(SpringerBriefs in Animal Sciences) Rakesh Ranjan, Amita Ranjan (Auth.) - Fluoride Toxicity in Animals-Springer International Publishing (2015)

SPRINGER BRIEFS IN ANIMAL SCIENCES
Rakesh Ranjan
Amita Ranjan
Fluoride Toxicity
in Animals
SpringerBriefs in Animal Sciences
More information about this series at http://www.springer.com/series/10153
Rakesh Ranjan · Amita Ranjan
Fluoride Toxicity
in Animals
13
Rakesh Ranjan Amita Ranjan
National Research Centre on Camel Department of Veterinary Pharmacology
Bikaner, Rajasthan and Toxicology
India College of Veterinary and Animal
Sciences, Navania
Rajasthan University of Veterinary
and Animal Sciences
Bikaner, Rajasthan
India
ISSN 2211-7504 ISSN 2211-7512 (electronic)

SpringerBriefs in Animal Sciences
ISBN 978-3-319-17511-9 ISBN 978-3-319-17512-6 (eBook)
DOI 10.1007/978-3-319-17512-6
Library of Congress Control Number: 2015936363
Springer Cham Heidelberg New York Dordrecht London

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Preface
It is our great pleasure to present the book, Fluoride Toxicity in Animals. Animals
living in areas where fluorosis is endemic in the human population invariably s uffer
from the toxic effects of excess fluoride intake. Nevertheless, there has been lim-
ited research on fluorosis in animals whereas fluorosis in the human population has
received more attention from biologists, environmental scientists, and management
authorities worldwide.
This book has been written for higher undergraduate and graduate students of
toxicology, veterinary science, animal nutrition, environmental science, public
health workers, animal welfare activists, public health veterinarians, medical pro-
fessionals, and all others interested in the subject. A brief account of physical and
chemical properties of fluorine and different fluoride compounds is given along
with their relative significance in fluoride toxicity. Important natural and anthro-
pogenic sources of fluoride toxicity in animals are described to help identify the
problem. Basic features of fluoride absorption, distribution, metabolism, reten-
tion, excretion, and fluoride tolerance of different animal species are given in brief.
Methods for sample collection, preservation, and fluoride analysis in biological
and environmental samples are described. Important aspects of mitigation and pre-
vention of fluorosis in animals are given in Chap. 7 to help animal health workers
and management authorities.
No book can be claimed to be perfect and complete in all aspects. The scope
of improvement is always left. We sincerely look forward to readers for critical
suggestions.
We express our gratitude to our colleagues, officers, students, scientists, and
teachers for their valuable support. Thanks to Dr. D. Swarup and Dr. R.C. Patra
for providing the opportunity to work in the field of fluoride toxicity under their
guidance.
In addition we want to thank Lars Koener, Ursula Gramm, Amit Cyril Tirkey
and reviewers of the book for their support and suggestions in publishing this book
in a nice shape.
v
vi Preface
We also express thanks to our parents, family members, and friends, as without
their whole-hearted support it would have been impossible to complete this
manuscript.
Rakesh Ranjan
Amita Ranjan
Contents
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Fluorine Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Physical and Chemical Properties . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Distribution of Fluorides . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Is Fluoride Essential for Health? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Fluoride and Human Health. . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.2 Fluoride and Animal Health. . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Fluoride Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.1 Fluorosis in the Human Population. . . . . . . . . . . . . . . . . . . 5
1.3.2 Fluorosis in Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2 Sources of Fluoride Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.1 Natural Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.1 Forage, Grasses, and Grains. . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.2 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1.3 Volcanic Activities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2 Anthropogenic Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.1 Mineral Mixture and Other Feed Supplements . . . . . . . . . . 15
2.2.2 Airborne Fluoride. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2.3 Industrial Effluents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2.4 Agrochemicals and Household Products. . . . . . . . . . . . . . . 17
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3 Fluoride Kinetics and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.1 Absorption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.1 Gastrointestinal Tract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1.2 Respiratory Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1.3 Dermal and Other Routes. . . . . . . . . . . . . . . . . . . . . . . . . . . 24
vii
viii Contents
3.2 Distribution and Retention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3.2.1 Transplacental Passage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.2 Cerebrospinal Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.3 Skeleton and Other Calcified Tissues. . . . . . . . . . . . . . . . . . 26
3.2.4 Teeth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2.5 Exoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2.6 Hair and Fingernails. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2.7 Soft Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2.8 Egg. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3 Elimination and Excretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3.1 Urine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3.2 Feces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3.3 Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3.4 Perspiration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3.5 Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4 Toxic Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.1 Acute Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.2 Chronic Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2.1 General Health Effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2.2 Effects on Calcified Tissues. . . . . . . . . . . . . . . . . . . . . . . . . 37
4.2.3 Effects on Soft Tissues (Nonskeletal, Nondental Effects). . . 42
4.3 Molecular Mechanism of Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . 46
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5 Fluoride Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5.1 Fluoride Tolerance in Different Animal Species . . . . . . . . . . . . . . . . 54
5.1.1 Laboratory Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.1.2 Domestic Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.1.3 Wild Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5.1.4 Poultry and Other Birds. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
5.1.5 Insects and Other Invertebrates . . . . . . . . . . . . . . . . . . . . . . 59
5.1.6 Fish and Other Aquatic Animals . . . . . . . . . . . . . . . . . . . . . 59
5.2 Factors Affecting Fluoride Tolerance. . . . . . . . . . . . . . . . . . . . . . . . . 60
5.2.1 Animal Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.2.2 Dietary and Nutritional Factors. . . . . . . . . . . . . . . . . . . . . . 61
5.2.3 Chemical Form of Fluoride . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.2.4 Dose, Duration, and Continuity of Fluoride Intake. . . . . . . 63
5.2.5 Environmental and Other Factors. . . . . . . . . . . . . . . . . . . . . 64
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6 Fluoride Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.1 Titrimetry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.2 Colorimetric/Spectrophotometric Methods. . . . . . . . . . . . . . . . . . . . 70
Contents ix
6.3 Gas Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

6.4 Neutron or Proton Activation Technique. . . . . . . . . . . . . . . . . . . . . . 70
6.5 Potentiometric Analysis/Ion Selective Electrode (ISE) Method . . . . 71
6.5.1 Working of Ion Selective Electrode. . . . . . . . . . . . . . . . . . . 71
6.5.2 Factors Affecting Performance of ISE. . . . . . . . . . . . . . . . . 72
6.5.3 Sample Collection and Preservation. . . . . . . . . . . . . . . . . . . 73
6.5.4 Electrode Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
6.5.5 Checking Electrode Operation. . . . . . . . . . . . . . . . . . . . . . . 75
6.5.6 Preparation of Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.5.7 Analytical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.5.8 Fluoride in Acid Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . 77
6.5.9 Fluoride in Alkaline Solution. . . . . . . . . . . . . . . . . . . . . . . . 77
6.5.10 Points to Remember During Fluoride Analysis. . . . . . . . . . 78
6.5.11 Total Ionic Strength Adjustment Buffer (TISAB) . . . . . . . . 78
6.5.12 Electrode Filling Solution . . . . . . . . . . . . . . . . . . . . . . . . . . 79
6.5.13 Storage of Ion Selective Electrode. . . . . . . . . . . . . . . . . . . . 80
6.5.14 Fluoride in Aqueous Samples. . . . . . . . . . . . . . . . . . . . . . . . 80
6.5.15 Fluoride in Soft and Calcified Tissues. . . . . . . . . . . . . . . . . 81
6.5.16 Fluoride in Vegetation and Fodder Samples. . . . . . . . . . . . . 81
6.5.17 Fluoride in Soil, Feed, and Mineral Mixture. . . . . . . . . . . . 82
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
7 Mitigation and Prevention of Fluorosis. . . . . . . . . . . . . . . . . . . . . . . . . . 85

7.1 Minimizing/Withdrawing Excess Fluoride Intake. . . . . . . . . . . . . . . 86
7.1.1 Search for Safe Groundwater Source. . . . . . . . . . . . . . . . . . 86
7.1.2 Use of Surface Water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
7.1.3 Rainwater Harvesting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
7.1.4 Water Defluoridation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.1.5 Precipitation-Based Techniques. . . . . . . . . . . . . . . . . . . . . . 89
7.1.6 Adsorption and Ion-Exchange-Based Techniques. . . . . . . . 90
7.1.7 Reverse-Osmosis-Based Techniques. . . . . . . . . . . . . . . . . . 91
7.1.8 Distillation-Based Techniques. . . . . . . . . . . . . . . . . . . . . . . 91
7.1.9 Electrocoagulation/Electrolysis-Based Techniques. . . . . . . 91
7.2 Preventive and Therapeutic Measures . . . . . . . . . . . . . . . . . . . . . . . . 91
7.2.1 Minerals, Drugs, and Other Chemicals . . . . . . . . . . . . . . . . 91
7.2.2 Vitamins and Antioxidants. . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.2.3 Plant Products/Herbal Medicines. . . . . . . . . . . . . . . . . . . . . 93
7.3 Minimizing Industrial Fluoride Emissions. . . . . . . . . . . . . . . . . . . . . 96
7.4 Generating Public Awareness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
About the Authors
Dr. Rakesh Ranjan Ph.D. is presently working as Senior Scientist with ICAR-Na-
tional Research Centre on Camel, Bikaner, Rajasthan, India. Earlier, he has served
College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences
University, Ludhiana, India for about 9 years as assistant professor in the depart-
ment of Veterinary Medicine. He is working on animal fluorosis for the past 11 years.
Dr. Ranjan currently serves on the editorial board of Environmental Pollution, published
by Canadian Center for Science and Education, Toronto, Canada and Indian Journal
of Veterinary Medicine, published by Indian Society for Veterinary Medicine. He is
life member of International Society of Fluoride Research (ISFR), New Zealand and
has presented his research highlights on fluoride toxicity in animals in several national
and international conferences including conferences of ISFR, held at Toronto Canada
(2008) and Szczecin, Poland (2012). He was selected for Raman Fellowship, UGC,
Government of India for postdoctoral research in USA. He has published more than 60
research and extension papers and one book chapter. He has been awarded with Intas
best review article award, ISVM appreciation award and best oral and poster presenta-
tion awards in several international and national scientific conferences.
Dr. Amita Ranjan Ph.D. is presently working as assistant professor (Veterinary
Pharmacology and Toxicology), College of Veterinary and Animal Sciences, N avania,
under University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India. Prior
to that, she was research associate in Department of Teaching Veterinary Clinical
Complex, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana,
Punjab, India, where she worked on toxicity testing of synthesized nanoparticles.
Toxicity of agrochemicals and other environmental pollutants in animals are areas
of her research interest. She is a life member of Indian Society for Veterinary
Pharmacology and Toxicology. She is recipient of award of honour from chief minis-
ter, for securing second position in the Bihar state in matriculation examination. She
received Radha Harihar Prasad memorial gold medal during graduation and senior
research fellowship, Indian Council for Agricultural Research, Government of India
during Ph.D. She has published several research papers in different international and
national journals of repute and authored one book on pet care and management.
xi
Chapter 1
Introduction
Abstract Fluorine is a highly reactive halogen element that can form compounds
with most of the elements except oxygen, nitrogen, and noble gases such as helium,
neon, and argon. It is believed that fluorine in small quantities is required for the
maintenance of health in humans and animals, although conclusive evidence is
lacking. High fluoride intake is toxic for humans and animals. Wild and domestic
herbivores appear highly susceptible, although natural cases of toxicity have been
reported in a wide range of terrestrial invertebrates and vertebrates. Aquatic inverte-
brates and fish are also susceptible to high doses of fluoride exposure. Characteristic
lesions in bone and teeth appear frequently in spontaneous cases of chronic toxicity
in domestic and wild ruminants. Fluoride toxicity in the human population has been
widely investigated, but limited reports on fluorosis in animals are available. Animals
living in areas where fluorosis is endemic in the human population invariably suffer,
although the susceptibility varies with species. Therefore, more systematic studies on
animal fluorosis should be undertaken to safeguard animal health and welfare.
Fluorine is a highly electronegative and reactive halogen element belonging to

Group 7A (VIIA) of the periodic table. It was first synthesized in elemental form
in the year 1886 by the French scientist Henri Moissan. There is one stable isotope
of fluorine with atomic number 9 and atomic weight 18.9984. It exists as a dia-
tomic molecule F2 in its elemental form. There are several radioactive isotopes of
fluorine with atomic weight 17, 18, 20, 21, and 22. The isotope 22F has the longest
half-life of 109.7 min (Weast 1986).
Fluoride is the ionic form of fluorine. In biological science the terms “fluo-
rine” and “fluoride” are often used interchangeably. Fluorine, being the most reac-
tive nonmetal, almost never exists in nature in an elemental state. It reacts with
electropositive elements to form fluoride. Although several chemical compounds
containing fluorine either exist naturally or have been synthesized for indus-
trial, agricultural, or domestic purposes, hydrogen fluoride gas, fluorosilicic acid,
sodium silicofluoride, and sodium fluoride are major compounds responsible for
fluoride toxicity in animals.
© The Author(s) 2015 1

R. Ranjan and A. Ranjan, Fluoride Toxicity in Animals,
SpringerBriefs in Animal Sciences, DOI 10.1007/978-3-319-17512-6_1
2 1 Introduction
1.1 Fluorine Chemistry
Before discussing the role of fluorine in the maintenance of health or production

of toxic effects, it is pertinent to mention physical and chemical properties of
important fluoride compounds and their distribution in the environment. Physical
and chemical properties of fluorine compounds have a great influence on fluorine
uptake, metabolism, excretion, and biological effects.
1.1.1 Physical and Chemical Properties
At room temperature elemental fluorine is a pale yellow-green gas with a char-

acteristic pungent odor. Fluorine can react vigorously with most of the elements
except oxygen, nitrogen, and the lighter noble gases such as helium, neon, and
argon (Banks and Goldwhite 1966). It reacts with hydrogen to form hydrogen flu-
oride (CAS No. 7664-39-3), which is a colorless corrosive gas or liquid with boil-
ing point 19.5 °C and vapor pressure more than 1 atmosphere. The odor detectable
limits are 0.033–0.1333 mg/m3, and the irritating concentration is 4.17 mg/m3.
Hydrogen fluoride fumes readily dissolve in water to form hydrofluoric acid which
can etch glass. Hydrogen fluoride, both in gaseous and liquid form, is highly irri-
tating and can cause severe burns upon contact with skin and mucous membranes.
Hydrogen fluoride has multitudinous industrial applications. The highest con-
sumption of hydrogen fluoride is in the synthesis of fluorocarbons, which are used
as refrigerants, solvents, and aerosols. Other important industrial applications of
fluoride compounds include sodium fluoride, fluorosilicic acid, and sodium fluo-
rosilicate. Sodium fluoride (CAS No. 7681-49-4) is an odorless white powder or
colorless crystals with a water solubility of 4 % at 15 °C and a pH (saturated solu-
tion) 7.4. Fluorosilicic acid is used primarily in water fluoridation, either directly
or after processing into sodium silicofluoride. Fluorosilicic acid is also used in the
aluminum industry for the synthesis of aluminum fluoride.
1.1.2 Distribution of Fluorides
Fluorine compounds are widely distributed and rank thirteenth among the ele-
ments in the order of abundance in the earth’s crust. The chemical formula and
fluorine concentration in important fluoride minerals are given in Table 1.1.
In water, inorganic fluorides (F) usually remain in ionic form under conditions of
relatively low pH and hardness and in the presence of ion-exchange materials such
as bentonite clays and humic acids. In unpolluted freshwater, fluoride concentration
usually varies from 0.01 to 0.3 mg/L, whereas in unpolluted seawater, it varies
from 1.2 to 1.5 mg/L (Camargo 2003). However, wide variations in groundwater
1.1 Fluorine Chemistry 3
Table 1.1 Chemical formula Mineral Chemical formula Fluorine concentration (%)

and fluorine concentration in
Sellaite MgF2 61
natural fluoride minerals
Villiaumite NaF 55
Fluorspar CaF2 49
Cryolite Na3AlF6 45
Bastnaesite (Ce, LaY)(CO3)F 9
Fluorapatite Ca5(PO4)3F 3.5
fluoride concentration may occur naturally without any human intervention, depend-
ing upon geological, chemical, and physical characteristics of the water-supplying
area, consistency of the soil, porosity of rocks, pH, temperature, complexing action
of other elements, and the depth of wells (WHO 2002).
Fluoride concentration in the atmosphere in unpolluted areas usually varies
between 0.02 and 2.0 µg/m3 (USEPA 1980). Atmospheric fluoride may be in gas-
eous or particulate forms. Gaseous forms include hydrogen fluoride, sulfur hex-
afluoride, silicon tetrafluoride, and hexafluorosilicic acid. Particulate forms include
sodium aluminum fluoride, calcium phosphate fluoride, sodium hexafluorosilicate,
aluminum fluoride, calcium fluoride, and lead fluoride. Hydrogen fluoride and
inorganic fluoride particulates (sodium and calcium fluoride) are major inorganic
fluorides present in the atmosphere, accounting for nearly 75 and 25 %, respec-
tively (WHO 2002).
1.2 Is Fluoride Essential for Health?
Whether fluoride is essential for maintenance of health is an issue of much

debate. An expert World Health Organization committee included fluorine in the
list of 14 elements that are essential for life (WHO 1973). The American Dietetic
Association considers that fluorine is an important element for all mineralized tis-
sues in the body (Palmer and Wolfe 2005). The Federal Register of the US Food
and Drug Administration also declared fluorine as an essential nutrient for human
health. However, it is almost impossible to provide experimental evidence support-
ing the essentiality of fluoride for maintenance of human and animal health. The
Food and Nutrition Board of the National Research Council (NRC) has therefore,
withdrawn the term “essential” and is now using terms “beneficial element” (BE)
and “apparent beneficial intake” (ABI) for fluoride.
1.2.1 Fluoride and Human Health
Fluoride is required for mineralization of bone and teeth, maintenance of fertil-

ity, hematopoiesis, and activation of certain enzymes, such as adenylate cyclase,
4 1 Introduction
acid and alkaline phosphatases, and isocitrate dehydrogenase (Kirck 1991). In

vitro studies suggest that fluorine helps in activities of histone methyltransferase,
stabilizes the interaction between guanosine triphosphatase (GTPase) and GTPase-
activating proteins, and affects the posttranslational assembly of glycosaminogly-
can chains in mineralizing bone cells (Kirck 1991).
It is widely accepted that a low level of fluoride intake decreases suscepti-
bility to dental caries in human beings (Treasure and Dever 1992). Anticaries
action of the fluoride is mediated by its incorporation into the tooth enamel as
fluor(hydroxyl) apatite which has high levels of fluoride and low levels of carbon-
ate and high acid resistance. In addition, fluoride can inhibit bacterial metabolism
of carbohydrates, hence it decreases production of acids (Bowden 1990). A few
decades back, use of fluorinated toothpaste for prevention of dental caries was a
great vogue, but soon it became obsolete due to increasing reports of ill effects
on oral health such as the occurrence of perioral dermatitis (Mellete et al. 1983;
McCaffery 2003). Daily intake of 1–3 mg F/kg body weight has a potential effect
on dental caries prevention over a short time span, however, prolonged intake of
equal doses may have deleterious health effects. Earlier, fluoride was also used
in the treatment of osteoporosis, although the therapeutic window was known
to be narrow (Dequeker and Declerck 1993). Some investigators even reported
increased risk of fracture in osteoporotic women treated with fluoride (Hedlund
and Gallagher 1989; Kurttio et al. 1999). Therefore, fluoride is not used at present
for prevention and treatment of osteofluorosis (Danielson 1992).
1.2.2 Fluoride and Animal Health
Many biologists doubt the essentiality of fluoride for maintenance of animal

