracycline sorption to soil 4 at three different concentrations of CaCl2 .
Ca2+ concentration suppresses sorption to soil 4, while high N a+ concentration has little
effects on oxytetracycline sorption.
To summarize oxytetracycline sorption to soils 23 and 4, it seems clear that Ca2+ concentrations of 2 to perhaps 15 mmol/L Ca2+ contribute very favorably to oxytetracycline
sorption to these soils at pH 7.5. This supports the hypotheses that Ca-oxytetracycline
complexes are important species that control sorption to clay minerals, Parolo et al. and
Aristilde et al. [7, 31]. Larger Ca2+ concentrations approaching 100 mmol/L strongly suppressed oxytetracycline sorption presumably through competition for cation exchange sites.
N a+ concentrations, on the other hand have little effect on oxytetracycline sorption.

17

4

y	=	0.0053x	- 0.1836
R²	=	0.817

3.5

3

Q (µg/g)

2.5
0 added NaCl

2

0.01 M NaCl
0.1 M NaCl

1.5

Linear (0.01 M NaCl)

1

0.5

0
0

200

400

600

800

Ce (µg/L)
Figure 1.9 The oxytetracycline sorption to the soil 4 at three different concentrations of
NaCl.

1.3.2

Sorption at Higher Concentrations

Since all the isotherms above are basically linear, the study used some higher oxytetracycline
concentrations to test whether a sorption maximum could be observed. Figure 1.11 shows
oxytetracycline sorption to soil 19 without adding additional N aCl or CaCl2 and initial
oxytetracycline concentrations ranging up to 5000 µg/L. The soil mass was about 500 mg.
The results show a significant oxytetracycline sorption to soil 19 without reaching maximum.

18

5

y	=	0.006x	+	0.4135
R²	=	0.91479

4.5
4

Q	(µg/g)	

3.5
0 added NaCl or CaCl2

3

0.1 M NaCl

2.5

0.1 M CaCl2
2

Linear (0 added NaCl or
CaCl2)

1.5
1
0.5
0
0

200

400

600

800

Ce	(µg/L)	
Figure 1.10 Oxytetracycline sorption to soil 4 at three different concentrations of CaCl2 or
N aCl.
In fact, sorption accelerates as oxytetracycline concentrations increase, with the isotherm
slope increasing from about 0.01 L/g (up to about 1000 µg oxytetracycline/L) to about
0.034 L/g at higher concentrations. These slopes are larger than most of those for soil 4 but
smaller for those for soil 23.
In order to double check the curvilinear nature of Figure 1.11, the study repeated this
isotherm while reducing the amount of soil in each batch from 500 mg to 200 mg. Figure
1.12 shows again the oxytetracycline sorption to soil 19 was significant without reaching
19

40
35

Q (µg/g)

30
25
20
15
10
5
0

0

500

1000

1500

2000

Ce (µg/L)
Figure 1.11 Oxytetracycline sorption to soil 19 when soil mass about 0.5 g with no added
CaCl2 and N aCl.
any maximum. The data in Fig. 1.12 affirm that the isotherm slope increases as sorption
increases over the observed range.
Figure 1.13 combines and compares the sorption data of Fig. 1.11 and Fig. 1.12, showing
oxytetracycline sorption to soil 19 when soil mass was about 500 mg versus 200 mg. The
results show that the sorption of OTC was significantly higher when the soil mass was 200
mg. This might be interpreted in terms of the accelerating (S-shaped) isotherms, for which
sorption is increasingly favorable as sorption increases. That is, when a smaller amount of
soil is suspended in oxytetracycline solution, much more sorption per unit soil mass must
occur in order to reach a given aqueous phase oxytetracycline concentration. At a given
aqueous-phase concentration then, the larger sorbed mass on the smaller quantity of soil

20

80
70

Q (µg/g)

60
50
40
30
20
10
0
0

500

1000

1500

2000

2500

Ce (µg/L)
Figure 1.12 Oxytetracycline sorption to soil 19 when soil mass about 0.2 g with no added
CaCl2 and N aCl.
seems to induce a larger equilibrium quantity of sorbed tetracycline compared to the system
containing a larger quantity of soil. Minerals like those found in these Saudi soils could be
used to remove tetracyclines from polluted water systems. A number of other palygorskite
rich materials have been already proposed for removing tetracycline from contaminated waste
streams [33, 46, 47].
The data of the current study showed that oxytetracycline (OTC) sorption was strongest
for soil 23 even though soil 23 has the lowest clay and organic carbon contents. In terms of the
data that the study gathered, soil 23 had the largest water soluble Ca2+ concentration (Table
1.3). This support the hypotheses that higher calcium content contributes to oxytetracycline
sorption through complexation. In the other hand, the precise mineralogies remain unknown

21

80000
70000

Q (µg/kg)

60000
50000
40000

~ 200 mg Soil

30000

~ 500 mg Soil

20000
10000
0
0

500

1000

1500

2000

2500

Ce (µg/L)
Figure 1.13 Oxytetracycline sorption to soil 19 at two different soil masses (0.5 g and 0.2 g).
and it may be the soil 23 contains a mineral component that has a greater affinity for
oxytetracycline.

1.3.3

Sorption Coefficient (Kd )

For comparison with other data from the literature, sorption coefficient (Kd ) values and
sorption per unit mass of soil organic carbon (Koc ) values for each soil sample were calculated
(Table 1.4). The results show that soil 23 has highest Kd and Koc values; 60 and 23000 L/kg
SOC, respectively.
Previously measured/estimated Kd values for oxytetracyclines were ranging from 420 to
1030 L/kg as reviewed by Kumar et al. [48]. For a smectite containing Drummer soil from
Indiana at pH 7.5 the Kd for three tetracyclines averaged about 2500 L/kg [24]. Another
review of tetracycline sorption, in the pH range from 5 to 8.5, to a wide variety of soils [49]

22

Table 1.4 Sorption coefficient (Kd ) and sorption per unit mass of soil organic carbon (Koc )
values for plain soils; and comparison between the three selected soils.
Sample# Ca2+ (meq/L)

N a+ (meq/L)

Clay (%) Koc (L/kg)

Kd (L/kg)

4
19
23

152
147
189

60
32
18

23
23∗
60

84
73
190

3800
5000∗
23000

∗ These

even

values were derived from averaging over the entire the data set,
though the slope was not uniform across all the data (Figure1.13).

found Kd values extending from 40 to 10000 L/kg. This means that the largest Kd value
found in this research (60 L/kg) barely overlaps with the range of previous observation of
soils.
The Saudi Arabian soils used in this study were relatively poor in organic matter which
may have reduced sorption compare to other soils. However, the combination of relatively
high clay content and divalent cations such as Ca2+ should enable tetracycline sorption. For
example, a Kd of about 60000 L/kg was recently observed [32] for sorption of tetracycline to
montmorillonite at pH 7.7. This means that a soil containing only 1% montmorillonite could
have Kd of 600 L/kg. From the literature, the study found that Al-Ahsa city, which is in
eastern region of Saudi Arabia, topsoils are rich in palygorskite and smectite as substantial
clay content [1, 34]. Both of these minerals could contribute to tetracyclines sorption.

1.4

Conclusions from Experimental Work

There is a tendency to think that sorption of the tetracycline antibiotics to soils increases with
decreasing pH, because the cationic forms are more strongly sorbed [21–23]. This tendency
would imply minimal tetracycline sorption to desert soils. The hypothesis of this study, based
on the work of Parolo et al. and Aristilde et al., is that sorption of tetracyclines to desert soils

23

could be significant at pH 7.5 because of sorption of Ca-oxytetracycline cationic complexes
to clay mineral surfaces. This hypothesis was supported by the data from the current study
at low concentrations because all three clayey soils at pH 7.5 sorbed significant quantities of
oxytetracycline and Ca-soils sorb more OTC than Na-soils. However, these desert soils have
concentrations in excess of 0.1 mol/L (Table 1.3), and at these high concentrations it looks
like Ca2+ competition suppresses OTC sorption more than N a+ . However, the soils still
have substantial affinities for tetracyclines. While supporting the major hypotheses, these
data raise many questions about mineralogy, mechanism, and other geochemical controls
which may need further investigations.

24

Chapter 2
Geochemical Speciation Modeling of
Tetracycline Sorption to K- and
Ca-Smectites

2.1

Introduction

In the 20th century, antibiotics were major new discoveries in terms of human and animal
medicine. In the world, including the United States, antibiotics protect the lives of millions
of humans and animals every year [50]. In 2010, the usage of antimicrobials in animal foods
reached approximately 13.5 million kg [51, 52].
As mentioned earlier, the tetracycline is a broad-spectrum antibiotic that was discovered
in early of 1950s [53]. The chemical structure of tetracycline has explained and showed
in Chapter 1 and Figure1.1. In the aqueous phase, the functional groups in a tetracycline
structure often become ionized and cause tetracycline to form cation, zwitterion (neutral),
or anion species, depending on solution pH. It is showed in Chapter 1 and Figure 1.2.
Tetracycline is used extensively in human and veterinary medicine since the effectively
treat a variety of common infectious diseases. Tetracycline usage comprised 42% of the total
antimicrobials used in food animal production , 5, 602, 281 kg of tetracycline in 2010 [52,54].

25

Since large fractions of tetracycline are not metabolized in the animals, significant amounts
of tetracycline are excreted with animal manures, either as the original compound or as its
bioactive metabolites [52, 55, 56]. Thus, tetracycline and its derivatives are transferred to
soil [57, 58], surface water [59–62], and groundwater [58, 63, 64].
In the environment, tetracycline speciation in aqueous solutions can be very complex.
In the simplest case, tetracycline in pure water already contains four species, depending
on pH. Also, it is showed in Chapter 1 and Figure 1.2. Typically, aqueous solutions will
also contain a variety of metal cations, including Ca2+ , M g 2+ , and K + . These cations may
form complexes with tetracycline, thus reducing the activity of uncomplexed tetracycline and
forming additional species that must be considered (Figure 1.3in Chapter 1). The activities
of metal ions may be altered by the presence of organic ligands that can complex the metals,
or by sorption of the metals to solid phases such as clay minerals or organic matter. Such
competitive complexation or sorption of metals will generally increase the activity of aqueous
uncomplexed tetracycline. Finally, tetracycline itself may sorb to a variety of soil minerals
(e.g., smectite clays, goethite, or magnetite) and to soil organic matter, thus decreasing
tetracycline activity in solution.
In order to understand this complexity of tetracycline speciation in environmental waters,
computational tools are needed to iteratively and quantitatively estimate the distribution of
tetracycline among its many possible species. Phreeqc is a tool that was developed over 35
years ago and continues to be improved [65] for thermodynamic modeling of aqueous environmental systems. Phreeqc is a public-domain model that has already been integrated with
saturated- and unsaturated-flow water transport modules [66], so it provides a framework
for future reactive-transport modeling.
Thermodynamic modeling, as in Phreeqc, is very useful because even the most sophis26

ticated analytical procedures such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), [67] only measures the total tetracycline concentration in solution
rather than the concentrations of the component, (e.g. changed) tetracycline species, which
may manifest different chemistries. Thermodynamic approaches allow a more powerful interpretation of already sophisticated analytical LC-MS/MS data by estimating the distribution
of tetracycline among its possible species. Therefore, Phreeqc modeling is a method for extracting the most value from analytical data sets in order to better understand complexation,
sorption, and bioavailability of antibiotics in the environment.
The objective was to develop a quantitative model for tetracycline speciation in even more
complex systems like soils. This current study builds upon previous work [3] by developing
new thermodynamic terms and modeling additional data to create a more comprehensive
Phreeqc model of the system. A main goal was to predict tetracycline sorption to K- and
Ca-clay minerals as a function of realistic geochemical variables. While many of the relevant
chemical equilibria have already been described, several new thermodynamic relationships
in particular have been presented and used by Parolo et al. [7] and Aristilde et al. [31] and
incorporated in this work to help fit Phreeqc results to the available data in order to describe
the competitive adsorption of tetracycline to K- and Ca-clay minerals. Finally, the fit of the
parameters to several independent datasets was tested.

2.2

Methods

The thermodynamic parameters used in this study are listed in Table 2.1. Those equations
and parameters that were newly developed for this study are highlighted in bold. In Werner
et al. [4], they measured tetracycline speciation and complexation by Ca2+ , and M g 2+ as

27

functions of pH. The study selected their parameters because they form a self-consistent set.