health. Perhaps the fluoride requirement is so small that deficiency is never
observed spontaneously or could hardly be produced in natural and/or experimen-
tal conditions (Wheeler and Fell 1983; Alberts 1998). In a study on the water flea
(Daphnia magna), it was observed that the growth rate is enhanced at extremely
low concentrations of <0.007 mg F/L water and the authors concluded that the
essential F level for the flea is 0.004 mg F/L, which is lower than the level found
anywhere in the natural environment (Dave 1984).
Attempts to demonstrate fluoride deficiency disorders in experimental and labo-
ratory animals, by and large, failed despite several decades of research. In a study,
mice fed low F diets developed anemia and impaired reproduction (Messer et al.
1972, 1973). However, later on, it was found that anemia and impaired reproduc-
tion were due to low dietary iron and no such symptoms developed when mice
were fed a diet containing low fluoride and sufficient iron (Tao and Suttie 1976).
Another study reported skeletal abnormalities in female goats and poor growth in
their offspring when they were fed a diet containing less than 0.3 ppm fluoride (on
a dry matter basis) for 10 generations (Anke et al. 1997). However, no further study
documenting similar findings appeared in the literature. Some experimental studies
1.2 Is Fluoride Essential for Health? 5
documented the beneficial effects of fluoride supplementation on animal health

and performance. For example, an increase in growth rate of broiler chickens
was recorded after supplementation of 80 µg F/g diet (Gutierrez et al. 1993).
Nevertheless, evidence arising from scientific studies to date has not been sufficient
to conclude that fluoride is essential for maintenance of animal health and produc-
tion; rather its toxic effects appear more important.
1.3 Fluoride Toxicity
Large doses of fluoride exert toxic effects in almost all living creatures. Fluoride
is a general tissue poison, even more poisonous than lead and just slightly less
poisonous than arsenic. The toxic potential of fluoride can be very well assessed
by the fact that a single dose of 5–10 mg/kg body weight causes acute toxic effects
and death may occur following a single oral fluoride intake at the rate of 16 mg/kg
body weight (WHO 2000).
Depending upon the quantity of intake and the chemical form of the fluo-
ride compound, fluoride toxicity can occur in acute, subacute, and chronic form.
Acute fluoride toxicity, although rare in occurrence, results mostly after accidental
ingestion of large doses of fluorine compounds, such as sodium fluoroacetate and
fluoroacetamide used as a rodenticide, sodium fluorosilicate used as an insecticide,
and sodium fluoride used as an acaricide. Because of the decrease in use of these
fluoride compounds for various household and agricultural purposes, acute fluo-
ride poisoning is now seldom reported. Prolonged intake of low but toxic doses of
fluoride compounds induces chronic fluoride toxicity, often referred as “fluorosis”.
Chronic fluoride toxicity is more common and important for human and domestic
animals and is often characterized by pathological changes in teeth (dental fluoro-
sis) and bone (osteofluorosis). Ingestion of fluoride compounds (through food and
water) appears the major route of fluoride uptake. When drinking water contains
excess fluoride and serves as a major source of excess fluoride intake, the chronic
fluoride toxicity produced is referred as “hydrofluorosis”. Water fluoride concen-
trations above 1.5 ppm, have been reported to cause osteo and dental fluorosis in
a wide range of domestic animals including cattle (Bos taurus), buffalo (Bubalus
bubalis), horse (Equus caballus), donkey (Equus asinus), dromedary camel
(Camelus dromedarius), sheep (Ovis aries), and goat (Capra hircus) (Ranjan et al.
2009; Choubisa 2013, 2014).
1.3.1 Fluorosis in the Human Population
Naturally occurring high fluoride concentration in drinking water is the most

important cause of fluorosis in the human population. Over 200 million people
throughout the globe are estimated to consume water with fluoride concentration
6 1 Introduction
above the safe level, that is, 1.5 mg/L (Fig. 1.1; Amini et al. 2008). UNICEF
identified 27 countries where the fluoride concentration in drinking water is high
enough to cause fluorosis in humans and livestock. The list includes Algeria,
Argentina, Australia, Bangladesh, China, Egypt, Ethiopia, India, Iran, Iraq, Japan,
Jordan, Kenya, Libya, Mexico, Morocco, New Zealand, Pakistan, Palestine,
Senegal, Sri Lanka, Syria, Tanzania, Thailand, Turkey, Uganda, and United Arab
Emirates. The list appears incomplete as water fluoride concentration exceeding
1.5 mg/L also occurs naturally in Canada, the United States, Ghana, England, and
Norway (Weinstein and Davison 2004). There are two well-known high fluoride
belts in the world: one extends along the East African Rift from Eritrea to Malawi
and another belt starts from Turkey and extends up to Iraq, Iran, Afghanistan,
India, northern Thailand, and China (Ayoob and Gupta 2006). The Americas,
China, and Japan also have similar belts. In China, over 26 million people were
reported to suffer from fluorosis. Coal burning for domestic cooking has been
identified as an important cause of endemic fluorosis in the human population in
China (Guijian et al. 2007). Some other sources of fluoride uptake include tea,
processed food, fluoridated drinking water, and fluoridated toothpaste, gels, and
mouthwash. In Mexico, 5 million people (about 6 % of the total population) are
affected by hydrofluorosis (UNICEF statement on fluoride). Poland, Finland,
Czech Republic, and several African countries including Ivory Coast, Senegal,
North Algeria, Uganda, Ethiopia, as well as Northern Mexico and Central
Argentina also have naturally elevated levels of fluoride in drinking water.
Fig. 1.1 World map showing countries where fluoride concentration in potable water is above
1.5 ppm. (With kind permission from C. Annette Johnson, Eawag, Swiss-Federal Institute of
Aquatic Science and Technology, Dubendorf, Switzerland)
1.3 Fluoride Toxicity 7
1.3.2 Fluorosis in Animals
Almost all terrestrial and aquatic animals are susceptible to high doses of fluoride,
although the tolerance level varies from one species to another. For terrestrial ani-
mals, important sources of excess fluoride intake include drinking water, soil, or
vegetation naturally containing excess soluble fluoride compounds or contaminated
with fluoride compounds emitted by volcanic eruptions or industrial activities.
Many invertebrates are highly susceptible to fluoride toxicity. Several fluoride
compounds including sodium fluoride, cryolite, and sodium fluorosilicates were
widely marketed and used as insecticides and/or pesticides during the nineteenth cen-
tury. Sodium fluoride was patented for use as a pesticide in the year 1896. However,
use of these compounds gradually declined after discovery of more effective syn-
thetic compounds and due to the risk of accidental acute poisoning in humans and
domestic animals. Honeybees and silkworm larvae are highly susceptible to fluoride
toxicity; the silk industry in several countries has been badly affected by industrial
fluoride pollution (Bourbon 1967; Weinstein and Davison 2004). Industrial fluoride
emissions may have great influence over changes in insect populations or behavior in
nearby areas. However, it is difficult to conclude that these effects are solely attribut-
able to fluoride as other pollutants released simultaneously by industries such as sul-
phur dioxide, nitrogen oxides, and heavy metals are also toxic to invertebrates.
Aquatic invertebrates and vertebrates including fish are also susceptible to
fluoride toxicity. Net-spinning caddisfly larvae and upstream-migrating salmon
are highly sensitive to water fluoride concentration. High fluoride concentration
adversely affects behavior, growth, and reproduction in fish, although typical bony
changes as observed in terrestrial mammals do not appear. In general, freshwater-
or softwater-dwelling aquatic animals have lower fluoride tolerance than marine or
hardwater-dwelling animals (Camargo 2003).
Among terrestrial vertebrates, herbivores are more susceptible than carnivores
and other animals occupying an upper position in the food pyramid. Domestic
and wild herbivores are more exposed to environmental contaminants as they are
nonselective eaters and can consume contaminated feed, forage, and water. Poor
quality mineral mixture and feed supplements may also cause sporadic outbreaks
of fluorosis in organized livestock farms. It is interesting to note that the oldest
known spontaneous cases of fluoride toxicity were recorded in domestic animals,
when outbreaks of lameness and mortality were observed in sheep after a vol-
canic eruption in Iceland in the year 1895. It took several decades to identify that
the underlying cause of mortality in the sheep was excess fluoride present in the
volcanic ash that contaminated forage in the pastureland. In a collection of bones
and teeth of sheep that died during that period, Roholm (1937) recognized gross
changes that were typical of fluorosis and observed very high fluoride concentra-
tion in the bones. The early description of the incidence of fluorosis in animals in
the last century was published in 1912 from Italy as a disease in cattle resembling
osteomalacia (Swarup and Dwivedi 2002). The disease was suspected to be caused
by fluoride-rich waste released by a superphosphate fertilizer factory.
8 1 Introduction
The occurrence of fluorosis in wild and domestic animals has been reported
from several countries across the globe (Ranjan 2007). Cattle and sheep have
attracted more attention of researchers worldwide, perhaps due to their large popu-
lation and greater economic importance, however, other animals including water
buffaloes, horses, goats, pigs, and wild cervids also suffer from the toxicity prob-
lem naturally. It appears that animals living in areas where fluorosis is endemic
in the human population are also at risk of developing fluorosis. Unfortunately,
fluorosis in animals has not thus far been given due importance by researchers and
management authorities worldwide. Hence, there is a wide scope and urgent need
to investigate toxic effects of excess fluoride in animals and develop strategies for
prevention and remediation.
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CA (2008) Statistical modeling of global geogenic fluoride contamination in groundwaters.
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Anke M, Gurtler H, Glei M, Anke S, Jaritz M, Freytag H, Shafter V (1997) Effects of fluoride
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Proceedings of the ninth symposium on trace elements in man and animals. NRC Press,
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Ayoob S, Gupta AK (2006) Fluoride in drinking water: a review on the status and stress effects.
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Bowden GH (1990) Effects of fluoride on the microbial ecology of dental plaque. J Dent Res
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Choubisa SL (2014) Bovine calves as ideal bio-indicators for fluoridated drinking water and
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Danielson C (1992) Hip fractures and fluoridation in Utah’s elderly population. J A Med Assoc
268:746–748
Dave G (1984) Effects of fluoride on growth, reproduction and survival in Daphnia magna.
Comp Biochem Physiol C 78:425–431
Dequeker J, Declerck K (1993) Flouride in the treatment of osteoporosis: an overview of thirty
years clinical research. Schweizerische Medizinische Wochenschrift, J Suisse de Medicine
123:2228–2234
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of arsenic, fluorine, and selenium from indoor burning of Chinese coal. Rev Environ Contam
Toxicol 189:89–106
Gutierrez O, Marrero AI, Terry I, Cairo J (1993) Utilization of fluoride as growth promoter in
broiler rations: performance measurements. Cuban J Agric Sci 27:319–323
Hedlund LR, Gallagher JC (1989) Increased risk of hip fracture in osteoporotic women treated
with sodium fluoride. J Bone Miner Res 4:223–225
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Kirck KL (1991) Biochemistry of the elemental halogens and inorganic halides. Plenum, New
York, pp 19–68
Kurttio P, Gustavsson N, Vartiainen T, Pekkanen J (1999) Exposure to natural fluoride in well
water and hip fracture: a cohort analysis in Finland. Am J Epidemiol 150:817–824
McCaffery K (2003) Fluoride and dermatitis. J Am Dent Assoc 134:1166
Mellete JR, Aeling JL, Nuss DD (1983) Perioral dermatitis. J Assoc Military Dermatol 9:3–8
Messer HH, Armstrong WD, Singer L (1972) Fertility impairment in mice on a low fluoride
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mice. J Nutr 103:1319–1326
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on health. J Am Diet Assoc 105:1620–1628
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mitted to Indian Veterinary Research Institute, Izatnagar, India
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tle. Indian J Anim Sci 79:546–549
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and some experimental investigations. HK Lewis Ltd, London, pp 141–143
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mal health. Indian Council of Agricultural Research, Pusa, New Delhi
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Chapter 2
Sources of Fluoride Toxicity
Abstract Sources of excess fluoride intake for animals are diverse and include
drinking water, fluoride compounds used for household and agricultural purposes,
forage and grasses contaminated with industrial fluoride emissions or volcanic
ash, and occasionally, poor quality mineral mixture and feed supplements. Soil
rich in soluble fluoride may also be responsible for fluorosis in grazing animals,
particularly when growing vegetation is small and scanty. Toxicity arising due to
airborne fluoride is rare and oral intake remains the major route of excess fluoride
uptake. A water fluoride level as low as 1.5 ppm can cause chronic fluoride toxic-
ity in several species, although the literature suggests higher water fluoride toler-
ance levels in most domestic animals. Volcanic-ash–contaminated pasture has been
reported to cause mortality outbreaks in grazing animals in several countries.
Fluoride (F) toxicity can arise due to excessive fluoride intake from a variety of
natural or manmade sources. Important F sources for animals include vegetation/
forage contaminated by fluoride-rich industrial effluents or windblown or rain-
splashed soil with high fluoride content, water high in fluoride content either
naturally or due to industrial contamination, mineral mixtures and other feed
supplements containing excess fluoride, vegetation grown on soils high in fluo-
ride content, and a combination thereof (Shupe 1980; Swarup and Dwivedi 2002;
Fig. 2.1). Fluoride is well absorbed by several routes, however, ingestion is the
major mode of fluoride uptake for most animals (Shupe 1980). Soluble fluoride is
distributed over the earth’s surface and atmosphere as a result of natural processes
such as erosion, hydraulic leaching, volcanic activity, and to a lesser extent by
mining and manufacturing processes. Animals when grazing over soluble fluoride-
rich soil can ingest toxic doses of fluoride, especially if the pasture is overgrazed
and animals are grazing small plants close to the soil (Shupe and Olson 1971). In
this chapter, important sources of acute and chronic fluoride toxicity for animals
are described in brief.

12 2 Sources of Fluoride Toxicity
Water
(drinking)
Feed supplements Vegetation

(Bone & fish meal) (grazing)
Animals
Volcanic ash and

Mineral mixture
gases
Industrial F Agrochemicals
emission (gaseous
and particulates) Containing fluoride
Fig. 2.1 Sources of fluoride toxicity in animals
2.1 Natural Sources
Fluorosis in animals can occur due to high fluoride concentration naturally occurring
in dietary substances including feed, fodder, concentrate ration, and drinking water.
2.1.1 Forage, Grasses, and Grains
Soil fluoride concentration is believed to have little influence over fluoride concen-
tration in vegetation, inasmuch as most of the fluoride in soils cannot be assimi-
lated readily by plants. However, a direct relation is reported to exist between F
concentration in the soil and selected forage. Fluoride in forage increases by 3 ppm
for each 100 ppm increase in soil F up to approximately 2200 ppm (Mascola et al.
1974). Other factors modulating fluoride concentration in fodder plants include
height of the plant and season. Fluoride content increases with decrease in plant
height. The fluoride concentration is higher in plants grown during winter and
spring in comparison to those grown during summer and autumn (Mascola et al.
1974). Fluoride uptake by plant roots from soil occurs by passive diffusion, and
2.1 Natural Sources 13
thereafter F is carried to the shoot by transpiration. Most of the plants do not

accumulate fluoride in toxic concentrations and the level usually remains below
10 mg per kg dry weight. The tea plants (family Theaceae) can exceptionally accu-
mulate high F concentrations. However, tea plants or their by-products are not a
usual ingredient of animal feed. Many plant species, particularly Acacia georgi-
nae and Dichapetalum cymosum (a South African shrub known as “Gifblaar”) can
assimilate soil F and convert them into fluoroacetate, which is extremely toxic
for animals (Shupe et al. 1984). Fluoroaceate is converted in vivo in mitochon-
dria into fluorocitrate through condensation of fluoroacetyl-Co-A with oxaloac-
etate by the enzyme citrate synthetase which normally supplies acetyl-Co-A
into the citric acid cycle. Fluorocitrate is a strong inhibitor of aconitase, which is
an important enzyme of the Krebs cycle. Thus fluorocitrate inhibits the biochemi-
cal pathway for energy production in the organism (Patocka and Strunecka 2002).
It is interesting to note that gaseous fluoride compounds can enter leaves
through stomatal pores and in high concentration they may induce toxicity signs in
plants, including chlorosis, peripheral necrosis, leaf distortion and malformation,
and abnormal fruit development. Long-term exposure to F concentrations greater
than 0.2 μg/m3 may cause injury to plants (WHO 1984). It has been observed that
air-contaminated forage loses a portion of its fluorine to the atmosphere after cut-
ting and during storage as hay (NRC 1960).
The mean fluoride concentration of mineral soils is around 0.2–0.3 g/kg, whereas
that of organic soil is usually lower. The fluoride content of topsoil increases by
the addition of fluoride containing phosphate fertilizers, pesticides, and irriga-
tion water or by deposition of gaseous and particulate emissions from industry. In
New Zealand, it was found that long-term use of phosphorus fertilizers resulted in
an increase in total surface soil (0–75 mm depth) fluoride concentration up to 217–
454 mg per kg (Loganathan et al. 2001). Fluoride uptake by grazing animals through
plant consumption is expected to be much lower than F uptake directly by soil
ingestion because most plants do not accumulate F. Therefore, reducing soil inges-
tion by maintaining good pasture cover can reduce the risk of fluorosis in herbivores
(Loganathan et al. 2008). Forage and grasses grown in industrial areas are often con-
taminated by fluoride-rich industrial effluents or by windblown or rain-splashed soil
having a high fluoride concentration. Most industrial fluoride emissions are airborne
and fluoride-rich dust, ash, and fumes may contaminate soil, water, and vegetation
not only in the industrial vicinity, but also up to a considerable distance from the
source of emission (Radostits et al. 2000). In such conditions F concentration in
plants depends upon (1) the amount of fluorine released into the atmosphere, (2) the
distance between the F emission source and the pasture area, (3) the type of vegeta-
tion, and (4) the distribution pattern as affected by the wind and topography (NRC
1960). Plants grown in areas where F is emitted into the atmosphere may contain
500–1000 ppm fluoride. Hence, animals should not be allowed to graze in pasture
close to industries such as brick-kiln plants, rock phosphate processing plants, alu-
minum industry, and so on. Fodder plants grown in polluted areas should be har-
vested, soaked, and washed with water before offering to animals. This practice will
substantially reduce the available F to animals.
Grains usually do not contain toxic concentrations of fluoride. Corn, wheat, oats,
and barley grown on F-rich soil have very low F concentrations (less than 10 ppm)
and nearly equal those grown on soil low in F (NRC 1960). Nevertheless, sorghum
(Sorghum bicolor) consumption in human beings has been found to increase the risk
and severity of osteo and dental fluorosis (Krishnamachari and Krishnaswamy 1973;
Hari-Kumar et al. 2007). Sorghum grown in fluorotic areas contains a high molybde-
num concentration. Molybdenum reduces urinary fluoride excretion and enhances flu-
oride retention (Stookey and Muhler 1962). A sorghum-based diet therefore increases
F retention in human beings (Lakshmaiah and Srikantia 1977) and rats (Lakshmi and
Lakshmaiah 1999). It is surprising to note that sorghum plants are more susceptible to
hydrogen fluoride gas exposure than wheat plants (Mac-Lean et al. 1984).
2.1.2 Water
The fluoride concentration of natural groundwater depends upon geological factors,