2.2.1

Treatment of Cation-Exchange in Phreeqc

For modeling cation exchange on clay minerals, the study followed the general method of
Appelo and Postma [5]. In using this method, all cation exchange equilibria are referenced to
exchange with N a+ . That is, a Na-saturated clay is chosen as the thermodynamic “zero”,
and the energetic state of all other cations in the exchange complex are relative to this
“zero”. Thus, the cation exchange parameters in Table 2.1 for cation I i+ are referenced to
this equation [5]:

N a+ + 1/i . I − Xi ⇐⇒ N a − X + 1/i . I i+

(2.1)

The Phreeqc database compiles all such reactions as half-reactions of the form [5]:

N a+ + X − = N aX

logk = 0

(2.2)

Where X − is a site of cation exchange on the clay mineral. The logk parameter in Phreeqc
is the logarithm (base 10) of the equilibrium constant (Kexch ) for this cation-exchange halfreaction. Since logk = 0, Kexch must equal 1, and so if any other cation-exchange halfreaction is added to that of N a+ , the Kexch and the logk for the overall reaction will simply
be the Kexch and the logk for the non-N a+ ion. Therefore, all cation exchange equilibria
are referenced to exchange with N a+ [5].
To illustrate how parameters in the database work, considered the exchange of K + for
Ca2+ on a clay mineral. The K + half-reaction (Table 2.1) is:

28

K + + X − = KX

logk = 0.7

(2.3)

If we multiply the previous reaction by 2, we will obtain the following equation:

2K + + 2X − = 2KX

logk = 1.4

(2.4)

Also, the Ca- exchange reaction in Phreeqc (Table 2.1) is:

Ca2+ + 2X − = CaX2

logk = +0.8

(2.5)

logk = −0.8

(2.6)

Reversing the previous reaction:

CaX2 = Ca2+ + 2X −

If we combine reactions 2.4 and 2.6, we will get the following equation:

2K + + Ca − X2 −→ 2KX + Ca2+

logk = +0.6

(2.7)

Therefore, since [4G = −RT lnk = −RT (2.3)logk] and R = 8.3145 J/K.mol, T = 298K
hence RT = 2.48 kJ/mol. Thus, 4G = −(2.48)(2.3)(0.6) = −3.4 kJ/mol.
For the cation exchange half reaction that involves only one exchange site:

K + + 1/2Ca − X2 −→KX + 1/2Ca2+

logk = +0.3

(2.8)

So, 4G = −(2.48)(2.3)(0.3) = −1.7 kJ/mol.
To check the K + and Ca2+ parameters in Table 1.1, the competitive cation exchange

29

between K + and Ca2+ was calculated at three different ionic strengths (Figure 2.1). The
results were as expected that potassium is less competitive than calcium at low ionic strength,
and as ionic strength increases the potassium becomes more competitive with calcium. And
then these parameters have been used in many modeling studies of cation exchange involving
K + and Ca2+ [68–70].
1.20E+00

1.00E+00

K+ on CEC

8.00E-01

IS = 0.01 M

6.00E-01

IS = 0.1 M
IS = 0.8 M
4.00E-01

2.00E-01

0.00E+00
0

0.2

0.4

0.6

0.8

1

1.2

K+ in Solution
Figure 2.1 Calculated equilibria for K- Ca-exchange using Phreeqc and the cation exchange
coefficients of Appelo and Postma [5] and Table 2.1 at three different ionic strengths. IS=
ionic strength, and CEC= cation exchange capacity.

30

2.2.2

Experimental Data on Tetracycline Exchange with K + or
Ca2+ on Clay Minerals

The work of Yunjie Ding [6] measured the cation exchange equilibria of tetracycline with Kand Ca-smectites. This is a robust data set that provides sorption data for K + , Ca2+ , and
tetracycline as well as pH, the change in pH during experiment, and metal release from the
clays.
Ding [6] measured metal release by K + -clays and Ca2+ -clays during exchange reactions
with tetracycline. Such measurements were only possible for systems with no added background electrolyte (labelled ‘no ionic strength’ below), and could be useful for determining
whether tetracycline adsorbs to clays in forms other than the H4 T ec+ cation (Figure1.2).
For example, when tetracycline sorbs to calcium smectite, there are at least three possible
reaction stoichiometries. First, if two tetracycline molecules sorb to the clay and one Ca2+
ion appears in solution, then a reaction similar to

2H4 (T ec)+
+ CaX2 = 2H4 (T ec)X + Ca2+
(aq)

(2.9)

is inferred. Second, if tetracycline sorbs to the clay but the amount of Ca2+ ions in
solution remains constant, then a reaction like

H3 (T ec)0(aq) + CaX2 = [CaH3 (T ec)X2 ]

(2.10)

could be inferred. Third, note that Figure1.3 shows that the CaH2 (T ec)+ complex should
be an important tetracycline species in calcium systems [4]. Sorption of that complex should
result in one Ca2+ consumed from solution along with each two tetracycline sorbed, by the

31

reaction:

2CaH2 (T ec)+
+ CaX2 = 2[CaH2 (T ec)X] + Ca2+
(aq)
(aq)

(2.11)

Thus, the stoichiometry of changes in the inorganic cation concentration in solution as a
function of tetracycline sorption can be used to constrain interpretations of the operant sorption mechanisms. Indeed, it may be feasible to use cation-release data and apply Equations
2.9 – 2.11 to estimate the speciation of tetracycline and calcium on the exchange complex.
In each initial tetracycline solution, the following equilibrium was established at the initial
pH:

+
+
H4 (T ec)+
⇐⇒ H3 (T ec)o(aq) + H(aq)
⇐⇒ H2 (T ec)−
+ 2H(aq)
(aq)
(aq)

(2.12)

If the cationic species H4 (T ec)+ sorbs to the clay and is removed from solution, and if
we assume constant pH, then the above equilibrium will be re-established. If the amount of
H4 (T ec)+ removed from solution was Ci , then an amount of H + approximately equal to Ci
will need to be removed from solution in order to re-establish the above equilibrium. Thus,
proton consumption is an approximation of the amount of H4 (T ec)+ sorption under a given
condition, and should be reflected as a loss of protons from solution and an increase in the
solution pH after tetracycline sorption by the clay.

32

2.3
2.3.1

Results and Discussion
Possible Impurities in the Clay or Deionized (DI) Water

To test the performance of the tetracycline parameters, the pH values for all initial solutions
were calculated using Phreeqc. To do so, each initial concentration of tetracycline that
was used by Ding [6] was equilibrated and the pH was calculated. The calculated pH was
significantly lower than the observed pH, especially when the tetracycline concentrations
were low. Ding used tetracycline HCl to make his initial solutions, so the following equations
apply:

H4 T ecCl−→H4 T ec+ + Cl−

(2.13)

H4 T ec+ −→H + + H3 T ec0

(2.14)

Equation 2.14 shows that the solution pH should certainly be below 7 for all concentrations of tetracycline HCl in pure water. However, Ding observed many pH values above 8. A
hypothesis that is consistent with Ding’s data is that a basic impurity was present in all his
systems. One possibility is carbonate impurities in the SWy-2 clay he used [71], and another
possibility is impurities in the water. The deviations between observed and expected pH
values were systematic and a good fit to all data was obtained by adding 0.08 mmol/kgw
of N aHCO3 and 0.02 mmol/kgw of N a2 CO3 . The calculated and observed pH values as a
function of initial tetracycline concentrations are plotted in Figure 2.2. There is no evidence
for the nature of the high pH contaminant in the clay or water used by Ding [6], but in order
to fit pH properly, this buffer was used in all subsequent calculations.
33

10
9

Initial pH

8
7

Ding's data for Ca
Phreeqc Ca

6

Ding's data for K
5

Phreeqc K

4
3
0

100

200

300

400

500

Initial tetracycline concentration µmol/L
Figure 2.2 Comparison of modeled and experimental [6] pH values of initial tetracycline
solution.
Many complexation constants have been determined for tetracycline interactions with
cationic metals, but the main focus of this study was to develop some new thermodynamic
parameters for cation exchange reactions involving tetracycline. A provisional set of these
parameters are listed and highlighted in Table 2.1.
One important parameter for clay minerals is the cation exchange capacity (CEC), the
total quantity of cationic charge that can be reversibly sorbed per unit mass of clay. For
the clay mineral SWy-2 used by Ding [6], the CEC is known to be 78 cmol/kg. However,
sorption of tetracycline to both K-SWy-2 and Ca-SWy-2 with no other salt in the system
showed sorption plateaus at 42 − 43 cmol of tetracycline per kg clay p.79 of [6]. A plausible
reason for this discrepancy is explained by Ding p.88 of [6]:
34

“The basal spacing of smectites were 14.7 Å with tetracycline loadings < 135 µmol/g ...
the distance between two adjacent clay sheets was 5.1 Å ... molecular dynamics simulation
results indicate that at low loading rate (< 135 µmol/g), tetracycline lays parallel to clay
surfaces, .... When sorption approaches to 420 µmol/g, which is the sorption plateau in
Figure III-2, the clay sheets expands to 7.5 Å. At this distance tetracycline adapts a tilted
position in clay interlayers. Molecular dynamic simulation results for sorption at 420 µmol/g
indicate that tetracycline molecules adapt vertically tilted position.”
The present study also adopts the hypothesis that the clay interlayer “fills up” at 42 − 43
cmol(+)/kg at which point the interlayer is plausibly “full” and causes a sorption plateau.
Further sorption of tetracycline is possible but requires the interlayer to rearrange so that
tetracycline can adopt a tilted configuration. Realistically, loading of clay minerals in nature
by tetracycline should be less than 42−43 cmol(+)/kg, so confining our fitting efforts to this
lower- tetracycline region of the sorption curve seems appropriate from an environmental,
practical standpoint. Ding [6] showed that tetracycline concentrations greater than 100
µmol/L (44 mg tetracycline/L) were required to achieve the sorption plateau even in the
absence of competing salts. For comparison, observed concentrations of tetracycline in liquid
manures may approach 40 mg tetracycline/L [72], but concentrations will generally be much
more dilute in environmental waters or soil solutions.
One benefit of this approach is that such a model might fit all smectite clays and thus be
widely applicable: All smectites have CEC > 42 − 43 cmol(+)/kg but all possess roughly
the same interlayer surface area (about 750 m2 /g) [73], and so many smectites should display plateaus or inflections in their tetracycline sorption near 42 − 43 cmol(+)/kg. For
example, Figueroa et al. [27] studied sorption of oxytetracycline and tetracycline to N amontmorillonite. Their oxytetracycline sorption isotherm at pH 5.5 and 10 mmol N aCl
35

showed a plateau at about 440 − 480 cmol(+)/kg—their Fig. 7a labels that plateau as 44
cmol(+)/kg, but this seems to be in conflict with the reported Kd of 5500 L/mol(+) (see
their Fig. 3 at 10 mmol N aCl and pH 5.5). At 0.1 mmol/L tetracycline and with a clay
of CEC = 80 cmol(+)/kg, the Kd predicts sorption of 440 mmol tetracycline per kg clay,
exactly 10x the plateau pictured in their Fig. 7a. Another paper (Li et al., [74]) also commented that the sorption results of Figueroa et al. [27] seem low by a factor of about 10.
Furthermore, the same authors (Li et al., [74]) observed an anomalous result that up to 42
cmol(+)/kg of tetracycline could sorb to SWy-2 smectite without any significant change in
the desorbed inorganic cations in solution. Again, this phenomenon and the high pH they
observed, are indirect evidence of carbonate impurities in the SWy-2 smectite clay itself.
Therefore, the present study fixed the quantity of cation exchange sites in the “exchange”
module of Phreeqc at 0.42 moles of exchange sites per kg clay, and the study hypothesizes
that this value should work for most smectite clays in the environment.
Aristilde et al. [75] observed maximum sorption of oxytetracycline (OT C + ) by smectite
to be 44 cmol(+)/kg even at OTC solution concentrations approaching 1 mmol/L at pH 4
and in a background of 0.01 mol/L N aN O3 . On the basis of X-ray diffraction analysis and
molecular simulations, they argued that OT C + was segregated into only half the smectite
interlayers, with the other half of the interlayers filled with N a+ , a phenomenon known as
demixing.
This study calculated tetracycline/K + exchange in 4 different ionic strength situations
that also included several tetracycline concentrations and different pH values. A first situation was a dilute solution (no added salt other than tetracycline HCl and the carbonate
buffer discussed above), while the remaining three cases added an additional 0.01 M , 0.1 M ,
and 0.8 molal as KCl. Note that this study uses different units at the highest ionic strength,
36

since molarity and molality begin to significantly diverge at such concentrations.
The study adjusted the new thermodynamic parameters to fit Ding’s results [6]. To
do so, this study systematically examined several values of the tetracycline/K + exchange
parameters before acceptable results were reached. Then, after a number of attempts, the
research achieved a good fit for tetracycline/K + exchange.
Figure 2.3 shows the comparison between the Phreeqc predictions and Ding’s data [6]
in dilute solution (no added salt other than tetracycline HCl and sodium carbonate) for
tetracycline sorption by K-smectite, and the result was fit very well. Note that Essington
et al. [76] modeled the sorption of chlortetracycline (CT C) sorption by Na-smectite. They
concluded that cation-exchange of CT C + for N a+ was the dominant sorption process below
pH 5, but that sorption above pH 5 was only slightly smaller in magnitude and needed to
invoke strong sorption of the zwitterionic (neutral CT C 0 ) form of CTC. The present study
suggests that interlayer sorption of the N a+ -CT C 0 complex is a species they could have
considered.
Figure 2.4 compares the Phreeqc predictions with Ding’s data [6] at 0.01 M as KCl for
tetracycline sorption by K-smectite. The result was good, but the prediction of Phreeqc
was slightly higher than Ding’s data. Figure 2.5 shows the comparison between the Phreeqc
predictions and Ding’s data [6] at 0.1 M as KCl ionic strength. In this case, tetracycline
sorption by K-smectite was predicted very well. Figure 2.6 shows the comparison between
Phreeqc prediction and Ding’s data [6] at 0.8 molal as KCl ionic strength for tetracycline
sorption by K-smectite. Again, the result was good, but the prediction of Phreeqc was
slightly lower than Ding’s data.