consistency of the soil, porosity of rocks, pH and temperature of the soil, complexing
action of other elements, depth of wells, leakage of shallow groundwater, and chemi-
cal and physical characteristics of water (Li et al. 2014). Both surface and groundwa-
ter may have high F concentration in a particular locality, but the level is often higher
in groundwater than the surface water. When groundwater percolates through rocks
containing fluoride-rich compounds, fluoride leaches out and concentration may
increase far above the safe level. The mean fluoride content of rocks varies between
0.1 and 1.0 g/kg. Important fluoride-containing minerals are fluorspar, cryolite, and
apatite. However, in most soils, F is associated with micas and other clay minerals
that make them less soluble and hence less toxic. Sodium fluoride and magnesium
fluoride are also found as natural minerals. Fluoride contamination occurs through
a natural process in which fluoride-bearing rocks crumble and break down, but the
process can be speeded up if the chemistry of the aquifer is disturbed. Climatic con-
ditions may also influence rate and degree of fluoride dissolution in water from rocks
and soil. High F concentration in shallow zone groundwater is largely due to the geo-
chemical F deposition in the vicinity of the groundwater extraction structures. The
toxicity potential of fluoride-rich water is also influenced by the ambient tempera-
ture; alkalinity; and calcium, copper, and magnesium concentrations in water.
Water having a fluoride concentration up to 1.0 mg/L is safe, levels in between
1.1 and 2.5 mg/L are marginally contaminated, whereas above 2.6 mg/L are con-
sidered highly contaminated and dangerous for drinking purposes for human beings
(Susheela 1999). It is estimated that total fluoride intake by a person when potable
water contains 1 ppm fluoride would be from 0.05 to 0.10 mg per kg of body weight
per day (NRC 1960). Most terrestrial animals, particularly domestic herbivores, are
supposed to tolerate F in water higher than these threshold levels. However, many
reports indicate the appearance of toxic symptoms in cattle, buffalo, and other live-
stock reared in areas where mean water fluoride concentration is equal to or slightly
above 1.5 ppm (Choubisa 1999; Maiti et al. 2003; Ranjan et al. 2009).
2.1 Natural Sources 15
2.1.3 Volcanic Activities
Domestic and wild animals often suffer from severe fluorosis by ingestion of water
and plants contaminated with volcanic ash (Araya et al. 1990; Bellomo et al. 2007).
Volcanic ash usually has a very high soluble fluoride concentration and can be depos-
ited over a large geographical area. In volcanic areas, the fluoride concentrations in
water and pasture grass may remain high for years even after cessation of volcanic
activities (Araya et al. 1993). The fluoride concentration in volcanic ash emitted by
the Hekla volcano, Iceland, was up to 2000 ppm and the forage covered by ash had
fluoride concentrations of 350–4300 µg/g (Thorarinsson 1979). In Chile in 1988, fluo-
ride-bearing ash arising from the Lonquimay volcano affected more than 10,000 farm
animals and resulted in the death of more than 4000 goats, sheep, cattle, and horses
(SEAN 1989). Likewise, the death of thousands of sheep in 1995–1996 was attrib-
uted to ashfall from the Ruapehu volcano eruptions in Mexico (Armienta et al. 2011).
In Argentina, the ash (“tephra”) released from the Puyehue–Cordon Caulle volcanic
eruption in June 2011 reached up to a distance of 1400 km. Severe fluorotic dental
lesions were observed in wild red deer who died after the volcanic eruption, which
was attributed to consumption of grasses contaminated with volcanic ash (Flueck and
Smith-Flueck 2013). In Iceland, thousands of sheep, cattle, horses, and other domestic
and wild animals died due to repeated volcanic eruptions over the years (Armienta
et al. 2011). Occasionally, acute toxicity in animals may occur after inhalation of
hydrogen fluoride released from volcanoes (Weinstein and Davison 2004).
2.2 Anthropogenic Sources
Anthropogenic sources are also responsible for fluoride toxicity in animals. Some
of them have been described in this chapter.
2.2.1 Mineral Mixture and Other Feed Supplements
There are several reports documenting mineral supplements as a major source of

fluoride toxicity in livestock (Griffith-Jones 1977; Hillman et al. 1979; Singh and
Swarup 1995). In a cattle herd numbering 120 animals, introduction of a com-
mercial salt lick with fluoride concentration 1400 mg per kg resulted in the devel-
opment of fluorotic lesions after one year. The fluoride concentrations in water
and pasture samples were within the normal range suggesting the salt lick as the
sole source of excess fluoride intake (Schultheiss and Godley 1995). In northern
Australia, up to 15 % of cattle in a herd revealed signs of lameness due to use of
fertilizer-grade monoammonium and diammonium phosphate as a mineral supple-
ment. The affected animals were given large quantities of the mineral supplement
thinking that the lameness was due to a phosphorus deficiency as it was an endemic
problem in that area (Jubb et al. 1993). Chronic ingestion of gypsum that was
included in a feed supplement and also ingested from fertilizer dumps in paddocks
resulted in the death of 226 cattle at a farm over a period of 18 months. Here, direct
toxicity of gypsum as well as the effect of excess fluoride was incriminated as the
cause of heavy mortality in the cattle herd (Bourke and Ottaway 1998).
The starting material for almost all chemically processed phosphates in mineral
mixture and animal feed supplements is rock phosphate which contains approxi-
mately 13–14 % phosphorus and 3–4 % fluoride. The rock phosphate is first con-
verted into phosphoric acid which is used for chemical synthesis of various types
of feed phosphates. Phosphoric acid is synthesized from rock phosphate either by
heating the rock phosphate in an electric furnace (dry process) or by treating the
rock phosphate with sulphuric acid (wet process). The phosphoric acid produced
by the electric furnace process has a very low F concentration and can be directly
used for chemical synthesis of feed phosphates. In phosphoric acid produced by
the dry heat process, the P:F ratio is usually greater than 2000:1 (Thompson 1980).
However, the energy requirement per unit of phosphorus produced is approximately
eight times greater in the dry process as compared to the wet process. Hence, the
cost of production of phosphoric acid by the dry process is almost double that of the
wet process. Wet process production methods therefore account for approximately
93 % of the total industrial production of phosphoric acid (Thompson 1980). The
phosphoric acid produced by the wet process should always be defluoridated before
use in feed phosphate synthesis. Defluoridation of phosphoric acid is done by addi-
tion of silicon dioxide and heat is supplied in the form of steam. The defluorida-
tion process increases the cost of the feed phosphates, hence some manufacturers
skip this step and use the phosphoric acid produced by the wet process directly for
synthesis of feed phosphates. This may lead to high fluoride concentrations in min-
eral mixtures and animal feed supplements. Livestock, particularly dairy cattle may
suffer from fluorosis after consuming such mineral mixtures or feed supplements
(Singh and Swarup 1995). The phosphoric acid which has a P:F ratio of at least
100:1 is considered safe for production of feed phosphates for livestock.
2.2.2 Airborne Fluoride
The mean F concentration in ambient air in unpolluted or nonindustrial areas is

generally less than 0.1 µg/m3. The levels may be slightly higher in areas near alu-
minum smelters or other industries, but should not exceed 2–3 µg/m3. The current
“ceiling” value for F (in the form of hydrogen fluoride) in ambient air, as recom-
mended by the National Institute for Occupational Safety and Health (NIOSH),
Washington, DC, is 2.5 mg/m3 (Weinstein and Davison 2004).
Fluoride is released in the atmosphere by natural processes and human activities in
both gaseous and particulate forms. Gaseous forms include hydrogen fluoride, silicon
tetrafluoride, hexafluorosilicic acid, and sulfur hexafluoride. Particulate forms include
sodium aluminum fluoride (cryolite), aluminum fluoride, calcium fluoride, sodium
hexafluorosilicate, lead fluoride, and calcium phosphate fluoride (fluorapatite). Natural
2.2 Anthropogenic Sources 17
dispersal of gaseous and particulate fluoride into the air has been recognized in regions
of volcanic activity (USEPA 1980). Other natural sources include dust from soil and
seawater droplets carried up into the atmosphere by winds (WHO 1984). Coal contains
a substantial amount of arsenic, silicon, and fluorine. Hence, coal-fired power plants are
important sources of anthropogenic hydrogen fluoride emissions. Fluoride concentra-
tion in coal may range 4–40 g/kg (McDonald and Berkeley 1969). According to an esti-
mate, total air emissions of hydrogen fluoride by electrical utilities in 1998, 1999, 2000,
and 2001 were 64.1, 58.3, 58.3, and 55.8 million tons, respectively. In many parts of
China, coal burning for household purposes was documented as the cause of endemic
fluorosis in the human population (Guijian et al. 2007). Small kilns used for making
bricks and tiles are another source of airborne fluorosis in China and India. The fluoride
concentration in coal and mud used for making bricks and tiles may exceed 10,000 mg/
kg, hence a high emission of fluoride in the air occurs after firing (WHO 2000).
2.2.3 Industrial Effluents
More than 28 industries release fluoride-rich fumes and effluents into the environ-
ment (Swarup and Dwivedi 2002). Important among them are the aluminum indus-
try, steel production plants, superphosphate plants, ceramic factories, coal-burning
power plants, brickworks, glassworks, and oil refineries (WHO 1984). Livestock
including cattle (Swarup et al. 2001), buffaloes (Patra et al. 2000), sheep (Sahoo et al.
2003), goats (Sahoo and Ray 2004), and camels (Karram and Ibrahim 1992) living in
the vicinity of such industries often suffer from chronic fluoride toxicity. Pasture con-
taminated with rock phosphate dust emitted from a fertilizer factory resulted in the
occurrence of fluorosis in sheep living in adjoining areas (Zumpt 1975). Wild herbi-
vores are also reported to suffer from industrial fluorosis (see Chap. 5).
Hydrofluoric acid is used in the alkylation process for the production of high-
octane fuels by refineries of crude petroleum. Waste products generated from such
refineries are mostly disposed of by land treatment, wherein waste products are
applied onto or into the soil for biodegradation of organic wastes, immobilization
of inorganics, and avoidance of bioaccumulation of hazardous compounds (Loehr
and Malina 1986). This practice results in an increase in soil fluoride concentra-
tion and may cause fluorosis in animals living on such soil. For example, fluorotic
lesions along with high bone fluoride concentration in cotton rats (Sigmodon hispi-
dus) living in such petroleum-waste–treated areas was reported from some parts of
the United States (Paranjpe et al. 1994; Rafferty et al. 2000).
2.2.4 Agrochemicals and Household Products
Both organic and inorganic fluoride compounds are used for agricultural and
domestic purposes. For example, sodium fluorosilicate can be used as a rodenti-
cide and sodium fluoride as a feed premix for treatment of roundworms in pigs.
A few decades back, many fluoride compounds including sodium fluoride, cryo-
lite, and sodium fluorosilicates were widely used for pest control in agriculture.
Accidental exposure to these compounds can cause acute toxicity in man and ani-
mals. Mass poisoning and death of about 800 dogs occurred after consumption of
poultry meat contaminated with fluoroacetate or fluoroacetamide in Israel (Egyed
1979). But these compounds are now rarely used and hence are of little signifi-
cance as a source of fluoride toxicity in animals.
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Chapter 3
Fluoride Kinetics and Metabolism
Abstract Fluoride, after absorption from the gastrointestinal tract, respiratory

tract, or skin and mucous membrane reaches different organs and body tissues
through blood circulation. Following oral intake, unabsorbed fluoride is excreted
through feces, and 50–70 % of absorbed fluoride is excreted through urine, per-
spiration, saliva, milk, and egg (in birds) and rest is retained in the body. Calcified
tissues, mainly bone and teeth, act as a natural sink for fluoride and contain about
99 % of the total body fluoride burden. Fluoride accumulation in soft tissues is
very low, with kidneys having the highest concentration. The placenta appears to
protect the fetus from the toxic effects of fluoride as evident from low transplacen-
tal fluoride passage in many animal species. Normal cerebrospinal fluid contains
very low fluoride concentration, but it increases marginally during chronic fluoride
toxicity. The exoskeleton in invertebrates, skeletal tissues in fish, and hair and fin-
gernails in vertebrates also accumulate fluoride and may act as bioindicators of the
fluoride burden in animals.
Fluoride absorption, distribution, deposition in body tissues, and excretion are

governed by many factors, although the basic features remain identical in humans
and different species of animals. Figure 3.1 depicts the pathways of fluoride distri-
bution, retention, and excretion after its oral intake.
3.1 Absorption
Fluoride compounds can be absorbed via different routes including the gastroin-
testinal tract, respiratory tract, and through skin and mucous membranes.

22 3 Fluoride Kinetics and Metabolism
Fluoride ingestion
Gastro-intestinal tract
Bone & Teeth
(99% of retained F) Absorption
Faeces
Soft tissues & Blood Blood (6-10%)
(1% of retained F) 75% Blood plasma
20% Blood cells Urine
5% Protein bound (50-70%)
Excretion Sweat & Saliva

Retention (Up to 13%)
(30-50%) (50-70%)
Milk
(<1-2%)
Fig. 3.1 Fluoride metabolism in animal body after its oral intake
3.1.1 Gastrointestinal Tract
The gastrointestinal tract is the major route of fluoride uptake, except for animals
raised in polluted or industrial environments where fluoride compounds are present
in high concentration as vapors, fumes, or dust. In animals, fluoride is absorbed
mostly in the stomach (in simple-stomach animals), rumen, abomasum (in rumi-
nants), and upper intestine by a passive process, although its excretion into the
mucosa of the intestine is by active transport (Parkins 1971). The rate of active
transport of fluoride in intestinal mucosa decreases with the increasing age of the
animal (USEPA 1980). Permeation of fluoride through the gastric mucosa is pH
dependent; the higher acidity of stomach contents increases F absorption. In the
stomach under acidic conditions, fluoride ions can reversibly combine with hydro-
gen ion to form hydrogen fluoride (HF) or hydrofluoric acid, which is an uncharged
molecule and can readily pass through biological membranes. Hydrofluoric acid
has pKa 3.45. Hence, any fluoride present at a pH below 3.45 will exist mainly in
the undissociated form as HF. At pH above 3.45, as in blood plasma, tissue fluid,
and the like, fluoride exists mainly in ionized form. In ruminants, only about 50 %
of ingested fluoride is absorbed in the rumen, because the rumen pH is around 5–6,
which is not suitable for fluoride absorption (Weinstein and Davison 2004).
The rate and extent of F absorption in the gastrointestinal tract is also regulated
by several other factors, the most important among them being the chemical nature
and solubility of fluoride compounds. In general, inorganic F compounds are more
soluble and hence rapidly and extensively absorbed from the gastrointestinal tract.
Sodium fluoride has the highest bioavailability, whereas calcium fluoride, magne-
sium fluoride, and aluminum fluoride have poor bioavailability. Bioavailability
of fluoride from bone and fish meal is nearly 50–60 %. Other factors modulating
F absorption include species, sex, and age of the animal and presence of dietary
components including minerals (divalent cations), fat, protein, and fiber. In human
beings, only 50 % of the fluoride from fish protein concentrate is absorbed because
3.1 Absorption 23
most of the fluoride is in the form of highly insoluble fluorapatite. High fat and
high cereal products in the diet reduce or delay fluoride absorption. Absorption of
fluoride ingested along with milk is slower than that ingested with water (USEPA
1980). Almost all fluoride naturally present in water is absorbed when the gastro-
intestinal tract is empty. However, the rate and extent of F absorption decreases in
the presence of milk or other food items. It was observed that F from sodium fluo-
ride or disodium mono-fluorophosphate was absorbed up to 100 % when ingested
along with water in a fasting state. However, a decrease in F absorption by 20–30 %
occurs when they are ingested along with milk or baby formula (Whitford 2005). In
simple-stomach animals, high protein in the diet favors F absorption by stimulat-
ing gastric acidity. High Ca, Mg, Al, and B in the diet reduce fluoride absorption
(Cerklewski 1997). The decrease in absorption is due to the binding of fluoride with
these cations, which ultimately increases fecal excretion of fluoride.
Fluoride absorption in the gut of herbivorous insects is very low and only about
0.1–9 % of the ingested fluoride is retained. This happens because their gut pH is
usually alkaline exceeding 7, and there is an absence of calcified tissues that can
act as a sink for fluoride (Weinstein and Davison 2004).
3.1.2 Respiratory Tract
Fluoride released into the air from volcanoes and industrial activities exists both
in gaseous and particulate forms. Common gaseous forms of fluoride include
HF, silicon tetrafluoride (SiF4), carbon-tetrafluoride (CF4), and hexafluorosilicate
(H2SiF6). On the other hand, sodium fluoride (NaF), aluminum fluoride (AlF3),
calcium fluorosilicate (CaSiF6), fluorspar (CaF2), and cryolite (Na3AlF6) are com-
mon particulate forms of fluoride emitted by industries. Volcanic eruptions are
major natural sources of airborne fluoride in many parts of the world. HF is the
major gaseous form of fluoride released by volcanoes. The annual global release
of HF from volcanic sources ranges from 60 to 6000 kilotonnes, of which approx-
imately 10 % may be introduced directly into the stratosphere (WHO 2002). In
addition, other fluorine compounds (such as SiF4, H2SiF6, and F2) are also present
in volcanic gases. These gaseous and particulate fluoride compounds, depending
upon their solubility and particle size, may be absorbed partially or completely
through the respiratory tract (WHO 2002). HF has high water solubility and is
rapidly absorbed in the upper respiratory tract. The particulate F compounds are
deposited in the nasopharynx, the tracheo-bronchial tree, and the alveoli, and
their absorption depends upon their water solubility and particle size. The larger
particles may be coughed up and reach into the gastrointestinal tract where they
are absorbed or excreted in feces. In a study in rats, controlled exposure of HF
(8.7 mg HF/m3 in the air for 2 h/day for 6 months) had no apparent effect on
appearance, behavior, and weight gain, but resulted in F accumulation in hair,
bone, and teeth (Stolarska et al. 2000). Fluoride absorption from the respiratory
tract may have some significance in animals living in areas near industries emit-
ting gaseous fluoride.
The respiratory tract appears to be an important route of fluoride uptake in