37

500
450
400

Cs (µmol/g)

350
300
250

Phreeqc prediction

200

Ding's exprimental data

150
100
50
0
0.00E+00 5.00E-02 1.00E-01 1.50E-01 2.00E-01 2.50E-01

Cw (mmol/L)
Figure 2.3 Sorption of tetracycline to the K-smectite clay in dilute solution (no added KCl).
Next, the study attempted to create tetracycline/Ca2+ exchange parameters that were
consistent with the well-fitting tetracycline/K + parameters. For example, this study fixed
the H4 T ec+ /K + and H4 T ec+ /Ca2+ parameters at the same values that were effective inK-clay systems and only varied the exchange parameters for Ca2+ /tetracycline complexes
such as Ca2 HT ec2+ /Ca2+ and CaH2 T ec+ /Ca2+ .
In the aqueous phase where tetracycline and cations such as calcium are present, the
tetracycline will form complications with cations depending on solution pH phase (Figure
2.7).
The current study focuses on three sorption parameters: H4 (T ec)+ , CaH2 (T ec)+ and
Ca2 H(T ec)2+ . Summing these three terms results in total Tec sorption for Ca-systems, in
this model. Given that extensive modeling supports the use of log k = 3.8 for sorption of the

38

350
300

Cs (µmol/g)

250
200

Phreeqc prediction

150

Ding's exprimental data

100
50
0
0.00E+00 5.00E-02 1.00E-01 1.50E-01 2.00E-01 2.50E-01

Cw (mmol/L)
Figure 2.4 The sorption of tetracycline to the K-smectite clay at 0.01 M KCl ionic strength.
250

Cs (µmol/g)

200
150

Phreeqc prediction

100

Ding's exprimental data
50
0
0.00E+00 5.00E-02 1.00E-01 1.50E-01 2.00E-01 2.50E-01

Cw (mmol/L)
Figure 2.5 Sorption of the tetracycline to the K-smectite clay at 0.10 M KCl as ionic
strength.

39

90
80

Cs (µmol/g)

70
60
50
40

Phreeqc prediction

30

Ding's exprimental data

20
10
0
0.00E+00

1.00E-01

2.00E-01

3.00E-01

4.00E-01

Cw (mmol/L)

Speciation Fraction

Figure 2.6 Sorption of tetracycline to the K-smectite clay at 0.80 molal KCl ionic strength.
7.00E-04
6.00E-04
5.00E-04
4.00E-04
3.00E-04
2.00E-04
1.00E-04

0.00E+00

3

4

5

6

7

8

9

10

11

pH
H4(Tec)+

H3(Tec)0

Ca2H(Tec)+2

CaH2(Tec)+

Figure 2.7 Ca2+ and tetracycline speciation and complication in aqueous phase as a function
of pH solution which calculated using Phreeqc model [7]. These calculations were done with
0.00076 mol/L tetracycline in calcium chloride with ionic strength of 0.004 M .
H4 (T ec)+ species, this study explored the parameter space offered by the CaH2 (T ec)+ and
Ca2 H(T ec)2+ terms. When the Ca2 H(T ec)2+ term was commented out in the database

40

(meaning that Ca2 H(T ec)2+ sorption was not allowed in Phreeqc), the log k for CaH2 (T ec)+
was varied across values from 3 to 7. With a log k for CaH2 (T ec)+ of 7, Phreeqc predicted
tetracycline sorption (H4 (T ec)+ plus CaH2 (T ec)+ ) that fit the experimental data very well
(Figures 2.8 and 2.9). At higher ionic strengths, the Phreeqc predictions were increasingly
too large compared to experiment (results not shown), implying that any larger values of
log k for CaH2 (T ec)+ were unrealistic at high ionic strengths, even though results at low
ionic strengths were promising.
500
450

Cs (umol/g)

400
350
300
250
200

Phreeqc predictions

150

Ding's data

100
50
0
0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

Cw (mmol/L)
Figure 2.8 Sorption of tetracycline to Ca-smectite in dilute solution (no added salt other
than tetracycline HCl and sodium carbonate) when log k value for CaH2 (T ec)+ was set to
7.

41

450

Cs (umol/g)

400
350
300
250
200

Phreeqc predictions

150

Ding's data

100
50
0
0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

Cw (mmol/L)
Figure 2.9 Sorption of tetracycline to Ca-smectite at 0.01 M ionic strength when log k value
for CaH2 (T ec)+ was set to 7.
This study’s other significant Ca-related variable is the log k for sorption of Ca2 H(T ec)2+
complexes by smectites, and so this variable was explored while commenting out the CaH2 (T ec)+
term. Again, with log k = 3.8 for sorption of the H4 (T ec)+ species, the log k for sorption of
Ca2 H(T ec)2+ was varied from 3 to 6.6. With a log k for Ca2 H(T ec)2+ of 6.6, Phreeqc predicted tetracycline sorption (H4 (T ec)+ plus Ca2 H(T ec)2+ ) that followed the experimental
trends fairly well (Figures 2.10 and 2.11). At higher ionic strengths, the Phreeqc predictions
were much too large compared to experiment (results not shown), implying that any larger
values of log k for Ca2 H(T ec)2+ were unrealistic at high ionic strengths, even though results
at low ionic strengths were promising.