factory workers, wherein prolonged occupational exposure to even moderately
high fluoride concentration in ambient air can cause osteo and dental fluorosis.
However, for herbivorous animals living in areas where the air is polluted with
particulate or gaseous fluoride compounds, the respiratory tract is not the sole
route of fluoride absorption. Most of the airborne F emissions contaminate soil,
water, and vegetation a long distance from the source of emission (Radostits
et al. 2000). Fodder plants and grasses grown in polluted areas can accumulate
a large amount of fluoride. High F concentrations in forage and grasses growing
in the vicinity of industrial F emissions are therefore quite common (Swarup and
Dwivedi 2002). Thus, fluoride uptake from soil and vegetation can significantly
contribute to the total fluoride burden in herbivores living in such areas.
Fluorine gas is extremely irritating and causes nasal and eye irritation (even at
low levels), and death due to pulmonary edema (at high levels) after inhalation.
Likewise, acute inhalation of HF following facial splashes with hydrofluoric acid
can cause bronchiolar ulceration, pulmonary hemorrhage/edema and death. In
addition, renal and hepatic damage also occurs in animals after exposure to excess
fluorine or HF gas (ATSDR 2003) suggesting substantial absorption of F through
the respiratory tract.
3.1.3 Dermal and Other Routes
Dermal exposure of fluorine, HF, and hydrofluoric acid can cause serious damage
to skin and mucous membranes due to irritation (itching or burning sensation) and
burn. The severity of the damage is directly related to the concentration and dura-
tion of exposure. In rabbits, a 1-min exposure to 2 % hydrofluoric acid did not pro-
duce skin lesions; however, necrotic lesions were observed after a 1–4-h exposure.
When hydrofluoric acid is applied directly over the skin, it is absorbed quickly
through the epidermis causing extensive damage and increased fluoride concen-
tration in the systemic circulation (ATSDR 2003). Dermal absorption of other
fluoride compounds, particularly those occurring in solid state, are perhaps very
low. The dermal route of absorption does not appear to play any significant role
in chronic fluoride toxicity in vertebrates. Even in invertebrates, such as silkworm
larvae that are highly sensitive to fluoride toxicity, the gastrointestinal tract is the
major route of F absorption.
Many insects including honeybees, when exposed to a polluted environment,
deposit fluoride on the external body surface as dry deposits, rather than in the
body tissues (Davies 1989). The effect of this fluoride deposition is not clearly
known. The role of fluoride pollution on honeybee depopulation in industrial
vicinities is a matter of much debate (Weinstein and Davison 2004).
Dermal and other routes of fluoride absorption have not been investigated
widely in aquatic animals, although many aquatic invertebrates and fish are highly
sensitive to fluoride. Studies of fluoride accumulation in body tissues and its toxic
3.1 Absorption 25
effects in fish and other invertebrates are numerous, but data on the relative contri-
bution of dermal, gastrointestinal tract, and other routes of fluoride absorption are
lacking. In fish, fluoride compounds can also enter through fluoride-permeable gills
(Bielawski 1971). The contribution of gills towards total fluoride uptake is also not
clear and the gastrointestinal tract is supposed to be the major route of F uptake.
3.2 Distribution and Retention
Irrespective of the route of exposure, absorbed fluoride reaches into blood cir-
culation which acts as a transport medium and carries F to different body parts.
Fluoride in blood exists in two forms, organic and inorganic fluorides. The inor-
ganic F is only important from a toxicological point of view, as it is the only active
form. Values of total F in blood plasma are of little biological interest because
there is no known conversion of organic to inorganic forms (Venkateswarlu 1975).
In blood, about 75 % inorganic fluoride remains in plasma, about 5 % bound with
plasma proteins, and rest is present in or on erythrocytes. Out of total plasma inor-
ganic fluoride, 15–70 % remains in ionic form (Singer and Armstrong 1964). The
ionic form does not diffuse through the cell membranes; the diffusion occurs as
HF which is in a nonionic state. In human serum at least 50 % of the total serum
F is in nonionic form, but the ionic form may predominate following excess F
uptake (Swarup and Dwivedi 2002).
The time lag between ingestion and peak plasma concentration varies with sol-
ubility of the fluoride compound, emptiness of the stomach, and the nature of the
F carrier. For example, if a given quantity of sodium fluoride is ingested with milk,
it will be absorbed slowly and the time taken to reach peak plasma concentration
will be longer than when ingested with water. In human beings, it takes about
30–60 min to reach peak plasma fluoride concentration after ingestion of sodium
fluoride with water (Carlson et al. 1960). From plasma, F migrates across cell
membranes of nearly all soft tissues which have a steady-state tissue-to-plasma
concentration ratio ranging from 0.5 to 0.9. Exceptions to this are brain and fat
which have a considerably lower ratio and kidneys which have a higher ratio
because fluoride is concentrated in the renal tubular fluid (Whitford et al. 1976).
The peak plasma F concentration starts declining due to two main reasons:
uptake by calcified tissue and excretion in urine. Fluoride sequestration by calci-
fied tissues from the plasma pool occurs through exchange for other anions, such
as hydroxyl, citrate, and carbonate ions. In general, the clearance of fluoride from
plasma by the skeleton is inversely related to the stage of skeletal development.
Skeletal uptake, however, can be positive or negative depending on the level of
fluoride intake, hormonal status, and other factors (Whitford 1994a). Plasma F
concentration is not regulated by homeostasis and F metabolism is modulated by
several factors including chronic and acute acid-base disturbances, hematocrit,
altitude, physical activity, circadian rhythm and hormones, nutritional status, diet,
and genetic determinants (Buzalaf and Whitford 2011).
3.2.1 Transplacental Passage
In human beings, fluoride passes freely through the placenta when fluoride concen-
tration in maternal blood is low; but placenta regulates fluoride transfer and protects
the fetus when maternal blood fluoride concentration is high (USEPA 1980; Mohanty
et al. 2011). This protective mechanism is perhaps a passive process and the fetus is
protected by the bulk of the maternal fluid space, quick clearance, slow placental fluo-
ride diffusion, and ectoptic calcification of the placenta (Erickson and Malmnas 1962).
In cattle, research findings on transplacental fluoride transport are conflicting. One
study demonstrated that fluoride does not pass from dam to offspring in an amount
sufficient to induce fluorotic changes (Shupe et al. 1963). Similar observations were
recorded in Holstein cows given added sodium fluoride in the diet (Shupe et al. 1992).
However, in another study when the pregnant cattle were exposed to high fluoride
intake, fluoride accumulated in the bone and teeth of the fetus and dental and bony
lesions appeared in the newborn calves (Krook and Maylin 1979). In calves born to
a fluoride-intoxicated cow, congenital fluorosis is manifested as brown discoloration
of enamel, enamel hypoplasia, brown mottling of bone, severe retardation of cartilage
cell differentiation, atrophy of osteoblasts, osteopenia, atrophy of bone marrow cells,
serous atrophy of bone marrow fat, and severely stunted growth (Maylin et al. 1987).
Transplacental passage of fluoride has not been widely investigated in different
animal species. In horses, it is unlikely that enough fluorine passes the placental bar-
rier to induce fluorotic changes in offspring (Shupe and Olson 1971). The effect of
variation in placental types on transplacental F transport in different animal species is
not clear thus far. Absence of congenital fluorotic lesions in most of the animal species
suggests that, despite differences in structure and mineral transport mechanism, all
types of placenta provide some protection to the fetus from excess maternal fluoride.
3.2.2 Cerebrospinal Fluid
Fluoride concentration in the CSF in human beings and horses is 50 % or less than
that of plasma (Whitford 1994a). Transport of fluorine, as with other halogen and
ionic substances in CSF, is an active process. The blood–brain barrier appears to
protect the brain from flooding of excess fluoride. Fluoride concentration in the
CSF increases marginally only in patients with severe fluoride intoxication or the
breakdown of the blood–brain barrier (Hu and Wu 1988).
3.2.3 Skeleton and Other Calcified Tissues
Approximately 99 % of the body F burden is associated with the calcified tissues.

Bone, as in the case of lead, acts as a natural sink for fluoride. Bone fluoride lev-
els can be used to quantitate long-term fluoride exposure. Bone is composed of
3.2 Distribution and Retention 27
water (45 %), inorganic salts, mainly Ca and P (35 %), and organic matrix or oste-
oid (20 %) consisting of collagen and noncollagenous glycoproteins and proteo-
glycans. The inorganic part of the bone comprises mainly mineral hydroxyapatite,
and forms crystals that are deposited upon and parallel to the collagen fibers in a
regular manner. These crystals are surrounded by a hydration shell that permits
free flow of fluoride ions between the extracellular fluid and interior of the crystal
(Swarup and Dwivedi 2002).
Fluoride deposition in bone occurs in two phases. In the first phase, fluoride ions
diffuse from extracellular fluid into the hydration shell and displace hydroxyl groups
in the bone crystal surface to make a mixed fluorohydroxyapatite (Hodge and Smith
1970). This is a rapid and reversible process. Steady-state equilibrium exists between
the F concentration in the extracellular fluids and F deposited on bone surface layers
(Florkin and Stotz 1971). In the second phase, F is deposited in the denser and deeper
parts of the bone. This is a slow process as interlacunar apatite crystals of bone are
closely packed and have little fluid. Here F is incorporated deeper into the interlacu-
nar apatite crystal at a rate nearly equal to deposition of phosphorus as bone phos-
phate. Once F is incorporated deeper into the apatite crystals, it cannot be removed
from the bone without resorption of the crystal. This happens during the osteoclastic–
osteoblastic cycle of bone remodeling (Hodge and Smith 1970). Fluoride deposition
in both surface and deeper parts of bone and teeth is maximum during the growth
phase as crystallites are small and have more hydration facilitating F diffusion.
Bone fluoride concentration increases as animals grow older, even in animals
on a low fluoride diet (Shupe et al. 1963). Fluoride uptake and concentrations dif-
fer among different types of bone and parts of a particular bone (Kay 1975). In
the long bones, F concentration is higher at the ends and lower in the shaft; higher
in the periosteal and endosteal region and lower in the middle region (Weidmann
and Weatherell 1970). Fluoride concentration is higher in bones that have a high
cancellous component (ribs, vertebrae, and sternum) than the bones having a more
cortical component (compact bones; Marier et al. 1963). This occurs because F
deposition depends upon the metabolic activity and vascularization of the bone
(USEPA 1980). During chronic toxicity F deposition in the skeleton proceeds rap-
idly at first and then more slowly until eventually a saturation stage is reached.
Beyond this saturation point, flooding of the soft tissues with fluorine occurs caus-
ing metabolic breakdown and death of the animal (Underwood and Suttle 1999). In
animals, bones can be classified on the basis of F concentration as: 300–400 ppm-
normal; <4500 ppm-innocuous F concentration (compact bone); 4500–5500 ppm-
marginal osteofluorosis; 5500 ppm (compact bone) or >7000 ppm (cancellous
bone) toxicosis; and 15,000–20,000 ppm-saturation (Swarup and Dwivedi 2002).
F is not irreversibly bound to bone; it is released either after cessation of excess
F uptake or when the diet cannot sustain mineralization of the skeleton (e.g., dur-
ing lactation). Fluoride is released from bone in two phases. The first phase is a
rapid process that takes weeks and involves anion exchange in the hydration shell.
The second phase is slower with an average half-life of 8 years and occurs due to
osteoclastic resorption of the bone. F is released slowly from compact bones in
comparison to trabecular bones (WHO 1984).
Fish also accumulate F in skeletal tissues; the concentration varies from one
species to another even when living in the same water reservoir. Carnivorous fish
accumulate higher concentrations of F in the skeleton. When raised in F polluted
water sources, the concentration may reach close to 1000 mg/kg dry bone tissue
(Pinskwar et al. 2003). The bioaccumulation factor (ratio of tissue fluoride level
and aqueous fluoride level) in skeletal tissues of one- to three-year-old brown
trout, Salmo trutta, from the Madison River in Yellowstone National Park ranged
from 114 to 133 ppm (Neuhold and Sigler 1960). Because bone and calcified tis-
sues of fish accumulate a considerable amount of fluoride, bone and fish meal can
constitute a significant source of fluorine for farm animals.
3.2.4 Teeth
Fluoride deposition in teeth primarily occurs during formative stages. The F content
of enamel is acquired partly during development and partly from the oral environ-
ment after eruption. Dentin fluoride concentration is almost equal to bone F concen-
tration and tends to increase with age. Fluoride concentration in enamel is lower than
in dentin. Enamel fluoride concentration reflects the level of fluoride exposure during
tooth formation, whereas dentin and bone fluoride concentrations are generally pro-
portional to the long-term F uptake (Weatherell 1969). The tooth surface continues to
accumulate F from the oral environment; hence F concentration of the outer surface
of the enamel and the inner surface of dentine tends to increase with age.
3.2.5 Exoskeleton
The exoskeleton of invertebrates acts as a sink for fluoride. Some krill species
accumulate very high fluoride in their exoskeleton under natural (unpolluted) con-
ditions and the level may be as high as 6000–12,876 µg/g dry weight (Sands et al.
1998). The exoskeleton in the cephalothorax region in crayfish accumulates more
F than other regions (Gonzalo and Camargo 2012).
3.2.6 Hair and Fingernails
Some studies on human and laboratory animals suggested that F concentration in

hair may serve as an unobtrusive way to determine the body F burden (Stolarska
et al. 2000; Parimi et al. 2013). F concentration in the hair of persons not exposed to
high F uptake ranges from 1.81 to 2.32 ppm; and it may be as high as 5460 ppm in
persons occupationally exposed to fluoride pollution (Kokot and Drzewiecki 2000).
Fluoride concentration in fingernail clippings in humans can also serve as
a biomarker of F exposure, although it only reflects F intake about 3–6 months
before (Ayoob and Gupta 2006). In rat, nail fluoride concentration was reported as
a biomarker for acute fluoride exposure (Buzalaf et al. 2004).
3.2 Distribution and Retention 29
3.2.7 Soft Tissues
In general, fluoride concentration in soft tissues is very low and does not vary with
age. It is maintained within a narrow limit and shows only marginal variation even
during excess F uptake or fluctuations in plasma F levels. Intracellular F in soft
tissues is readily exchangeable with F present in extracellular fluid (Carlson et al.
1960). In a large-scale survey of cattle suffering from industrial fluorosis, it was
observed that F accumulated in the liver, lung, kidney, cerebrum, cerebellum, thy-
roid, and pituitary, but not in the heart, musculature, and spleen (Oelschlager et al.
1972). Kidneys contain higher F levels than other soft tissues, perhaps because
they are the major route of F excretion (Shupe 1980). Normal fluoride concentra-
tion in vital organs such as the liver, heart, and thyroid gland of cattle and sheep
usually varies between 2 and 5 μg/g on a dry matter basis and increases up to two-
to threefold even after prolonged exposure to excess fluoride (Suttie et al. 1958).
The pineal gland, a small gland present in the center of the brain, exceptionally
accumulates a high fluoride concentration. In a study on aged human cadavers, the
mean F concentration in the pineal gland was 297 ± 257 mg F/kg, whereas in
muscles it was 0.5 ± 0.4 mg F/kg wet weight (Luke 2001). This gland is not pro-
tected by the blood–brain barrier and has a very high blood perfusion rate, second
only to the kidneys. High F accumulation in the pineal gland may result in low
circulating melatonin and accelerated sexual maturity in females, as observed in
an experimental study in gerbils (Luke 1997).
Fluoride concentration in deboned poultry meat varies between 0.3 and 2.7 mg/kg
(Jedra et al. 2001). In the common carp (Cyprinus carpio), fluoride accumulation
was highest in the gills, followed by the liver, brain, kidney, muscle, and intestine
after exposure to high fluoride concentration in water for 90 days (Cao et al. 2013).
The bioaccumulation factor in different soft tissues varies between 2.3 and 6.2. In
freshwater and marine fish muscle, F concentration ranges from 0.6 to 26 mg/kg wet
weight (Lall 1994; Camargo 2003). Fluoride concentration in the muscle of crab,
shrimp, and prawn varies from 1 to 4 mg/kg (Soevik and Braekkan 1981). As soft
tissues accumulate little fluoride, meat meal, unless contaminated with some other
F compounds or bone, does not appear as a significant source of fluorine for farm
animals (Underwood and Suttle 1999).
3.2.8 Egg
F concentration in egg increases when birds are fed a high fluoride diet (Phillips
et al. 1935). Fluoride is mainly deposited in the eggshell and fluoride levels in
albumen and yolk are low (Guenter and Hahn 1979). Hence, eggshell fluoride
concentration is a sensitive indicator of fluoride exposure (Carriere et al. 1987).
Fluoride concentration in the yolk is higher than albumen. Nevertheless, F con-
centration in the egg appears not to exceed the safe limit for human consumption,
even when birds are raised on diet containing toxic doses of fluoride.
3.3 Elimination and Excretion
Unabsorbed fluoride from the gastrointestinal tract is eliminated through feces,

whereas a part of F present in the systemic circulation is excreted through different
routes. Once fluoride uptake is reduced, fluoride deposited in bone starts mobi-
lizing slowly and is excreted in urine and feces. In almost all mammals, the kid-
ney plays a key role in fluoride excretion. Other routes of fluoride elimination and
excretion include feces, saliva, milk, and perspiration.
3.3.1 Urine
The urinary excretion of fluoride is a pH-dependent process. The renal clearance

of fluoride is higher with alkaline urine (Ekstrand et al. 1978, 1980). However, the
extrarenal clearance of fluoride remains negligible during the production of alka-
line urine (Ekstrand et al. 1978).
The renal handling of fluoride is characterized by unrestricted filtration through
the glomeruli followed by a variable degree of tubular reabsorption. The F reab-
sorption perhaps occurs along the entire nephron by nonionic diffusion as HF. The
extent of reabsorption is inversely related to tubular fluid pH, hence the urinary
excretion of fluoride closely correlates with urinary pH (Jarnberg et al. 1981). At
a urinary pH of 5.0 the ratio of clearance of fluoride and glomerular filtration rate
may be as low as 5 %, and it may exceed 65 % at urinary pH 6.5. About 35–45 %
of the filtered fluoride is reabsorbed in the proximal renal tubules regardless of
the final urinary pH, although in acidosis the majority of the fluoride reabsorption
occurs in the distal nephron. Kidney damage significantly reduces urinary elimina-
tion of fluoride in rats (Dote et al. 2000).
Typical normal bovine urine contains less than 10 mg F/L. Concentrations
greater than 10 mg/L support a clinical diagnosis of fluorosis in cattle (Osheim
and Rasmusson 1998).
3.3.2 Feces
A fraction of the ingested fluoride not absorbed in the gastrointestinal tract is

excreted in the feces. In simple-stomach animals, approximately 6–10 % of the
total ingested fluoride is excreted in feces. The fecal fluoride excretion is higher
in ruminants. In cattle, fluoride concentration in feces is usually higher than the
forage because a greater proportion of the dry matter is absorbed than fluoride
(Weinstein and Davison 2004). The fecal elimination of fluoride depends upon
several factors such as solubility of the fluoride compounds, presence of dia-
valent cations (Ca, Mg, B, etc.), concentration of other dietary components (fat,
fiber, protein, etc.), and age and physiological state of the animal. Solubility of the
3.3 Elimination and Excretion 31
ingested fluoride appears as the major determinant regulating fecal and urinary F
excretion. For example, in a study of rats fed with krill meal fluoride was excreted
mainly in feces, whereas those given sodium fluoride excreted fluoride mainly
through urine (Szewielow 1991). In another study, fecal fluoride elimination in
rats increased twice when calcium concentration in the diet increased from 0.4 to
1.4 % by weight (Whitford 1994b). Simultaneous intake of boron compounds has
also been reported to enhance fecal F elimination in buffalo calves, perhaps due to
formation of insoluble compound BF4 (Bharti et al. 2007).
3.3.3 Saliva
In humans, average salivary fluoride concentration is about 75 % of the plasma

F concentration and varies within the range of 0.01–0.06 ppm in individuals not
exposed to excess fluoride. Salivary fluoride concentration may increase 10 times
its baseline value when a person is consuming soluble F compounds in the diet
(Toth et al. 2005). Salivary fluoride concentration peaks rapidly (1–15 min) after
ingestion, but it returns to baseline values within 20–60 min (Macpherson and
Stephen 2001; Petersson et al. 2002). It was estimated that less than 1 % of the
ingested fluoride is excreted in human saliva (Carlson et al. 1960). Salivary fluo-
ride is assumed to play an important role in prevention of dental caries in human
beings. However, its role in animals is not clear thus far.
3.3.4 Perspiration
Fluoride concentration in perspiration in humans is approximately 20 % of plasma

levels (Henschler et al. 1975). It may serve as an important route of fluoride excre-
tion in humans working hard in hot and humid climates. A study revealed that
about 13–38 % of ingested fluoride may be excreted from the body in perspira-
tion under conditions of high temperature and humidity (McClure et al. 1945).
Therefore, sweating may play an important role in fluoride excretion in animals
such as horses that have a good number of functional sweat glands and a high
sweating rate. However, further study is required to verify this assumption and to
explore the significance of sweat glands in fluoride excretion in different animal
species under different climatic conditions.
3.3.5 Milk
Fluoride concentration in cattle and goat milk is usually low and ranges between
0.02 and 0.8 ppm (Tinanoff and Mueller 1978; Vlachou et al. 1992; Liu et al. 1995;
Kant et al. 2009). In human breast milk, fluoride concentration ranges from 5 to
10 µg/L (Fomon and Ekstrand 1999). The F concentration in milk shows good cor-
relation with F intake in dairy cows, but it never exceeds the safe levels for human
consumption (Stoddard et al. 1963). A decrease in total milk production in chronic
fluoride toxicity in dairy cows may also be responsible for an increase in milk F con-
centration. It is interesting to note that fluoride absorption from milk occurs slowly;
hence milk products have been suggested as a useful vehicle to deliver fluoride to
infants and young children for prevention of dental caries (Stephen et al. 1984).
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Chapter 4
Toxic Effects
Abstract Clinical manifestations of acute fluoride toxicity include hypersaliva-

tion, restlessness, body stiffness, convulsion, and respiratory and cardiac failure,
eventually leading to death. In chronic fluoride toxicity calcified tissues act as a
natural sink for fluoride and hence characteristic lesions develop in bone and teeth.
Dental lesions appear more frequently than bony lesions and are characterized by
discoloration, hypoplastic pits, chalkiness, fast attrition, and early loss. Intermittent
lameness, weakness, and the appearance of bony protuberances are common due
to lesions developing in bone. Toxic effects on soft tissues become more evident
when bones saturate with fluoride and their capacity to accumulate more fluoride is
lost. Excess fluoride can alter the cytoarchitecture and physiology of renal, hepatic,
gonadal, and neuronal tissues. Many studies suggested genotoxic, carcinogenic,
immunotoxic, and cytotoxic potential of fluoride, although the results were highly
variable and even contradictory.
Toxic effects of fluoride are modulated by dose and duration of fluoride uptake
and several other factors discussed in the chapter on fluoride tolerance. Here, path-
ological changes and clinical manifestations in acute and chronic fluoride toxicity
in different animal species are described.
4.1 Acute Toxicity
Acute fluoride toxicity is caused by exposure to large doses of highly soluble inor-
ganic fluoride compounds such as sodium fluoride or hydrogen fluoride gas or hydro-
fluoric acid. In acute fluoride poisoning, almost all organ systems are affected. Clinical
signs are mostly nonspecific and resemble poisoning caused by other gastrointesti-
nal irritants such as arsenic, mercury, barium, and oxalic acid (Polson and Tattersall
1979). The manifestations in humans include vomiting (sometimes blood-stained),

36 4 Toxic Effects
diffused spasmodic abdominal pain, diarrhea, cyanosis, severe weakness, dyspnea,

muscle spasm, paresis and paralysis, cardiovascular disorders, convulsions, and coma
terminating in death (WHO 1984). The minimum dose to produce adverse health
effects in humans is about 5 mg fluoride/kg body weight, and the minimum lethal
dose in adults varies from 32 to 64 mg fluoride/kg body weight (Whitford 1996).
Acute fluoride toxicity in animals is rare in occurrence and is caused by
accidental ingestion of fluoride-containing insecticides or other chemicals such as
sodium fluoride, sodium fluorosilicate, and hydrofluoric or fluorosilicic acid. Wistar
rats when given acute toxic doses of sodium fluoride exhibited depression, prostra-
tion, piloerection, scant droppings, profuse salivation, initial colorless lacrimation
followed by chromodacryorrhea, dyspnea, passage of dark reddish-black pasty feces,
gasping, cyanotic paws, hunched posture, soiled perineum, and loss of reflexes (Giri
et al. 2014). Clinical signs of acute fluoride toxicity in domestic animals include
restlessness, stiffness, anorexia, hypersalivation, nausea, vomiting, urinary and
fecal incontinence, reduced milk production, convulsion, weakness, severe depres-
sion, pulmonary congestion, and respiratory and cardiac failure (NRC 1980; Shupe
1980). Profuse salivation, lacrimation, nasal discharge, dyspnea, severe abdominal
pain, convulsion, and death within 2.5–58 h was reported in buffalo calves follow-
ing administration of 200–400 mg of sodium fluoride per kg body weight (Gujarathi
et al. 1991). Gastrointestinal signs arise due to the corrosive action of hydrofluoric
acid which is produced in the acidic environment of the stomach (Whitford et al.
1980; Whitford 1996). Cardiac changes and cardiac arrest can occur due to hypocal-
cemia and/or hyperkalemia that develop following exposure to high doses of fluoride
(Cummings and McIvor 1988). Respiratory signs are more prominent after inhala-
tion of fluoride compounds such as hydrogen fluoride gas.
4.2 Chronic Toxicity
Fluoride is largely a cumulative poison and its prolonged exposure in high albeit
sublethal doses induces chronic illness, often referred to as fluorosis. Clinical
signs of toxicity may not appear for several weeks or months following uptake of
excess fluoride (Underwood and Suitte 1999). Chronic fluoride toxicity is more
common than acute fluoride toxicity in both humans and animals. Dental lesions
are perhaps the first visible sign in humans and domestic animals, followed by
signs related to skeletal deformities. If exposure to toxic doses of F is continued,
impairment of soft tissues, reproductive performance, neurological deficit, and
other toxic effects become evident (Perumal et al. 2013).
4.2.1 General Health Effects
General health effects of fluorosis in animals include stunted growth, decrease in

milk and wool production, altered reproductive performance, and deterioration
4.2 Chronic Toxicity 37
in general health (Xin et al. 2006; Ranjan et al. 2009a). A significant decrease in
milk production is observed in advanced cases of fluorosis in dairy cattle (Suttie
et al. 1957; Eckerlin et al. 1986). Experimental studies have shown that fluoride
inhibits release of pituitary lactotrophic hormone in mice resulting in a decrease
in milk secretion (Yuan et al. 1991). Pain and restricted movement are due to
bony and dental lesions. Alteration in concentrations of volatile fatty acids and
rumen microflora are responsible for reduced feed intake and feed conversion
efficiency hence the progressive loss of appetite and body weight in cattle (Jones
1972; Kapoor et al. 2002). Other observable clinical signs include an increase in
respiration and heart rates and pallor of the mucous membranes (Maylin et al.
1987; Singh and Swarup 1995; Sharma et al. 1997). Newborn calves show lower
weight gain and retarded growth, whereas delayed postpartum estrus is observed
in breeding cattle (Udall 1965).
4.2.2 Effects on Calcified Tissues
Fluoride has a high affinity towards calcium and calcified tissues inside the body.
Bone and teeth account for about 99 % of the body fluoride burden and show
characteristic lesions in chronic fluoride toxicity.
4.2.2.1 Dental Fluorosis
Dental lesions are the most sensitive indicators of fluoride toxicity, particularly in
young animals. The basic features of dental lesions in animals include hypominer-
alized outer enamel, coronal cementum hyperplasia, disrupted subsurface pigment
band, hypoplastic pits, puckered incremental lines, periodic radiolucent regions, and
decreased microhardness of the outer enamel (Shearer et al. 1978). The tooth becomes
brittle, hence attrition and early tooth loss are common in occurrence (Fig. 4.1).
Excessive intake of fluoride interferes with amelogenesis and dentinogenesis
resulting in defective enamel and dentin formation (Swarup and Dwivedi 2002).
Ameloblasts, the dental pulp cells, and the odontoblasts are primary target cells
in chronic fluorosis. Fluoride has cytotoxic action on odontoblasts and pulp cells
(Rodriguez and Rosselot 2001; Chang and Chou 2001). Atrophy and necrosis of
the ameloblasts result in defects in enamel. The pulp cells may undergo fibrous
and osseous metaplasia. The odontoblasts become atrophic and brown discolora-
tion appears on dentin (Krook and Maylin 1979).
Mottled and defective enamel is an indication of inorganic fluoride e xposure
during the development of teeth, as adverse effects are not apparent in teeth that
have already erupted prior to the fluoride exposure (Susheela et al. 1999). In
cattle, the period during which the developing teeth (incisors) are sensitive to
fluoride extends from about 6 to 30 months of age (Swarup et al. 2001; Swarup
and Dwivedi 2002). Therefore, if cattle are exposed to high fluoride after attaining
adulthood, the frequency and severity of dental lesions will be low. Dental lesions
38 4 Toxic Effects
Fig. 4.1 Severe mottling, chalkiness, and attrition of teeth in a fluorotic cow
do not reflect the current high fluoride intake in adult animals. Once developed,
dental lesions largely remain irreversible, although fluoride concentration in teeth
may vary with age. Preventive and curative measures for fluorosis in goats in
terms of improvement in dental lesions were found effective only when they were
taken before eruption of permanent teeth (Wang et al. 1995).
The severity of fluorosis in animals can be classified on the basis of dental
examination, particularly the lesions on incisors (Swarup and Dwivedi 2002).
Shupe et al. (1979) proposed a 0–5 scale classification system for evaluation of the
degree of dental fluorosis in livestock (Table 4.1).
Table 4.1 Classification of dental lesions in fluorosis (Shupe et al. 1979)

Score Type Description
0 Normal Translucent, smooth, glossy white enamel, teeth are normal in shape
1 Questionable Slight deviation from the usual translucency of normal enamel. Cause is
effect not precise. May have enamel flecks; cavities, if present may be unilateral
or bilateral, but no mottling is evident
2 Slight effect Slight mottling of enamel; may have some discoloration, but no abrasion.
Teeth have normal shape
3 Mild effect Moderate mottling (large patches of chalky enamel), discoloration of
enamel; teeth may have slight abrasion
4 Marked Definite mottling, discoloration, hypoplasia and hypocalcification; may have
effect pitting of enamel; enamel may be cream colored; definite abrasion of teeth
5 Excessive Definite mottling, discoloration, hypocalcification, may have pitting of
effect enamel; enamel may be cream colored, excessive abrasion of teeth
Table 4.2 Classification of dental lesions in fluorosis (Krook et al. 1983)

Score Lesions
1 Hypercementosis with tooth ankylosis, cementum necrosis, and cyst formation
2 Delayed eruption of permanent incisor teeth
3 Necrosis of alveolar bone with recession of bone and gingiva
4 Oblique eruption of permanent teeth, hypoplasia of teeth with diastema
5 Rapid progression of dental lesions and tooth loss
Another system of classification of dental lesions in fluorotic cattle was described

by Krook et al. (1983) which includes five expressions of dental lesions (Table 4.2).
4.2.2.2 Osteofluorosis
Bone is considered a natural sink for fluoride. In chronic fluorosis in livestock,

the bones and joints become enlarged and extra bone growths or exostosis may
appear in different parts of the skeleton (Fig. 4.2). Calcification of ligaments
and related structures may occur, which is manifested clinically as stiffness and
Fig. 4.2 A heifer showing

stunted growth, enlarged
joints, and lameness. Plasma
fluoride concentration was
2.17 ppm
40 4 Toxic Effects
Fig. 4.3 A buffalo showing poor body condition, stiffness, and lameness
intermittent lameness (Fig. 4.3). The forelimbs are more affected than hind limbs,
and in o ccasional cases, the animal may move around on its knees and assume
“knee posture.” Palpable bony exostosis may appear on the metatarsal, metacarpal,
mandible, or ribs of cattle and buffaloes (Wheeler and Fell 1983).
Deposition of fluoride in high concentration in bone has been reported in
natural (Boivin et al. 1989) as well as experimental (Khandare et al. 2005)

fluorosis. Inorganic fluoride uptake by bone tissues is primarily through the

replacement of hydroxyl groups of calcium hydroxyapatite and incorporation of
inorganic fluoride as calcium fluorapatite. In fluoride toxicity, bony lesions show
wide variability including osteosclerosis (increase in bone density), periosteal
hyperostosis (increase in the size of the cartilage-capped protuberance at the end
of the bone), osteoporosis (decrease in the bone tissue density), osteomalacia
(failure of ossification due to decreased availability of calcium ion), or osteophy-
tosis (presence of bony outgrowth). The type and extent of these changes depends
upon several factors including nature, dose, and duration of fluoride exposure,
age and sex of the subject, hormonal responses, type of bone affected, and nutri-
tional status of the animal. Variation in response is perhaps a cumulative effect of
all these factors. Soriano (1968) opined that smaller doses of fluoride stimulate
osteogenesis and new bone formation, and larger doses result in increased bone
resorption and defective matrix formation. This hypothesis was strengthened by
the finding that a high level of fluoride resulted in a reduction in collagen synthesis
in humans (Nichols and Flanagan 1966). Poor nutrition, especially with a n egative
calcium balance was reported to exacerbate osteoporosis (Krishnamachari and
Krishnaswamy 1973; Wang et al. 1994). The severity of fluorotic lesions in bone is
also influenced by the type of bone and the stress and strain subjected on the bone
(Swarup and Dwivedi 2002).
Osteofluorosis can be clinically categorized into four distinct stages in human

beings. In asymptomatic preclinical stage I, patients have a slight increase in bone
density that could be detected radiographically and bone ash F concentrations
range from 3500 to 5500 mg/kg. In stage I skeletal fluorosis, occasional stiff-
ness or pain in the joints and some osteosclerosis of the pelvis and vertebra are
observed and bone ash fluoride concentration ranges from 6000 to 7000 mg/kg. In
stage III, bone ash fluoride concentrations exceed 7500–8000 mg/kg and patho-
logical changes and clinical signs include dose-related calcification of ligaments,
osteosclerosis, exostoses, possible osteoporosis of long bones, muscle wasting,
and neurological defects due to hypercalcification of vertebrae. Osteofluorosis
in advanced cases is manifested as kyphosis (abnormally increased convexic-
ity in the curvature of the thoracic spine as viewed from the side), scoliosis
(lateral curvature of the vertebral column), flexion deformity (the act of bending
or the condition of being bent) of knee joints, paraplegia (paralysis of the lower
part of the body including legs), and quadriplegia (paralysis of all four limbs).
In severe cases, paralysis can occur due to the pressure caused by osteophytes
(bony outgrowth) or narrowing of the intervertebral foramen and increase in size
of the body of the vertebrae or narrowing of the spinal canal. Men suffer more
than women (approximately in the ratio 10:1), perhaps due to more involvement
in strenuous work. A higher incidence of bone cancer in men than women was
reported in workers exposed to fluoride (Grandjean et al. 1992). Genu valgum
(knock knees: legs angle inward at the knee) and genu varum (bow legs: legs are
bowed outward at the knee) are two important forms of skeletal fluorosis observed
in children suffering from fluorosis in India. This is associated with excess fluoride
along with low dietary calcium intake (Kirshnamachari and Krishnaswamy 1973;
Krishnamachari 1976).
In animals, normal bone fluoride concentration varies between 200 and
300 mg F/kg and it rarely exceeds 1200 mg/kg in fat-free bone on a dry m atter
basis. After excess fluoride intake, fluoride deposition in the skeletal tissues
proceeds rapidly at first and then more slowly, until eventually a saturation stage
is reached. When bone fluoride concentration reaches 15,000–20,000 mg/kg, the
capacity to accumulate fluoride by bone is lost. Bony lesions and radiographic
changes usually appear when the bone fluoride concentration exceeds 5000 mg/kg.
Radiographic changes closely parallel gross pathological changes in bone.
Inasmuch as response of bony tissues varies widely, it is quite obvious that the
radiographic appearance will show wide variations. The spectrum of radiographic
features in fluorosis include increased bone density, blurring or haziness of
trabeculae, compact bone thickening, periosteal bone formation, and ossification
of the attachments of tendons, ligaments, and muscles (Wang et al. 1994). These
changes are more appreciable in the axial skeleton (Christie 1980; Lian and Wu
1986). Despite wide variation, radiography can serve as a valuable tool for the
diagnosis of fluorosis. Raj Roholm proposed a method for diagnosis of skeletal
fluorosis in humans using bone radiographs. He classified bony changes detectable
in radiographs into three distinct stages (Roholm 1937). Later on, Fritz described
two additional phases of radiographic changes that could help early diagnosis of
42 4 Toxic Effects
osteofluorosis in humans (Frankel 1979). In cattle, the fluorosis can be classified

into three stages based upon bone fluoride concentration (Swarup and Dwivedi
2002). In stage I, the fluoride concentration may go up to 2500 ppm without
showing any structural or functional changes in bone. Stage II is characterized
by a bone fluoride level between 1500 and 5000 ppm. In this stage too, no gross
or radiographic changes are evident, but the microarchitecture of the bone shows
alterations. In stage III, bone fluoride concentration goes above 5000 ppm and
gross lesions become apparent.
4.2.3 Effects on Soft Tissues (Nonskeletal,

Nondental Effects)
Excess fluoride uptake can produce deleterious effects on several organs includ-
ing the gastrointestinal tract, lungs, heart, kidneys, and liver (Perumal et al. 2013).
Toxic effects of fluoride on noncalcified tissues and organs are diverse including
cytotoxicity, genotoxicity, immunotoxicity, carcinogenicity, and teratogenic effects
(El-Nasser et al. 1995; Kleinsasser et al. 2001; Zhan et al. 2006). Many of these
effects appear to be associated with free radicals and oxidative stress (Ranjan et al.
2009b; Ekambaram et al. 2010), although some workers have raised doubt over it
(Reddy et al. 2003).
4.2.3.1 Cytotoxicity
The cytotoxic potential of fluoride is due to its capacity to inhibit several cellu-
lar enzymes, particularly those related to energy metabolism (Barbier et al. 2010).
However, Tokunaga et al. (2003) opined that the cytotoxic potential of fluoride is
mediated by inhibitory action on glucose uptake by the cells and not by enzyme
inhibition. Fluoride exposure disrupts collagen synthesis and leads to the break-
down of collagen in various tissues and organ systems including bone, tendon,
muscle, skin, cartilage, lungs, kidneys, and trachea (Susheela and Mukherjee
1981; Sharma 1982).
4.2.3.2 Renal and Hepatic Toxicity
In healthy individuals, about 50 % of ingested fluoride is excreted by the kidneys.

The renal system, therefore, appears at high risk of fluoride toxicity. The highest
accumulation of fluoride in the kidneys among various soft tissues can be attributed
as one of the possible reasons (Whitford 1996). In fluoride-exposed rats, kidney
damage has been reported at levels as low as 1 ppm in drinking water (Varner et al.
1998). Fluoride toxicity induces a wide range of structural and functional changes
in the renal system. Fluoride inhibits various enzyme systems in the kidneys
(Jankaurkar 1974) and decreases Na, K, and ATPase activities (Suketa and Terui
1980). The increase in serum urea nitrogen and creatinine is well documented in
fluoride toxicosis in humans and animals (Singh and Swarup 1995; Shivashankara
et al. 2000). Histopathological observations in fluoride toxicosis include cloudy
swelling, degeneration of tubular epithelia, centrilobular necrosis, atrophy of
glomeruli, periglomerular fibrosis, and tubular necrosis (Shashi et al. 2002).
The liver is the organ responsible for detoxification of many endogenous
and exogenous toxins. Several studies have revealed an increase in activities of
the liver-specific enzymes in the serum of fluorotic animals (Maiti et al. 2004;
Upadhyay et al. 2005). An increase in activities of serum transaminases, sorbitol
dehydrogenase, and decreased levels of serum total protein and albumin reflecting
altered liver function were reported in fluorotic cattle (Arya et al. 1990) and goats
(Tsunoda et al. 1985). Hepatocellular necrosis, degenerative changes, hepatic
hyperplasia, extensive vacuolization in hepatocytes, and centrilobular necro-
sis were reported in experimental fluoride toxicity in rabbits (Shashi and Thapar
2000). Histopathological changes of a similar kind have also been reported in
experimental fluorosis in calves (Kapoor et al. 1993).
4.2.3.3 Reproductive Toxicity
Reproductive toxicity of fluoride has been widely investigated in laboratory and

domestic animals. Experimental studies suggest that fluoride toxicity is associ-
ated with morphometric and histological changes in testis (Susheela and Kumar
1991) as well as ovaries and hence reduced fertility in both males and females
(WHO 2002). In males, excess fluoride uptake disrupts spermatogenesis as well
as hormones regulating sexual maturation and reproduction such as testosterone,
estrogen, melatonin, and thyroid hormones (Long et al. 2009). Disruption in ster-
oidogenesis (Ghosh et al. 2002) and spermatogenesis was reported in various ani-
mal species including ram (Zakrzewska et al. 2002), rat (Sprando et al. 1998), and
rabbit (Kumar and Susheela 1994). Adverse effects on sperm motility, sperm mor-
phology, and semen hyaluronidase activity was also recorded in in vitro studies on
bovine semen (Schoff and Lardy 1987; Tanyildizi and Bozkurt 2002).
Females appear less susceptible than males, as reproductive performances did
not vary significantly even after exposure to excess fluoride for three generations
in mice (Tao and Suittie 1976). It is assumed that chronic fluoride toxicity has
no adverse effect on reproductive efficiency of female cattle (USEPA 1980). The
absence of teratogenic effects of fluoride in the female rat was also observed in
several studies (Collins et al. 1995; Heindel et al. 1996; Al-Hiyasat et al. 2000;
WHO 2002). It seems that ill effects observed on female reproduction are second-
ary to impairment of general body health rather than direct effect of fluoride on
reproductive organs. However, some recent studies documented a reduction in
reproductive hormone concentrations and abnormalities related to receptor pro-
tein expression after fluoride exposure leading to decreased fertility in female mice
(Zhou et al. 2013a, b).
44 4 Toxic Effects
4.2.3.4 Neurotoxicity
Several clinical and experimental studies have shown that fluoride induces
change in cerebral morphology and biochemical processes in nervous tissues that
affect neurological development as well as cognitive processes, such as learn-
ing and memory (Valdez-Jimenez et al. 2011; Liu et al. 2014). The developing
brain is sensitive to excess fluoride as evident by poor mental development, and
low intelligence, learning capacity, and memory power in children and young
laboratory animals exposed to excess fluoride (Xiang et al. 2003; Wang et al.
2004). Neurotoxic effects of fluoride on the adult brain resulting in diminished
mental acuity, memory impairment, hyperactivity, and cognitive deficits have also
been observed in several experimental studies (Ross and Daston 1995; Verner
et al. 1998). Concurrent iodine deficiency or exposure to other neurotoxic pollut-
ants may exacerbate the neurological manifestations in fluoride toxicity (Wang
et al. 2004).
Fluoride is a normal constituent of cerebrospinal fluid. The blood–brain
barrier does not allow the free movement of fluoride ions from blood to CSF.
However, fluoride concentration increases marginally during subacute or chronic
fluoride toxicity in CSF (Hu and Wu 1988) and brain tissues (Ross and Datson
1995). Fluoride may have direct cytotoxic effects on brain tissues or may act
indirectly through enhanced generation of free radicals and peroxides in CSF
(Strunecka et al. 2007). In addition, an in vitro study in rat hippocampal neurons
indicated that fluoride could induce cell-cycle arrest and DNA damage result-
ing in neurotoxicity and neuronal death (Zhang et al. 2008). Neuropathological
changes in experimental fluoride toxicity comprise degenerative changes that
are more evident in the hippocampus, amygdala, motor cortex, and cerebel-
lum (Shivarajashankara et al. 2002). Fluoride intoxication decreased synthesis
of cholesterol, free fatty acids, proteins, amino acids, and RNA in the brain of
rabbits (Shashi 1992; Shashi et al. 1994), indicating metabolic disturbances may
contribute to neurotoxic effects. Fluoride alters concentrations of
monoamine
neurotransmitters such as noradrenaline (NA), dopamine (DA), and serotonin