42

500
450

Cs (µmol/g)

400
350
300
250
200

Phreeqc predictions

150

Ding's data

100
50
0
0.00E+00 5.00E-02 1.00E-01 1.50E-01 2.00E-01

Cw (mmol/L)
Figure 2.10 Sorption of tetracycline to Ca-smectite in dilute solution (no added salt other
than tetracycline HCl and sodium carbonate) when log k value for Ca2 H(T ec)2+ was set to
6.6.
The next question for this study, then, was to characterize the combined behavior of
the parameters for CaH2 (T ec)+ and Ca2 H(T ec)2+ sorption. Somewhat surprisingly, both
parameters needed to be maintained near their individual maxima. That is, if log k values
for both terms were set to 4, then sorption was greatly underpredicted under conditions of
medium-to-high pH and low ionic strengths (Figure 2.12).
A better fit of the sorption data was obtained when the parameters for CaH2 (T ec)+ and
Ca2 H(T ec)2+ sorption were each set to 6.6 (Table 2.1). Figure 2.13 shows the comparison
between these Phreeqc predictions and Ding’s data [6] in dilute solution (no added salt other
than tetracycline HCl and sodium carbonate) for tetracycline sorption by Ca-smectite. The
result was fit well, but the prediction of Phreeqc was a little bit lower than Ding’s data.
43

450
400

Cs (µmol/g)

350
300
250
200

Phreeqc predictions

150

Ding's data

100
50
0
0

0.05

0.1

0.15

0.2

0.25

Cw (mmol/L)
Figure 2.11 Sorption of tetracycline to Ca-smectite at 0.01 M ionic strength when log k value
for Ca2 H(T ec)2+ was set to 6.6.
Figure 2.14 compares the Phreeqc predictions for tetracycline sorption by Ca-smectite with
Ding’s data [6] at an ionic strength of 0.01 M as CaCl2 . Also, the agreement between model
and experiment was fit well, but the model predictions were again a little bit lower than the
experimental data. This parameter set does a decent job of predicting tetracycline sorption
at low ionic strengths. However, just as with the individual CaH2 (T ec)+ and Ca2 H(T ec)2+
parameters, when both were set to 6.6, the Phreeqc predictions for tetracycline sorption at
high-Ca ionic strengths of 0.10 and 0.82 molal were much too large compared with experiment
(see below).

44

500

Cs (µmol/g)

450
400
350
300
250
200

Phreeqc predictions

150

Ding's data

100
50
0
0.00E+00 5.00E-02 1.00E-01 1.50E-01 2.00E-01

Cw (mmol/L)
Figure 2.12 Sorption of tetracycline to Ca-smectite in dilute solution (no added salt other
than tetracycline HCl and sodium carbonate) when log k values for both CaH2 (T ec)+ and
Ca2 H(T ec)2+ species were set to 4.
Figure 2.15 shows the comparison between the Phreeqc predictions and Ding’s data [6] at
an ionic strength of 0.10 M CaCl2 when the log k values for CaH2 (T ec)+ and Ca2 H(T ec)2+
sorption were each set to 6.6 (Table 2.1). The Phreeqc prediction of tetracycline sorption
followed the experimental trend, but the predictions from Phreeqc were higher than Ding’s
data. Nevertheless, the study did many attempts to vary these two log k values, and followed
several different approaches to reach better Phreeqc predictions of the experimental data.
Given that the log k for H4 (T ec)+ sorption is set to 3.8, if the 0.1-M sorption data are
to be fit, then the current research found that the log k values of both CaH2 (T ec)+ and
Ca2 H(T ec)2+ species should be reduced from 6.6 to 5.6. Therefore, Phreeqc was capable of
predicting the experimental data of Ding when CaCl2 was at 0.10 M, but the fit required
different parameters than at lower ionic strengths. Under these conditions, Figure 2.16 shows
45

500

Cs	(µmol/g)

450
400
350
300
250
200

Phreeqc prediction

150

Ding's exprimental data

100
50
0
0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

Cw	(mmol/L)
Figure 2.13 Sorption of tetracycline to Ca-smectite clay in dilute solution (no added salt
other than tetracycline HCl and sodium carbonate).
very good Phreeqc predictions of tetracycline sorption, and the Phreeqc results were very
close to Ding’s observation data.
Figure 2.17 shows the comparison between the Phreeqc predictions and Ding’s data [6]
at an ionic strength of 0.82 molal CaCl2 when the log k values for CaH2 (T ec)+ and
Ca2 H(T ec)2+ sorption were each set to 6.6 (Table 2.1). The Phreeqc sorption predictions
were far higher than Ding’s data, illustrating that many Ca-tetracycline complexes form and
sorb when calcium is plentiful. The study again did many attempts to vary the CaH2 (T ec)+
and Ca2 H(T ec)2+ log k values, and followed several different approaches to reach better
Phreeqc predictions of the experimental data. Again, fixing the log k for H4 (T ec)+ sorption
to 3.8, if the 0.82-molal sorption data are to be fit, then the current research found that
the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species should be reduced from
6.6 to 4.4. Under those conditions, Phreeqc was again capable of predicting the experimen46

450

Cs	(µmol/g)

400
350
300
250
200

Phreeqc prediction

150

Ding's exprimental data

100
50
0
0.00E+00 5.00E-02

1.00E-01

1.50E-01

2.00E-01

Cw	(mmol/L)
Figure 2.14 Sorption of the tetracycline to Ca-smectite clay at 0.01 M CaCl2 ionic strength.
tal data (Ding) gathered at 0.8 M CaCl2 , but the fit again required different parameters
than at lower ionic strengths. Then, Figure 2.18 shows very good Phreeqc predictions of
tetracycline sorption, and the Phreeqc results were very close to Ding’s observation data.
A truly self-consistent model would require only one thermodynamic equilibrium constant
for each reaction. The need for the present study to change the log k values for sorption
of Ca-tetracycline complexes at high ionic strengths indicates that something important
(important reactions may be neglected, activities may not modeled well, etc) is still.

47

450
400

Cs (µmol/g)

350
300
250
200

Phreeqc prediction

150

Ding's exprimental data

100
50
0
0

0.05

0.1

0.15

0.2

0.25

Cw (mmol/L)
Figure 2.15 Sorption of tetracycline to Ca-smectite at 0.10 M ionic strength strength when
the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set to 6.6.
Phreeqc allows cation exchange sorption to Ca-clay minerals for three species of tetracycline, H4 (T ec)+ , CaH2 (T ec)+ , and Ca2 H(T ec)2+ species. Formation of each of these
species depends strongly on pH, and so their contributions to overall tetracycline sorption
also depend on pH. In the solid phase at an ionic strength of 0.01 M , Figure 2.19 shows
the Phreeqc calculated contributions of these three sorbed tetracycline species to the total
sorption. Note that above pH of 7 the Ca2 H(T ec)2+ species dominate sorption, indicating
the importance of this species for tetracycline sorption in desert soil environments. Aristilde et al. [31], show the importance of tetracycline complexation with Ca2+ or M g 2+ for
tetracycline sorption to clay minerals at high pH, and Parolo et al. [7] emphasize the importance of this Ca2 H(T ec)2+ species when calcium-tetracycline ratio is large. Werner et al. [4]