(5-HT), which could lead to impaired neurotransmission (Liu et al. 2014).
A decrease in nicotinic acetylcholine receptors in the hippocampus is assumed to
be a possible reason behind cognitive deficits (Guan et al. 2001; Long et al. 2002).
An increase in levels of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in
certain parts of the brain including the striatum, hippocampus, and neocortex was
observed, suggesting their role in the impairment of learning and memory process
(Pereira et al. 2011). Other possible mechanisms may be changes in synaptic
structures in the brain (Chen et al. 2003).
4.2.3.5 Immunotoxicity
Fluoride adversely affects both humoral and cellular immunity (Krishnamoorthy

et al. 2014). The immunotoxic potential of fluoride is mediated via different
mechanisms. It depletes the energy reserves and ability of the white cells to
destroy foreign agents by phagocytosis (Gabler and Leong 1979). It also inhib-
its antibody formation (Jain and Susheela 1987) and may divert the immune
system to attack the body’s own tissues (Gibson 1998). Fluoride is also reported
to activate alveolar macrophages enhancing production of chemokines and pro-
inflammatory cytokines and inducing PMNs infiltration in the lung (Hirano et al.
1999). In vitro studies have shown that fluoride induces apoptosis in permanent
cell lines (Tokunaga et al. 2003). However, it is believed that the concentrations
required to produce inhibitory effects on lymphocyte and polymorphonuclear
leukocyte functions is too high and could not be achieved in blood and tissue fluid
during chronic fluoride toxicity (Challacombe 1996).
Fluoride is also assumed to cause skin eruptions such as atopic dermati-
tis, eczema or urticaria, gastric distress, headache, and weakness, particularly in
hypersensitive individuals (Challacombe 1996). However, the potential of fluoride
to cause skin allergy and allergic reactions in other organs has been rejected by
some authors (Spittle 1993). In several animal species including rabbits, guinea
pigs, and horses, allergic skin lesions have been reported; but similar findings have
not been reported yet in cattle and sheep (Justus and Krook 2006).
4.2.3.6 Genotoxicity and Carcinogenicity
The genotoxicity potential of fluoride has been tested in several in vitro and in
vivo studies with inconsistent and highly variable results (WHO 2002). It is gener-
ally believed that fluoride is not mutagenic in prokaryotic cells, although it may
increase the frequency of gene-locus mutations in cultured mammalian cells (NRC
1980). Some studies indicated that fluoride may induce chromosomal aberrations,
micronuclei, and sister-chromatid exchange in mammalian cells, but the results
were inconsistent perhaps due to differences in research methodology such as
differences in harvest time used to accommodate cell-cycle delay. Chromosomal
aberration could not be detected in cells exposed in vitro to fluoride concentra-
tions less than 10 μg/ml, suggesting 10 µg/ml as the threshold fluoride concen-
tration for the clastogenic activity of fluoride (NRC 1980). The chromosomal
aberrations induced by sodium fluoride consist primarily of breaks/deletions and
gaps, with very few exchanges. Some studies also indicated that fluoride increases
unscheduled DNA synthesis in mammalian cells. However, later on it was

observed that these may be possibly due to artifacts such as formation of precipi-
table complexes of magnesium, fluoride, and [3H] thymidine triphosphate (Skare
et al. 1986).
The results of in vivo studies on the genotoxic potential of fluoride in labora-
tory animals are reported to vary with the route of fluoride administration. The
studies in which fluoride was administered orally to rodents revealed no effect
upon sperm morphology or frequency of chromosomal aberrations, micronu-
clei, sister-chromatid exchange, or DNA strand breaks (WHO 2002). However,
few studies indicated that sodium fluoride may induce genotoxic effects after its
46 4 Toxic Effects
intraperitoneal administration in laboratory animals (Mohammed and Chandler

1982; Pati and Bhunya 1987).
The carcinogenic potential of fluoride is also an issue of much debate. A dose-
related increase in the incidence of osteosarcoma in male rats was observed in
experimental studies (NTP 1990). Likewise, a positive correlation between water
fluoridation and incidence of osteosarcoma was reported in the human popula-
tion (Lee 1993). On the other hand, in a large-scale statistical survey of 36 types
of cancer in the United States, it was observed that 23 had positively significant
(63.9 %), 9 not significant (25.0 %), and 4 negatively significant (11.1 %) correla-
tions with the percentage of people supplied with optimally fluoridated drinking
water. The authors therefore concluded that the likelihood of fluoride acting as a
genetic cause of cancer requires reconsideration (Takahashi et al. 2001). An exten-
sive study was conducted at the Utah State University Agricultural Experiment
Station in about 200 cattle over a period of 25 years to explore the carcinogenic
potential of fluoride. It was observed that cattle after exposure to dietary fluoride
levels in excess of 25 ppm (on dry weight basis) for most of their life span did
not show any significant sign of soft tissue damage or neoplasia and authors
concluded that environmental fluorides are not a significant factor in mamma-
lian carcinogenesis (Shupe et al. 1992). Considering conflicting reports, it would
be reasonable to conclude that further study is required for arriving at any final
conclusion.
4.2.3.7 Other Effects
Fluoride is claimed to have thyrotoxic effects (Liu et al. 2002). A study concluded
that chronic fluorosis in cows was associated with hypothyroidism (Cinar and
Selcuk 2005). Experimental studies suggest that follicular epithelial cells of the
thyroid gland undergo structural and functional changes after fluoride exposure
which ultimately disrupt thyroid hormone synthesis (Bouaziz et al. 2004; Rahman
and Fetouh 2013). Electrocardiogram changes along with a decrease in heartbeat
were also reported in dogs (Kilicap et al. 2004) and sheep (Donmez and Cinar
2003).
4.3 Molecular Mechanism of Toxicity
Fluoride exerts diverse effects on cellular processes in a time, concentration,

and cell-type–dependent manner. Fluoride at micromolar levels promotes cell
proliferation, but at millimolar concentrations inhibits several enzymes; modu-
lates intracellular redox homeostasis, calcium homeostasis, and gene expres-
sion; reduces protein synthesis; and induces apoptosis. It also affects secretion
and vesicular traffic that play an important role in many cellular signaling
processes. Free radicals and oxidative stress have been implicated in pathogenesis,
4.3 Molecular Mechanism of Toxicity 47
particularly in toxic effects on noncalcified tissues. Coexposure of other pol-

lutants including aluminum, arsenic, and lead also modulates the toxic potential
and mechanism of fluoride action. The molecular mechanism of fluoride toxicity
has been described in several scientific reviews (Barbier et al. 2010; Strunecka
et al. 2007; Perumal et al. 2013). Readers are advised to consult these articles for
detailed descriptions.
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Chapter 5
Fluoride Tolerance
Abstract Fluoride tolerance varies with species, age, and sex of the animal;
individual resistance; dose, duration, and consistency of fluoride uptake; and
dietary, nutritional, and environmental factors. The tolerance levels established
by experimental studies may be erroneous sometimes, due to poor correlation
between fluoride concentration in feed and water and actual bioavailability of
the fluoride. Rabbits, guinea pigs, rats, mice, and some wild rodents are highly
susceptible to fluoride toxicity. Among domestic animals, ruminants have less
fluoride tolerance than simple-stomach animals. Carnivores are even more toler-
ant than herbivorous simple-stomach animals. Dental and bony lesions similar to
those observed in domestic cattle and buffaloes may appear in wild cervids after
excess fluoride intake. Poultry are highly tolerant to fluoride. On the other hand,
insects, some other invertebrates, and soft water dwelling fish have low fluoride
tolerance.
The toxic effects of fluoride are modulated by several factors; hence it is often
difficult to set a rigid tolerance level for different animal species. The tolerance
levels of dietary fluoride (in feed and water) in livestock and poultry have been
established by experiential studies after administering soluble fluoride salts,
mostly sodium fluoride. Hence, in natural conditions the actual tolerance level
may be even two to five times higher than experimental values, as bioavailability
of dietary fluoride is usually less than sodium fluoride (USEPA 1980; Underwood
and Suttle 1999). When fluoride uptake is simultaneously through several routes,
such as from air, water, feed, fodder, salt-lick, medicine, and dermal absorption,
the actual tolerance level may differ from the literature value. This chapter deals
only with dietary tolerance levels, as ingestion is the major route for fluoride
uptake in most animals and contribution of inhalation and dermal absorption is
usually negligible (USEPA 1980).

54 5 Fluoride Tolerance
5.1 Fluoride Tolerance in Different Animal Species
The species of the animal has great influence on tolerance levels of fluoride. Here
fluoride tolerance of different animals is described in brief.
5.1.1 Laboratory Animals
Rabbits, rats, mice, and guinea pigs are all highly susceptible to fluoride toxicity
(Shupe and Olson 1971). That is why rabbits, rats, and mice are extensively used
in experimental studies on fluoride toxicity. A spontaneous outbreak of fluoride
toxicity with bone and teeth lesions was also reported in rabbits fed high-fluoride–
containing diets supplemented with rock phosphate as a mineral source (Romero
et al. 1991). Rabbits can tolerate up to 11 mg of fluoride per kg body weight per
day (Briggs and Phillips 1952). It is pertinent to note that sodium monofluoroac-
etate poisoned oats were widely used in Australia as bait to kill European rabbits
(Oryctolagus cuniculus L.) that caused significant loss of agricultural produc-
tion and were considered a serious threat to biodiversity of the natural ecosystem
(Twigg et al. 2003).
The no observed adverse effect level (NOAEL) and the lowest observed
adverse effect level (LOAEL; mg of F/kg of BW/day) for rats are 1.8–12.7 and
3.2–12.8, respectively; whereas for mice values are 2.7–9.1 and 4.5–5.7, respec-
tively (NRC 1980). The oral LD50 in mice is in the range 44.3–46 mg/kg, and
in rats it is 51.6 mg/kg (WHO 2000). In Wistar rats, the approximate lethal dose
(ALD) is 253.125 mg/kg BW (Giri et al. 2014).
The incisor teeth of rats and other rodents, unlike mammals, continue to grow
even in mature animals. At low doses of F exposure, incisors in rats initially lose
their normal orange pigment and luster and become chalky in appearance. At high
doses of F intake, enamel becomes pitted and corroded and upper and lower inci-
sors may appear either elongated or shortened due to alteration in the rate of wear
(Greenwood 1956).
5.1.2 Domestic Animals
Ruminants appear to be more susceptible than monogastric herbivores. Among

ruminants, the prevalence of dental and bony lesions in natural cases is generally
higher in grazers (cattle and buffaloes) than browsers (sheep, goats, and camels)
(Choubisa 2013a). The water buffalo is the most susceptible among domestic
ruminants, followed by cattle, sheep, and goats (Choubisa 2013b). Mature dairy
cattle and buffaloes consume more feed and water in relation to body weight
than mature beef cattle, hence their tolerance level is low (Table 5.1). A negative
5.1 Fluoride Tolerance in Different Animal Species 55
Table 5.1 Tolerance levels of fluoride in feed (on DM basis) and water for different animals
Animal species Fluoride in feed (ppm) Fluoride in water (ppm)
Beef and dairy heifers 30 2.5–4
Mature dairy cattle 30 3–6
Mature beef cattle 40 4–8
Sheep 50 12–15
Horse 60 4–8
Swine 70–100 5–8
Dog 100 –
Poultry 100 10–13
Source NRC (1974); Shupe et al. (1984); Swarup and Dwivedi (2002)
calcium balance, a frequent observation in lactating cattle and buffaloes, may be

another possible reason behind higher susceptibility to fluoride toxicity. Likewise,
lifetime fluoride exposure for finishing cattle is less than breeding cattle, hence
tolerance is high. As a rule of thumb, cattle can tolerate up to 1 mg F/kg body
weight for 5–10 years with only minor cosmetic effects, but 2 mg F/kg BW will
result in accelerated tooth wear and signs of osteofluorosis.
Sheep and goats can tolerate somewhat higher dietary levels of fluoride than
cattle, although clinical observations in hydrofluorotic areas may show wide vari-
ation. In a population of 780 goats and 564 sheep inhabiting areas where mean
water fluoride concentration was 3.2 ppm, none revealed any evidence of osteo
or dental fluorosis (Choubisa 1999). But later on, authors in a survey of 356 goats
and 248 sheep inhabiting areas where mean water fluoride concentration varied
between 1.5 and 1.7 ppm, observed that the prevalence of osteo and dental fluoro-
sis in goats (10.7 and 8.4 %) was higher than in sheep (7.3 and 5.6 %; Choubisa
et al. 2011). Clinical signs recorded in different stages of experimental fluoride
toxicity in sheep included anorexia, poor body condition, lameness, mandibular
thickening, excessive wear of molar teeth, and pale mucosa (Zhou and Zhai 1991).
As with cattle and water buffaloes, dental lesions appear early and more frequently
than bony lesions in natural cases of fluorosis in sheep and goats (Guo et al. 2002;
Singh et al. 2002).
The dromedary camel has high fluoride tolerance as evident from the absence
or very low frequency of dental and bony lesions in camels reared in hydro-
fluorotic areas (Choubisa et al. 2011; Choubisa 2013a). This is perhaps due to
their unique morphoanatomic and physiological adaptations such as low water
turnover rate, ability to survive without water for up to 20 days, and some other
unknown reasons. Nevertheless, dental and bony lesions of fluorosis may appear
in camels reared in areas where water fluoride concentration is as low as 1.45 ppm
(Choubisa 2013b). Anemia was reported as a significant hematological find-
ing in camels suffering from industrial fluoride pollution (Karram and Ibrahim
1992). Dental lesions in camels appear as nonstriated, vertical, homogeneous,
light-brown discoloration which is different from bovines where dental lesions
are characterized by the presence of horizontal striated deep-brown discoloration.
The frequency and severity of dental and bony lesions in camel calves are lesser
than in adults and aged animals, which is opposite to that observed in cattle and
water buffaloes (Choubisa 2013b).
Reports on the natural occurrence of fluorosis in horses are very few. In fluorosis-
endemic areas, horses do not suffer as frequently as cattle, sheep, or swine. Horses
are considered to have a higher tolerance to fluoride than other domestic animals.
However, horses grazing in areas where fluorotic lesions are common in cattle and
sheep may develop similar dental lesions (Shupe and Olson 1971). In a research
report, it was claimed that artificially fluoridated water was responsible for colic,
stiffness, and lameness observed in horses on a farm in Colorado, as the frequency of
these problems gradually reduced when water fluoridation was stopped (Burgstahler
2006). However, such claims require further investigation for scientific validation.
Swine perhaps have the highest fluoride tolerance among different domestic
animals. Domestic pigs are largely raised on high grain diets which usually have
low fluoride and high calcium concentrations. Dental lesions include enamel
hypoplasia and mottling, impaired milk production, and loss of body weight;
a disturbed estrous cycle was recorded in sows fed on diet containing more than
500 ppm fluoride concentration (NRC 1960). Pathological changes in teeth includ-
ing disturbed enamel formation along with high F accumulation was reported in
wild boars from the Czech Republic and Germany (Kierdorf et al. 2000, 2005).
Fluoride tolerance appears high in dogs as there is a paucity of reports on
occurrence of dental and bony lesions in dogs reared in fluorosis-endemic areas.
Adult dogs can tolerate a daily intake of 5 mg of fluorine as sodium fluoride
per kg BW for years, although a similar dose may induce dental lesions in pup-
pies (Greenwood 1956). In a study, the daily intake of 10–24 mg F/kg BW for
2–8 months in pups after weaning resulted in brown discoloration, erosions, and
fissures in the enamel of permanent molars and incisors together with bone lesions
(Grancher et al. 1988). The lethal dose of sodium fluoride for dogs is 47 mg/kg
BW. Fluoride concentration as high as 460 mg/kg in dog food (wet basis), given
for 2 years neither produced any bony lesions nor any adverse effects on repro-
ductive performance (Shellenberg et al. 1990). However, in another study, it was
observed that threshold F concentration for effects on structural and functional
changes in gastric mucosa was approximately 1 m mol/L, and maximum or near-
maximum effects were caused by 10 m mol F/L (Whitford et al. 1997).
5.1.3 Wild Animals
The behavior and feeding patterns of wild animals in captivity differ from those in
wild conditions. Some wild animals have a large territory where fluoride concentra-
tion in water and dietary substances may vary significantly. Hence, setting a tolerance
level on the basis of experimental studies may be erroneous. To date, knowledge of
fluoride toxicity in wild animals under natural conditions is largely based upon dental
and bony changes and bone fluoride concentration recorded in dead animals.
In general, herbivores, particularly those grazing grasses and small vegeta-

tion, are more susceptible than leaf and fruit eaters and carnivores. Grasses and
other small plants growing on soil containing a high fluoride concentration or in
industrial fluoride polluted areas often contain a higher fluoride concentration than
leaves of tall trees or fruits grown in the same area. Wild herbivores living in geo-
graphic areas where cattle and buffaloes suffer from chronic fluorosis also exhibit
signs of fluorosis, suggesting they are equally sensitive to fluoride. Carnivores suf-
fer less as they occupy the topmost position in the food pyramid and consume soft
tissues of animals which have low F concentration. In a study in Britain, Walton
(1984) observed that skeletal fluoride concentration in small mammals inhabit-
ing the vicinity of an aluminum smelter was increased, but no increase in bone
fluoride concentration was recorded in red foxes (Vulpes vulpes). Red foxes are
omnivorous and small rodents comprise a major part of their diet. Fecal examina-
tion of red foxes revealed that skeletal parts of the prey pass into feces undigested,
which may be the possible reason behind their low bone F concentration.
Cervids are highly susceptible to natural and industrial fluorosis. Dental fluorosis
due to industrial fluoride pollution has been reported in several deer species includ-
ing mule deer (Odocoileus hemionus hemionus), white-tailed deer (Odocoileus vir-
ginianus), and black tailed deer (Odocoileus hemionus columbianus) (Vikoren and
Gudbrand 1996), although the susceptibility to fluoride toxicity varies with species.
Roe deer (Capreolus capreolus) are reported as a sensitive bioindicator of indus-
trial fluoride pollution (Kierdorf et al. 1993). In almost all deer species, dental
lesions are more common than bony lesions in both natural and industrial fluorosis.
Moreover, dental lesions in deer skulls collected from the forest areas where fluoro-
sis was prevalent in domestic ruminants, very closely resembled those observed in
fluorotic cattle. The pathological changes in mandibular bone including periodontal
breakdown, periostitis, ostitis, and chronic osteomyelitis, resulted in early tooth loss
and a decrease in the life expectancy of wild deer (Schultz et al. 1998). High fluo-
ride concentration in bone and teeth of these skulls supported the hypothesis that the
dental and bony changes were linked with fluorosis.
Fluoride concentrations in normal deer vary from 160 to 560 ppm in mandibles
and 134–152 ppm in antlers (Newman and Yu 1976). In the bone of deer suffering
from fluorosis, fluoride concentration may reach up to 7000 ppm. In Argentina,
wild red deer (Cervus elaphus) developed severe dental lesions after a volcanic
eruption and bone fluoride levels reached up to 5175 ppm (Flueck and Smith-
Flueck 2013). The source of excess fluoride was ingestion of tephra (volcanic
ashes) deposited on the herbage. Fluoride accumulation is perhaps more rapid and
extensive in antlers than bone of the same animal (Suttie et al. 1985). However, a
study revealed that bone and antler fluoride concentration in fluorotic deer did not
differ significantly and authors concluded that antler material remains in equilib-
rium with fluoride elsewhere in the skeleton (Walton and Ackroyd 1988).
A population of eastern grey kangaroos (Macropus giganteus) living close to
an aluminum smelter in Australia exhibited clinical signs of lameness. A diagnosis
of fluoride toxicity was given after detailed gross and radiographic examination of
bone and teeth and fluoride analysis in bone (Clarke et al. 2006).
Minks have high fluoride tolerance. Captive minks raised only for pelts are usu-
ally euthanized at 7 months of age. They can tolerate up to 100 ppm fluoride in
their feed on wet weight basis or 270 ppm on dry weight basis without any adverse
effects on pelt quality, growth rate, and appearance of bony or dental lesions.
However, for breeding stock this level cannot be recommended as bony and dental
lesions may appear after 7 months ultimately affecting their reproductive perfor-
mance. The tolerance level of F in feed for breeding stock may be fixed as 50 ppm
on wet weight basis or 135 ppm on dry weight basis (Shupe et al. 1987).
Wild rodents such as the cotton rat (Sigmodon hispidus) are also susceptible
to fluoride toxicity and a high incidence of dental lesions along with high bone
fluoride concentration was reported in cotton rats living in areas where land-
treatment of petroleum waste products is practiced (Paranjpe et al. 1994; Rafferty
et al. 2000).
In wild birds, short-lived, seed-eating birds accumulate more fluoride in bone
than long-lived omnivore species. Bone fluoride concentration in wild birds living
in the vicinity of fluoride-polluted areas may reach as high as 11,000 µg/g (Henny
and Burke 1990).
5.1.4 Poultry and Other Birds
The fluoride tolerance of poultry is much higher than that of mammals (Shupe
1969). Spontaneous cases of fluorosis are rare in chicken, turkeys, and other poul-
try. Higher tolerance may be attributed to dietary factors, short life span, and some
other unknown physiological factors. In poultry, fluoride is accumulated in high
concentrations in bone without showing any pathological changes.
Chickens can tolerate up to 350 ppm F in their diet with no adverse effect
on growth (NRC 1960). When the fluoride concentration in the diet of chickens
reaches 700 ppm or more, growth is retarded, but there is no effect on egg pro-
duction (Halpin and Lamb 1932). A high concentration of fluoride may be found
in the egg yolk of poultry raised on a high F diet. Calcium and phosphorus con-
centration in bone decreases and generalized signs of lameness, leg weakness,
decreased weight gain, decrease in feed consumption, and feed efficiency may be
observed in advanced cases.
Turkeys are even more tolerant than chickens and can tolerate up to 300 to
400 ppm F in the diet (Phillips et al. 1955; NRC 1960). A decrease in appetite,
feed efficiency, and weight gain were major clinical findings recorded in an exper-
imental study when turkeys were maintained on a ration containing 400–800 ppm
F for 16 weeks (Anderson et al. 1955). The postmortem changes in these turkeys
included thickening of the duodenum mucosa suggesting local irritating effects of
excess fluoride intake.
The Adelie penguin (Pygoscelis adeliae) is a unique bird that does not show
any gross or radiographic symptoms of skeletal fluorosis despite having an excep-
tionally high bone fluoride concentration (up to 9000 µg/g). The major diet of
the Adelie penguin is krill, whose average body fluoride concentration is about
1232 µg/g. The krill body is covered with a chitinous crust which accumulates a
high fluoride concentration (as high as 5000 µg/g). However, high chitin concentra-
tion in the bone of penguins is assumed to prevent skeletal fluorosis, as chitin can
combine with fluoride preventing abnormal bone mineralization (Yin et al. 2010).
5.1.5 Insects and Other Invertebrates
The susceptibility of insects and other invertebrates to fluoride can be very well
understood by the fact that sodium fluoride, fluoroalcohols, and fluorophosphates
were widely used for control of insects, cockroaches, and poultry lice in the past
before the advent of DDT. Sulfuryl fluoride is an odorless, colorless, noninflamma-
ble, noncorrosive gas that was first registered as an insecticide and rodenticide in
year 1959, but is still being used in some countries as anti-insecticide fumigation.
Industrial fluoride pollution is assumed to have devastating effects on the silkworm
and honeybee industries (Weinstein and Davison 2004). In addition, fluoride pol-
lution has a significant impact on the overall insect population in industrial vicini-
ties, but the absolute effect of fluoride is difficult to assess due to simultaneous
exposure to other pollutants. Ground-dwelling invertebrates also accumulate fluo-
ride, but the impact of excess fluoride on their growth and population is not well
established and research findings are contradictory. In an experimental study, aver-
age whole-body fluoride concentration of Dikerogammarus villosus, a species of
amphipod crustacean was 27.6 µg/g dry weight which increased up to 16, 994 µg/g
72 h after exposure to 10 mg F/L. The authors therefore, concluded that the capac-
ity to rapidly accumulate a very high concentration of fluoride may be the reason
behind its high sensitivity to fluoride (Gonzalo et al. 2010).
5.1.6 Fish and Other Aquatic Animals
In general, aquatic animals (invertebrates and fish) living in hard or seawaters are
more tolerant to fluoride than those living in soft water with low ionic content.
A high concentration of calcium and chloride in the water decreases the bioavail-
ability of fluoride ions, thereby increasing fluoride tolerance (Camargo 2003).
Moreover, large body size within a given aquatic species is associated with greater
fluoride tolerance in both soft-water and marine animals (Camargo 2004). This
happens perhaps due to a higher fluoride elimination or immobilization rate in
larger individuals (Sigler and Neuhold 1972). The LC50 values for fluoride for
freshwater, brackish, and marine invertebrates at different stages of development,
exposure time, and temperature ranges from 10.5 to 308 mg F/L (WHO 2005).
In addition to fluoride concentration in an aquatic medium, susceptibility
to fluoride toxicity in aquatic animals also depends upon the exposure time and
water temperature. Fluoride concentrations in unpolluted freshwater usually range

from 0.01 to 0.3 mg F/L, whereas in unpolluted seawater it ranges from 1.2 to
1.5 mg F/L (Camargo 2003). Naturally elevated levels of fluoride may be found in
regions with high geothermal or volcanic activity or industrial units emitting flu-
oride-rich fumes or effluents. An increase in water temperature is associated with
increased risk of fluoride toxicity, possibly due to an increase in metabolic rate
and hence uptake of fluoride ions from the aquatic medium (Sigler and Neuhold
1972). Several experimental studies have also demonstrated a negative correlation
between chloride ion concentration in water and susceptibility to fluoride toxicity
in aquatic animals (Neuhold and Sigler 1962; Pimentel and Bulkley 1983).
Fluoride tends to accumulate in the exoskeleton of aquatic invertebrates and in
the bony tissues of fish. This may be helpful to aquatic animals dwelling in pol-
luted water by enhancing fluoride elimination from body circulation and increas-
ing hardness of the exoskeleton and bone. Fluoride accumulation in soft tissues
is low in most aquatic invertebrates and fish. Nevertheless, several aquatic inver-
tebrates appear highly sensitive to fluoride toxicity. Juvenile and young stages are
more sensitive than adults (Aguirre-Sierra et al. 2011). Industrial fluoride pollution
to freshwater reservoirs has been claimed responsible for the rapid decline in the
population of several invertebrates including snails, unionid mussels, and shrimp
species in several water reservoirs around the world.
Among freshwater or soft water invertebrates and fish, net-spinning cadd-
isfly larvae and upstream-migrating salmon are the most sensitive to fluoride.
The growth and migration of these freshwater organisms are adversely affected
when fluoride concentration reaches 0.5 mg/L or more. Hence, the safe fluo-
ride concentration recommended for protection of all stages of freshwater life
against the adverse effects of fluoride is 0.2 mg/L or less (Groth 1975; Damkaer
and Dey 1989). In the case of hard water with high ionic content, safe levels of
fluoride could be increased up to 1.0 to 1.5 mg F/L (Camargo 2003). Rainbow
trout (Oncorhynchus mykiss) and water flea (Daphnia magna) are widely used
in experimental fluoride toxicity studies. Sensitivity to fluoride toxicity has also
been reported in freshwater-dwelling unionid mussels, Alasmidonta raveneliana,
and several other invertebrate animals (Camargo and Tarazona 1990; Keller and
Augspurger 2005). A laboratory study revealed that 96 h LC50 and 240 h NOEC
values for Dikerogammarus villosus (also known as the killer shrimp, a species of
amphipod crustacean native to the Ponto-Caspian region of Eastern Europe) is 5.8
and 0.95 mg F/L, respectively (Gonzalo et al. 2010).
5.2 Factors Affecting Fluoride Tolerance
Fluoride tolerance in domestic animals is governed by several factors includ-

ing solubility of the fluoride compound, animal species, climatic conditions,
and management practices. The amount of fluoride ingested, duration of fluo-
ride ingestion, age at the time of ingestion, physiological status of the animal,
5.2 Factors Affecting Fluoride Tolerance 61
nutrition, general health status, stress, exposure to health hazards, immunity,

and individual response to fluoride are some other factors modulating the toxic
effects of fluoride (Shupe 1980).
5.2.1 Animal Factors
Within a species, fluoride tolerance varies with age and physiological and general
health status of the animal. Several experimental studies also indicate that genetic
determinants may influence an individual’s tolerance to fluoride (Everett et al.
2002; Mousney et al. 2006).
5.2.2 Dietary and Nutritional Factors
The effect of diet on fluoride retention was studied in normal humans by

Lakshmaiah and Srikantia (1977) wherein a significant increase in urinary fluoride
excretion was observed when Jowar (Sorghum bicolor) -based diets were shifted
to rice-based diets. Likewise, in rats increased fluoride retention in femur bone
was observed when grown on sorghum-based diets as compared to wheat- and
rice-based diets (Lakshmi and Lakshmaiah 1999). Some other studies conducted
in laboratory animals also suggested alteration in F retention and toxicity with a
change in dietary ingredients. Variation in nutritional values and levels of miner-
als, vitamins, protein, and fat in different kinds of diet are perhaps responsible
for these observations. Furthermore, the effect of the diet on the gastrointestinal
tract pH may be another contributing factor. For example, the acidic stomach envi-
ronment favors fluoride absorption because hydrogen fluoride is generated from
ingested fluoride compounds, which are easily absorbed from the gastric mucosa
(Whitford and Pashley 1984).
5.2.2.1 Minerals
There are several minerals present in the diet that can modulate fluoride toxicity.
Calcium, copper, zinc, and magnesium have a negative correlation with fluoride
deposition in the body, whereas molybdenum has a positive correlation (Khandare
et al. 2005a). A study on prevalence of dental fluorosis in the human population
suggested a negative correlation with calcium and copper content in drinking
water in fluorosis-endemic areas (Bhargavi et al. 2004). Other minerals affecting
fluoride tolerance include aluminum, phosphorus, and iron (Swarup and Dwivedi
2002). Molybdenum enhances urinary excretion of copper which ultimately
aggravates the toxicity potential of fluoride. In Andhra Pradesh, India where
both molybdenum and fluoride concentration in water is high, “genu valgum” in
the fluorotic human population is commonly observed, yet no similar disease is

reported from fluorotic areas of Punjab, India where Mo levels in diet were within
tolerance limit (Krishnamachari and Krishnaswamy 1974; Jolly et al. 1980).
Boron enhances urinary fluoride excretion by forming readily excretable BF4 com-
plex at digestive or metabolic levels, but has no capacity to mobilize fluoride accu-
mulated in calcified tissues (Elsair et al. 1980, 1981).
5.2.2.2 Calcium
Calcium deficiency is perhaps the most important dietary factor modulating skel-
etal and dental fluorosis (Ekambaram and Paul 2002; Khandare et al. 2005b). In
areas where water is hard due to the presence of calcium and magnesium salts or
dietary calcium intake is high, the prevalence of skeletal fluorosis is low (Singh
et al. 2011). Dietary calcium binds with fluoride to form insoluble complexes/
compounds inside the gastrointestinal tract, which is excreted through feces.
5.2.2.3 Protein
Fluoride inhibits biosynthesis of protein by inferring the binding of the amino

acyl-t-RNA adducts to the ribosomal RNA template (Hoerz and McCarty 1971).
Hence, protein supplementation helps in normalizing a fluoride-associated
decrease in protein level in various organs (Khandare et al. 2000). A diet rich in
protein and certain amino acids including glycine and glutamate has been reported
to have marked beneficial effects on fluoride-induced depression in steroido-
genesis and protein synthesis in testicles (Chinoy and Mehta 1999a, b). Protein
deficiency, on the other hand, aggravates the fluoride toxicity.
5.2.2.4 Fat
High dietary fat enhances the toxicity potential of fluoride in rats (Miller and
Phillips 1955). Dietary fluoride in the presence of fat causes delayed gastric emp-
tying, leading to increased fluoride absorption and toxicity (McGown et al. 1976).
5.2.2.5 Vitamins
Ameliorative effects of vitamins C, A, and E in fluoride-induced oxidative stress

in laboratory animals have been observed in several experimental studies (Chinoy
and Patel 2000; Chinoy and Memon 2001; Guney et al. 2007). Vitamin C acts
as a cofactor for hydroxylation of proline, an important amino acid required for
collagen synthesis. Collagen is the base material for synthesis of bone and tooth
matrix; hence vitamin C supplementation may help decrease severity of bony
5.2 Factors Affecting Fluoride Tolerance 63
and dental lesions in fluorosis. This may be a plausible explanation in man and
simple-stomach animals that are totally dependent on the dietary intake of vitamin C.
However, its role in ruminants requires further investigation as they can endog-
enously synthesize vitamin C in a quantity sufficient to meet their physiological
requirement. Moreover, vitamin C present in the diet is almost totally destroyed by
rumen microorganisms (Ranjan et al. 2013). Han et al. (2006) reported b eneficial
effects of antioxidant minerals including selenium, copper, and magnesium in
bovine endemic fluorosis.
5.2.3 Chemical Form of Fluoride
In general, fluoride compounds with high water solubility are more toxic. The
inorganic salts of F are usually more toxic than organic compounds barring a few
such as fluoroaceate and fluorocitrate. Sodium fluoride has the highest bioavaila-
bility after oral ingestion and hence is considered the most toxic inorganic fluoride
compound. Fluoride present in natural groundwater also has high bioavailability
and is equally toxic as feeding an equal amount of fluoride from sodium fluoride
through either drinking water or feed (NRC 1980). Likewise, most of the fluo-
rides emitted by industrial operations are as toxic as sodium fluoride (Swarup and
Dwivedi 2002). The toxic potential of rock phosphate, defluoridated rock phos-
phate, and dicalcium phosphate is next to sodium fluoride, followed by natural and
synthetic cryolite, calcium, and magnesium fluorosilicates and calcium fluoride
(Hobbs et al. 1954; Hobbs and Merriman 1962). Oral doses of 40 ppm sodium
fluoride caused inappetence in cattle, whereas 2400 ppm CaF2 was required to
produce similar effects (Ammerman et al. 1980). Therefore, total fluoride con-
centration in the diet sometimes may not show good correlation with the toxicity
problem observed in animals.
5.2.4 Dose, Duration, and Continuity of Fluoride Intake
Short-term high fluoride intake tends to produce systemic reactions, such as weight
loss and unthriftiness due to decreased appetite in cattle (Suttie et al. 1972). On
the other hand, dental lesions appear first after exposure to low or moderately high
fluoride concentration. The toxic effects of fluoride are also modulated by the dura-
tion and continuity of fluoride exposure. For equal total yearly fluoride intake, skel-
etal storage of fluoride remains almost similar when intake is either continuous or
intermittent (Suitte 1980). However, alternate periods of high and low exposure are
more damaging than constant intake, even when total fluoride intake during the
period is equal (Suttie et al. 1972). This happens because fluoride is released in
high concentration from bone and other storage sites during low intake and causes
more adverse effects on soft tissues (Underwood and Suttle 1999).
5.2.5 Environmental and Other Factors
Climatic conditions influence total water and food intake and hence the severity
of fluoride toxicity. The WHO (1984) guidelines suggest that in areas with a warm
climate, the optimal fluoride concentration in drinking water should be less than
1 mg/L, whereas in cooler climates it could go up to 1.2 mg/L. The higher total
water intake in a warm climate is the logic behind setting a lower threshold of F
in drinking water for human consumption. Likewise, domestic animals also con-
sume more water during the summer and, therefore, incidence as well as sever-
ity of toxic symptoms increases during the summer season in areas endemic to
hydrofluorosis. The contribution of heat stress on pathophysiology of F toxicity
may also affect the severity of the problem.
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Chapter 6
Fluoride Analysis
Abstract Fluoride analysis in biological and environmental samples is essential

for definitive diagnosis of fluoride toxicity and determining the source of excess
fluoride exposure. Several analytical techniques based on titrimetry, colorimet-
ric methods, enzymatic analysis, ion chromatography, proton activation analysis,
potentiometric analysis, and so on have been developed. However, potentiometric
analysis using an ion selective electrode is rapidly becoming a popular and widely
accepted method for determination of fluoride concentration in various biological
and environmental samples. Fluoride concentrations in aqueous samples may be
estimated directly, whereas preanalysis processing by alkali fusion, ashing, acid
extraction, and microdiffusion may be required for others to free the fluoride ions
from organic matrix/insoluble complexes. Various aspects of sample collection,
preservation, processing, and fluoride analysis by potentiometric technique are
described in detail in this chapter.
Fluoride analysis in biological and environmental samples can be performed

applying several techniques. In this chapter different analytical techniques are
described in brief and a detailed account of potentiometric analysis using an ion
selective electrode (ISE) is given. Estimation of fluorine and its compounds pre-
sent in the atmosphere in gaseous or particulate form is beyond the scope of this
chapter.
6.1 Titrimetry
In this technique, a titrant, usually containing a rare earth metal such as thorium or
zirconium is added to the solution containing fluoride. The fluoride ions react with the
titrant and then the solution is treated with an indicator dye, such as Alizarin Red S or
SPADNS (Trisodium 2-(parasulfophenylazo)-1,8-dihydroxy-3,6-napthalenedisulfonate