48

300

Cs (µmol/g)

250
200
150

Phreeqc prediction
Ding's exprimental data

100
50
0
0

0.05

0.1

0.15

0.2

0.25

Cw (mmol/L)
Figure 2.16 Sorption of tetracycline to Ca-smectite at 0.10 M ionic strength strength when
the log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set to 5.6.
argued for the dominance of CaH2 (T ec)+ species near neutral pH. Phreeqc used Werner
et al. complexation constants but our Phreeqc modeling for sorption of these complexes
to clay minerals indicates that CaH2 (T ec)+ species dominates tetracycline sorption all the
way down to pH of 4.5 (Figure 2.19). Sorption of tetracycline near pH of 4 is relatively
independent of calcium [31], which implies that the H4 (T ec)+ species dominates sorption.
Figure 2.19 also shows that sorbed concentration of H4 (T ec)+ and CaH2 (T ec)+ species are
comparable even at the lowest pH, implying that sorption processes in soil drive tetracycline
into the sorbed CaH2 (T ec)+ species at pH values well below those at which this species
would be prevalent in solution phase if the soil CEC were not present (Figure 2.7).
Then, the current results make it tempting to believe that a good overall fit has been found
using the modeling parameters compiled in Table 2.1. Thus, great care must be advised when
49

450

Cs (µmol/g)

400
350
300
250
200

Phreeqc prediction

150

Ding's exprimental data

100
50
0
0

0.05

0.1

0.15

0.2

0.25

0.3

Cw (mmol/L)
Figure 2.17 Sorption of tetracycline to Ca-smectite at 0.82 molal ionic strength when the
log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set to 6.6.
160

Cs	(µmol/g)

140
120
100
80

Phreeqc prediction

60

Ding's exprimental data

40
20
0
0.00E+00

1.00E-01

2.00E-01

3.00E-01

4.00E-01

Cw	(mmol/L)
Figure 2.18 Sorption of tetracycline to Ca-smectite at 0.82 molal ionic strength when the
log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set to 4.4.

50

Adsorbed species composition (%)

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
3.5

4.5

5.5

6.5

7.5

8.5

9.5

pH
H4(Tec)+

CaH2(Tec)+

Ca2H(Tec)2+

Figure 2.19 Contributions of three tetracycline species to the total sorption calculated using
Phreeqc model at an ionic strength of 0.01 M .
using goodness-of-fit as the main criterion for selecting thermodynamic modeling parameters.

2.3.2

Inorganic Cations Released When Tetracyclines Sorbed to
Clay

Ding measured the release of inorganic cations from clay for his systems where tetracycline
sorbed in the absence of added salt [6]. The smallest nonzero ionic strength the Ding used
was 0.01 M while the maximum observed inorganic cation release was 0.0002 M under his
experimental conditions, and so cation release can be only measured for the case of dilute
solution (no added salt other than tetracycline HCl). This study used Phreeqc to predict
K + and Ca2+ released to the solution phase from the smectite clays in dilute solution
(no added salt other than tetracycline HCl and sodium carbonate). Figure 2.20 shows
comparisons between Phreeqc predictions and Ding’s experimental data for both (a) the

51

amount of tetracycline sorbed to K-smectite clay and (b) the amount of K + released to
the solution. The experimental results show that the amount of K + released was increased
when tetracycline concentration was increased, and the K + released was always less than
or equal to the tetracycline sorbed. The Phreeqc results show that K + released was greater
than tetracycline sorption, especially at low concentrations. This is an artifact of using
the sodium carbonate buffer (Figure 2.2) to match the experimental pH values. The 0.12
mmol/L N a+ concentration in that buffer removed K + from the clay by cation exchange.
Perhaps potassium carbonate would have been a better choice for buffering the pH, because
would have allowed this study to measure K + released without competition from N a+ .

Tetracycline sorbed (µmol/g)

500
450
400
350
300

Phreeqc

250

Ding's data

200
150

K released in Phreeqc

100

K released in Ding's data

50
0
0.00E+00 5.00E-02 1.00E-01 1.50E-01 2.00E-01 2.50E-01

Tetracycline in solution at equilibrium (mmol/L)
Figure 2.20 Tetracycline sorption to K-smectite clay and K + released calculated using
Phreeqc in dilute solution (no added salt other than tetracycline HCl and sodium carbonate).

Figure 2.21 shows comparisons between Phreeqc predictions and Ding’s experimental
data [6] for both (a) the amount of tetracycline sorbed to Ca-smectite clay and (b) the
amount of Ca2+ released to the solution. But here, the Phreeqc results show that the
52

amount of Ca2+ released to the solution was very low compared to the experimental results,
for all amounts of tetracyclines sorbed to the clay. This implies that too much Ca2+ is
retained in the clay when tetracycline sorbed, so probably the log k for sorption of Catetracycline complexes is too large. When log k for the sorption of CaH2 (T ec)+ species was
reduced from 6.6 to 5.6, then the log k for sorption of the Ca2 H(T ec)2+ species had to be
increased from 6.6 to 10.2 to get reasonable fit of Ding’s experimental data as it shown in
Figure 2.22. Overall, the fit of the tetracycline sorption data was much better when both
these log k values were 6.6 as it shown in Figure 2.21, even though the calcium release data
were not fit well.

Tetracycline sorbed (µmol/g)

500
450
400
350
300

Phreeqc

250

Ding's data

200

Ca released in Phreeqc

150

Ca released in Ding's data

100
50
0
0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

Tetracycline in solution at equilibrium (mmol/L)
Figure 2.21 Tetracycline sorption to Ca-smectite clay and Ca2+ released calculated using
Phreeqc in dilute solution (no added salt other than tetracycline HCl and sodium carbonate)
when log k values of both CaH2 (T ec)+ and Ca2 H(T ec)2+ species were set to 6.6.

53

Tetracycline sorbed (µmol/g)

500
450
400
350
300

Phreeqc

250

Ding's data

200
150

Ca released in Phreeqc

100

Ca released in Ding's data

50
0
0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

Tetracycline in solution at equilibrium (mmol/L)
Figure 2.22 Tetracycline sorption to Ca-smectite clay and Ca2+ released calculated using
Phreeqc in dilute solution (no added salt other than tetracycline HCl and sodium carbonate)
when log k for CaH2 (T ec)+ species was set to 5.6 while log k for Ca2 H(T ec)2+ species was
set to 10.2.

2.3.3

Fraction of Tetracycline Apparently Sorbed as Uncomplexed
Cations

This study also used Phreeqc to calculate the percentage of tetracycline apparently sorbed as
uncomplexed cations. In Chapter three of Ding’s dissertation [6], Figure III −2 showed inorganic cations exchanged/released as a function of tetracycline sorption. This modeling study
calculated the relevant species and then compared these species to estimates from Ding’s
data. For K-smectite, the study assumed one H4 (T ec)+ molecule sorbed for each potassium cation exchanged/released. For Ca-smectite, the study assumed that two H4 (T ec)+
molecules needed to sorb in order to release one Ca2+ to solution as estimated from Ding’s
experimental data [6].

54

Figure 2.23 shows comparisons between tetracycline percentages sorbed to potassium
smectite clay as cations in Phreeqc versus the estimated percentages of tetracyclines sorbed
as cations from Ding’s experimental data. The results show that the Phreeqc prediction of
proportions of tetracycline sorbed as cations were very close to the estimated percentages
for tetracycline cation sorption from Ding’s experimental data. Moreover, the results show
that the percentages of tetracyclines apparently sorbed as cations increased when tetracycline

% Tetracycline apparently sorbed as cation

concentration was increased in both Ding’s experimental data and Phreeqc prediction results.
120
100
80

Estimated from Ding's data,
Fig. III-2

60

Phreeqc prediction

40
20
0
0

50

100

150

200

250

Tetracycline concentration in solution (µmol/L)
Figure 2.23 Percentage of tetracyclines sorbed as cations to K-smectite clay in dilute solution
(no added salt other than tetracycline HCl and sodium carbonate).

Figure 2.24 shows comparisons between tetracycline percentages sorbed to calcium smectite clay as cations in Phreeqc versus the estimated percentages of tetracyclines sorbed as
cations from Ding’s experimental data.
The results show that the Phreeqc prediction of proportions of tetracycline sorbed as
55

cations were fairly close to the estimated percentages for tetracycline cation sorption from
Ding’s experimental data. Again, the results show that the percentages of tetracyclines
apparently sorbed as cations increased when tetracycline concentration was increased in
both Ding’s experimental data and Phreeqc prediction results.
For calcium, there was greater discrepancy between modeling and experimental results
than the case of potassium. As noted above, Phreeqc under predicted the amount of calcium released when tetracycline sorbed, but apparently the relative proportions of calcium-

% Tetracycline apparently sorbed as cation

complexed versus uncomplexed tetracycline cations were more accurate.
120
100
80

Estimated from Ding’s data,
Fig.III-2

60

Phreeqc prediction

40
20
0
0

50

100

150

200

Tetracycline concentration in solution (µmol/L)
Figure 2.24 Percentage of tetracyclines sorbed as cations to Ca-smectite clay in dilute solution
(no added salt other than tetracycline HCl and sodium carbonate).

56

Table 2.1 Compilation of all chemical equations and equilibrium constants used in the present
study. All equations are written as they appear in the Phreeqc model database.
Section in Phreeqc
Reactions

log k

Tec H4 (T ec)+

−−b

H4 (T ec)+ = H4 (T ec)+

0.000d

H4 (T ec)+ = H3 (T ec) + H +

−3.45a

H4 (T ec)+ = H2 (T ec)− + 2H +

−11.45a

H4 (T ec)+ = H(T ec)2− + 3H +

−21.23a

H4 (T ec)+ = T ec3− + 4H +

−33.64a

Ca2+ + H2 (Tec)− = CaH2(Tec)+

3.4b

M g 2+ + H2 (T ec)− = M gH2 (T ec)+

3.9b

Ca2+ + H(Tec)2 − = CaH(Tec)

5.8b

M g 2+ + H(T ec)2− = M gH(T ec)

4.1b

Ca2+ + H3 (Tec) = CaH3 (Tec)2+

3.0d

K + + H2 (T ec)− = KH2 (T ec)

1.04e

K+ + H3 (Tec) = KH3 (Tec)+

1.04e

2Ca2+ + H(Tec)2− = Ca2 H(Tec)2+

8.671g

Database
SOLUTION MASTER
SPECIES

SOLUTION SPECIES

Fix H +

0.00d

PHASES
H+ = H+
EXCHANGE MASTER
X

X−

−−d

SPECIES

57

Table 2.1 (cont’d)
Section in Phreeqc
Reactions

log k

X− = X−

0.0f

H4 (Tec)+ + X− = H4 (Tec)X

3.8d

CaH2 (Tec)+ + X− = CaH2 (Tec)X

6.6d

#CaH3 (Tec)2+ + 2X− = CaH3 (Tec)X2

#4.8d

Ca2 H(Tec)2+ + 2X− = Ca2 H(Tec)X2

6.6d

M gH2 (T ec)+ + X − = M gH2 (T ec)X

2.0d

KH3 (Tec)+ + X− = KH3 (Tec)X

3.6d

N a+ + X − = NaX

0.0f

K + + X − = KX

0.7f

Ca2+ + 2X − = CaX2

0.8f

M g 2+ + 2X − = M gX2

0.6f

Database

EXCHANGE SPECIES

a

data from Werner et al. 2006 were used, but equations were added to arrive at these
values; b data from Gu and Karthikeyan, 2005; c data from MINEQL+ database (Version
4.5, 2002); d equation and value from this study; e data from Coibion and Laszio, 1979;
f data from Phreeqc database (Version 2.13.2-1727, 2007); g data from from Parolo et al.
2013.

58

2.4

Conclusions from Modeling of Tetracycline Sorption to K- and Ca-Smectites

The Phreeqc modeling software was self-consistently used to model K + /H4 (T ec)+ and
Ca2+ /H4 (T ec)+ exchanges simultaneously, in that one logk value (3.8) for sorption of
the H4 (T ec)+ species was shared in systems with background electrolytes of either KCl
or CaCl2 . The Phreeqc approach was able to model K + /H4 (T ec)+ exchange fairly well,
fitting datasets with KCl ionic strengths from dilute solution (no added salt) to 0.8 M .
Fitting these data required addition of one term for clay sorption of KH3 (T ec)+ complexes, using literature values for formation of the complexes in aqueous solution. Modelling
the Ca2+ -tetracycline system required cation-exchange parameters for both CaH2 (T ec)+
and Ca2 H(T ec)2+ species; these parameters were ionic-strength-dependent, since the same
set could not reproduce tetracycline sorption results over the entire range of CaCl2 ionic
strength. Near pH 7 and above, the Phreeqc modeling results indicated the importance of
Ca2 H(T ec)2+ species for tetracycline sorption in desert soil environments. While this work
developed several thermodynamic relationships for modeling tetracycline sorption to K- and
Ca-clays, this study may require further investigations.

59

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