70 6 Fluoride Analysis
salt). The change in color that occurs when excess thorium/zirconium reacts with
the indicator dye is detected either visually or by using instrumentation techniques
(Jacobson and Weinstein 1977). The pH and composition of the solution must be care-
fully controlled and interference of other substances is avoided by prior separation. It
is an accurate but cumbersome method. Furthermore, the accuracy is dependent on the
skills and experience of the analyst.
6.2 Colorimetric/Spectrophotometric Methods
Aluminum, iron, thorium, zirconium, lanthanum, or cerium react with an indicator

dye to form a complex having a low dissociation constant. Colorimetric/photometric
methods for fluoride analysis are based upon the bleaching of the colored complexes
of these metals with organic dye when fluoride is added (WHO 1984). The degree of
bleaching is directly proportional to fluoride ion concentration. SPADNS is the most
commonly used dye, but several other dyes including alizarin, eriochrome-cyanine
R, bromocresol orange, and the like can also be used. When the zirconium-SPADNS
complex is used the degree of color loss is measured at 570 nm. Fluoride test kits
based on SPADNS dye are being manufactured by several companies. The mini-
mum detection limit for such kits varies from 0.02 to 0.08 mg F/L water. These kits
give more accurate results when water F concentration ranges from 0.2 to 1.4 mg/L.
Samples with higher fluoride concentrations require dilution before analysis.
Aluminum ions, chloride ions, iron ions, turbidity, high alkalinity, coloring
substances, phosphate, and sulfate ions act as interfering agents in colorimetric
estimation methods. The water samples, therefore, should be processed (by acid
distillation) to remove these interfering agents before analysis.
6.3 Gas Chromatography
Gas chromatography is an excellent method for estimation of trace levels of fluo-

ride in biological samples. It requires a small sample volume and can measure both
bound/organic fluoride and free fluoride ions. In this technique, derivatization and
extraction are achieved using trimethylchlorosilane (TMCS) in toluene to produce
trimethylfluorosilane (TMFS). The TMFS peak heights in sample and standard solu-
tions are compared.
6.4 Neutron or Proton Activation Technique
Neutron or proton activation of fluorine-9 is a highly accurate method for detec-

tion of trace amounts of fluorine present in biological samples. This method
requires minimal sample volume and very little or no preanalysis sample
6.4 Neutron or Proton Activation Technique 71
processing. The emitted gamma rays or x-rays are measured using lithium-drifted
germanium detectors. Availability of the instrument is the major limiting factor for
large-scale use of this technique.
In addition to the above-mentioned techniques, several other techniques
including capillary electrophoresis, atomic absorption spectrophotometry, and

photon activation analysis, among others, can also be applied for fluoride analysis.
Techniques based on inductively coupled plasma atomic emission spectrometry
(ICP-OES) have also been developed for fluoride analysis (Kovacs et al. 2009).
Readers are advised to consult the literature for learning other analytical techniques
(Wang and Xu 1993; Wang and Zhou 1994; Wen et al. 1996; Yin et al. 2001).
6.5 Potentiometric Analysis/Ion Selective

Electrode (ISE) Method
Potentiometric analysis using an ion selective electrode is a simple, reliable, low-

cost technique, widely accepted as a standard method for fluoride analysis. This
technique has good sensitivity (detection limit up to 10−6 mol/dm3) and accuracy
provided ionic strength, temperature, pH, and concentration of certain interfer-
ing ions are regulated within a specific limit. Results are more accurate when the
fluoride concentration in solution is low (less than 15 mg/L). Because of all these
advantages, it is rapidly becoming the method of choice for fluoride analysis in a
wide variety of environmental and biological samples.
It is important to note that this method measures only the free fluoride ions,
rather than total fluoride concentration in the solution, unless the sample is
processed to convert all complex fluoride compounds into free fluoride ions.

Fluoride in any biological and environmental sample may be present in an
insoluble organic form that may not liberate fluoride ions during the analysis
without proper processing. Hence, preanalytical processing of samples is often
required to convert insoluble fluoride compounds into soluble inorganic form.
There are several methods by which samples can be processed for fluoride
analysis by the ISE method. The microdiffusion technique such as the acid-

hexamethyldisiloxane (HMDS) diffusion method as described by Taves (1968)
is the most accurate method. Methods involving acid or alkali digestion may not
convert all complex organic fluoride into ionic form that can be detected by ISE
(Venkateswarlu 1983). Open-ashing methods may result in the loss of volatile
fluoride compounds when the temperature exceeds 550 °C or they may result in
contamination with extraneous fluoride (Venkateswarlu 1983; Campbell 1987).
6.5.1 Working of Ion Selective Electrode
In potentiometric analysis, an ion selective electrode measures the electrode

potential (in mV) across a sensing membrane and this potential is referenced
against a silver/silver chloride half-cell (denoted Ag/AgCl) reference electrode,

which has a potential of 0.197 volt relative to the standard hydrogen electrode. The
output potential is proportional to the amount or concentration of the selected ion
(here fluoride ion) in the solution.
Fluoride selective electrodes are available in two different types, fluoride
combination electrodes and single junction reference electrodes. In the single
junction reference electrode, the reference and sensing electrodes are built into
one electrode. This kind of electrode is more useful as it can work even with a
small sample volume. The reference electrode has a porous plug located close
to the fluoride sensing membrane (about the size of a pinhead). The reference
electrode is filled with a solution (3 M KCl/saturated AgCl) called an e lectrode
filling solution which diffuses slowly in the solution in which electrode is
dipped. The electrode filling solution is usually supplied by the m anufacturer of
the ISE. The level of electrode filling solution in the electrode during fluoride
estimation should be monitored carefully. The sensing membrane in the fl uoride
ion selective electrode is made up of lanthanum fluoride (LaF3) doped with
Europium (II) fluoride, which is impervious to all ions except fluoride (and
hydroxyl ions).
6.5.2 Factors Affecting Performance of ISE
The ISE performance depends upon several factors including pH and temperature
of the working solution, presence of interfering ions in the test solution, and so on.
6.5.2.1 pH
The fluoride selective membrane is permeable only to fluoride ions. However,

hydroxyl ions (OH−) have an electric charge and ionic radius equal to fluoride
hence can penetrate the LaF3 crystal structure, and the instrument may give a
false high result when the test solution pH is high. For example, at pH 8.5, the
OH− concentration will be 0.054 mg/L; therefore the fluoride concentration will
be sensed higher by this amount. The fluoride selective electrode, however, has a
preference for the determinant (fluoride) ion over the interfering (hydroxyl) ions;
the ratio of these two is called the selectivity coefficient. For fluoride selective
electrodes, the selectivity coefficient for hydroxyl ion is 10−1.
Low pH (below 5.0) is also undesirable because fluoride ions combine with
hydrogen ions (H+) producing HF and HF2 which cannot diffuse through the fluo-
ride selective membrane. At pH below 5.0, the electrode will measure lower than
the actual concentration of the fluoride ions. Therefore, the pH of the sample is
very important and ideally it should be maintained close to 5.5.
6.5 Potentiometric Analysis/Ion Selective Electrode (ISE) Method 73
6.5.2.2 Temperature Variation
Variation in temperature during calibration and actual estimation can influence

the accuracy of the measurement. For example, a change in temperature by one
degree Celsius can result in a 2 % error in fluoride concentration. Therefore, the
temperature of the standard solutions and samples must be equal and should not
vary much during the calibration and analytical procedures.
6.5.2.3 Presence of Fluoride Complexing Ion
Certain ions including Fe3+, Al3+, Ca2+, Cu2+, and Mg2+ can interact with flu-
oride ions forming a salt that is impervious to the fluoride selective membrane.
Hence, the electrode will give an erroneous result when high concentrations of
these ions are present in the sample. However, the error is negligible at lower
concentrations of Fe3+ (up to 0.3 mg/L), Al3+ (up to 0.04 mg/L), Ca2+(up to
330 mg/L), Cu2+ (up to 0.4 mg/L), and Mg2+ (up to 500 mg/L) because TISAB
(total ionic strength adjustment buffer) added to the sample contains cheaters that
complex with these interfering ions.
6.5.3 Sample Collection and Preservation
Samples and reagents for fluoride analysis should be stored in polyethylene or

polypropylene containers. Glass vials or containers should be avoided as silica
present in glass may react with fluorine to give erroneous results. The method of
sample collection and preservation is summarized in Table 6.1.
6.5.4 Electrode Preparation
The fluoride selective electrode is filled with electrode filling solution (supplied/
recommended by the electrode manufacturing company) before fluoride analysis.
After filling the solution up to the recommended level, hold the electrode body
with one hand and push down the electrode cap with the thumb to drain out one
to two drops of the filling solution. The level of electrode filling solution during
F analysis should be at least one inch above the level of the sample. Care should
be taken that the O-ring of the electrode is moistened before estimation by tilting
the electrode, and the fill hole should always remain open during the analysis.
The ISE is now connected with the meter through the cable and the meter is
switched on.
74
Table 6.1 Collection and preservation of samples for fluoride analysis using ISE
Sample Sample size/volume Preservative Stability Comments
Water 50 ml 0.2 g ethylene 2 weeks when stored at 4° C. Remove coarse particles
dinitrilo-tetraacetic acid Up to one month or more if by simple filtration/
(EDTA), disodium salt kept at −20 °C sedimentation
Urine 50 ml midstream urine collected 0.2 g EDTA, 2 weeks when stored at 4 °C. As above
preferably during morning hours disodium salt Up to one month or more if
kept at −20° C
Blood 20 ml blood/plasma/serum Heparin at the rate of Plasma/serum samples can Plasma/serum is better than
20 I.U. per ml blood be stored for 2 weeks at 4 °C whole blood for storage
and up to one month or more
at −20 °C
Bone A piece of flat or long bone Remove flesh, dry and Bone ash can be preserved Muscle and fat layer is
(approximately 10 g) convert into ash after alkali up to one month or more removed carefully
fusion
Fodder Collect at least 20 samples (each Weigh the fodder and dry Can be stored at 4 °C for a See the guidelines of centre
equivalent to 100 gm dry matter) in shed. Can also be dried month d’expertiseen analyse
and pool them to make one in oven at 70–80 °C for environnementale du
composite sample 24–48 h Québec*
Feed/feed-supplement/ Collect 20 representative samples Take 100 gm composite Can be stored for several Ashing may be essential to
mineral mixture randomly, each about 100 g and sample. No added months at room temperature remove organic material
mix them properly to make a preservative is required
6
composite sample
Egg One whole egg No added preservative is 2 weeks when stored at 4 °C
required
*Sampling Guide for Environmental Analysis: Booklet 6—Forage Sampling for Fluoride Analysis, Centre d’expertiseen analyse environnementale du
Québec, http://www.ceaeq.gouv.qc.ca/documents/publications/guides_ech.htm
Fluoride Analysis
6.5.5 Checking Electrode Operation
Electrode operation can be checked by measuring the slope of the electrode.

Electrode slope is defined as the change in millivolts observed with every tenfold
change in fluoride concentration. For checking electrode operation, the meter is
set in mV (millivolt) mode. Take a beaker and place 100 ml mixture of TISAB
and deionized water solution in a suitable ratio. Add the fluoride standard to adjust
the fluoride concentration of the solution 1 ppm. Take the reading in mV. Again
take 100 ml mixture of TISAB and deionized water solution and add the fluoride
standard solution to adjust the fluoride concentration of the final solution 10 ppm.
Record the mV reading. There should be a 54–60 mV difference between the two
readings when the solution temperature is between 20 and 25 °C.
6.5.6 Preparation of Standards
Place sodium fluoride powder at 110 °C for 2 h in a desiccator before weighing.

Take 2.21 g dried sodium fluoride and dissolve in one liter deionized water. This
will give a stock standard solution with 1000 ppm fluoride concentration. A range
of standards of decreasing concentration are prepared by serial dilution of the
stock standard solution. The concentration range of standards used for calibration
of the instrument must be wide enough to accommodate the expected fluoride con-
centration in the test solution (sample). Readymade fluoride stock standard solu-
tions are also available on the market.
6.5.7 Analytical Techniques
Several analytical techniques can be used for fluoride analysis using ISE.
The selection of a technique depends upon sample volume, expected fluoride
concentration in the sample, chemical properties of the sample solution, and
individual choice. A brief description of important analytical techniques is given
below.
6.5.7.1 Direct Calibration
In this technique, the instrument is calibrated using a series of standard fluoride

solutions, and thereafter F in the test sample is analyzed directly. This technique
is suitable when the fluoride concentration in the test sample is fairly high, a
large variation in F is expected, or rapid analysis of a large number of samples is
required. Both standards and test solutions are mixed with TISAB to make their
ionic strength equal. Temperature of the standards and test solutions should be
nearly equal and the difference between temperatures should not exceed 2 °C. In
this method, the volume of the sample taken has no influence on the measurement.
Concentration of fluoride in the test solution is expressed as ppm or moles per liter
(one mole per liter fluoride is equivalent to 19,000 ppm). Details about calibra-
tion of the instrument are available in the literature (Buck and Cosofret 1993). It is
recommended to prepare and use four standards for calibration of the instrument.
Each standard should have ten times less fluoride concentration than the last. For
example, standards containing 1000, 100, 10, and 1 ppm fluoride may be used for
calibration. Readings can be taken in mV or directly in concentration mode.
6.5.7.2 Incremental Techniques
Incremental techniques are best suited when complexing or interfering ions in the
test sample are present in excess (50–100 times the test ion, i.e., fluoride ion). The
instrument can be run in mV or in concentration mode. Incremental techniques
can be further classified into the following categories:
(i) Known Addition

This technique is useful for test samples containing low fluoride concentration,
checking results of the direct calibration, or fluoride estimation in samples con-
taining a high concentration of interfering ions. Here, the electrode is immersed in
the sample and TISAB mixture and an aliquot of fluoride standard is added. The
fluoride concentration in the test sample is determined by calculating the differ-
ence in readings before and after addition of the fluoride standard solution.
(ii) Known Subtraction

This technique is based upon titration and utilizes the stable standard of any spe-
cies that reacts completely with the test ions (fluoride) present in the solution.
Here, standards of the reacting species (ions) are added to the test sample and
the difference in reading before and after addition is calculated. In this technique,
knowledge of the stoichiometric ratio between the standard (reacting ion) and
sample (test ion/fluoride) is necessary.
(iii) Analate Addition

The electrode is immersed in a mixture of fluoride standard solution and TISAB
and a measured quantity/volume of the test solution is added. The difference in
readings before and after addition of the test solution is calculated and the fluo-
ride concentration is determined. This technique is useful for determining fluoride
concentration in soluble solids or powders, viscous samples, samples with high
fluoride concentration, or when interfering ions are present in high concentra-
tion in the sample. However, it is not suitable for samples having a low fluoride
concentration.
(iv) Analate Subtraction

This method is used for analysis of those ions for which ion selective electrodes are
not available. For example, a fluoride selective electrode can also be used for analysis
of aluminum ions in the solution by detecting the amount of sodium fluoride required
to react completely with all aluminum ions present in the solution. Aluminum reacts
with fluoride ions to form aluminum fluoride, which is insoluble in water.
(v) Titration
In titration techniques, an ion selective electrode is used just as a litmus or universal
indicator for determining the end point of the titration. The advantage of using an
ion selective electrode is an increase in the accuracy of results, although it is a time-
consuming cumbersome process. This technique is seldom used for fluoride analysis.
6.5.8 Fluoride in Acid Solution
The pH of the acidic solutions (pH below 5) must be adjusted into a weakly acidic
to a weakly basic range before fluoride analysis. Addition of a strong base such as
sodium hydroxide is not recommended for pH adjustment because the total ionic
strength of the adjusted samples and standards will vary with the amount of added
sodium hydroxide. A sodium acetate solution (15 %) can be used for adjusting the
pH of acidic solutions.
Procedure
1. Prepare 15 % sodium acetate solution in deionized water.
2. Prepare a background solution containing all components of the test solution
except fluoride.
3. Prepare standards in the concentration range of the test samples by adding fluo-
ride to the background solution. Dilute each standard tenfold with the sodium
acetate solution (9 parts sodium acetate and 1 part standard).
4. If a standard prepared from the background solution gives the same reading
(after dilution with sodium acetate) as a standard prepared from pure sodium
fluoride, then use of the background solution is not essential. In such cases,
calibrate with standards prepared in deionized water.
5. Calibrate the electrode using standards and measure the fluoride c oncentration
in the sample after adding sodium acetate in a ratio similar to that used for
standard preparation.
6.5.9 Fluoride in Alkaline Solution
In alkaline solutions (pH above 9.5), the fluoride selective electrode responds to
both hydroxyl ion as well as fluoride ion and gives an erroneous result. Therefore,
the pH of the test solution is adjusted between 5 and 6 with a 4 M buffered potas-
sium acetate solution. Samples and standards are diluted in a 1:10 ratio with this
solution.
Procedure
1. Prepare a 4 M buffered potassium acetate solution: dilute 2 parts 6 M ace-
tic acid (CH3COOH) with one part distilled water. Mixing should be done in
a beaker placed in a water bath. Add 50 % KOH solution to the acetic acid
slowly, stirring constantly until pH becomes 5.
2. Prepare standard, calibrate electrode, and measure F concentration in test sam-
ple as described for fluoride in acid solutions.
6.5.10 Points to Remember During Fluoride Analysis
1. Samples, standards, and TISAB should be brought to the same temperature

before fluoride analysis. Temperature of the solution in which the electrode is
dipped should not exceed 100 °C. The temperature range of 20–25 °C is the
most suitable for fluoride analysis.
2. In all analytical samples and standards, TISAB must be added in equal ratio.
3. Samples and standard solutions should be stirred well during estimation.
4. After immersing the electrode in a solution, check the electrode sensing surface
for air bubbles. If air bubbles are present over the sensing surface, remove the
electrode and dip it again in the solution.
5. Rinse the electrode with deionized water between measurements and blot the
electrode dry with tissue paper. Do not wipe or rub the electrode sensing surface.
6. Verify calibration of the electrode every 2 h during measurement by placing the
electrode in the standard solution used for calibration. If the value has changed
by more than 2 %, recalibrate the instrument.
7. Adequate response time must be given to stabilize the reading. A long response
time is required when fluoride concentration in the test solution is low or large
variation in F concentration between two subsequent samples is present.
6.5.11 Total Ionic Strength Adjustment Buffer (TISAB)
TISAB is an essential component of all potentiometric assays using an ion selec-

tive electrode. It acts as buffer, increases the background ionic strength of the
solution to a relatively high level, and masks most chemical interference (interfer-
ing ions) present in the test solution. TISAB comes in variable strength (I–IV).
Requirement of a particular TISAB and ratio of sample versus TISAB depends
upon the expected fluoride concentration and presence of inhibitory ions in
Table 6.2 Use of different types of TISAB in fluoride analysis

TISAB Indications for use Sample: TISAB Comments
type ratio*
I When test solution has low 1:1 Can be used when traces of
ionic strength, concentration magnesium, calcium, chloride,
of fluoride complexing ions nitrate, sulfate, and phosphate
and fluoride are low (below are present in the sample
0.4 ppm)
II When sample has fairly high 1:1 Better to use when fluoride
ionic strength and high iron concentration in test solution is
and silicate ions more than 2 × 10−5 M
(0.4 ppm) and/or iron
and silicate ions are high
III When the sample contains 10:1 CDTA complexes with
more than 100 ppm alu- aluminum and iron ions. In a
minum or iron ions 1 ppm fluoride sample, it can
complex with 5 ppm Al or Fe
ions. However, there will be an
error of approximately 5 %
in the measurement of 1 ppm
fluoride in the presence of
200 ppm aluminum or iron ions
IV When sample contains 1:1 It can complex more than
100 ppm or more aluminum 100 mg/L of iron or aluminum in
or iron ions the presence of 1 mg/L fluoride.
A measurement of 1 mg/LF
gives rise to an error of 5 % in
the presence of 200 mg/L iron or
aluminum ions
*May vary with concentration of interfering ions, ionic strength, and pH of the sample
the sample. Different types of TISAB have been developed, which vary in their
chemical composition. However, they all contain three major constituents: an ion
strength adjusting salt, a complexing ligand, and one pH buffer. TISABs II, III,
and IV commercially supplied by different companies, also have variable ingredi-
ents except the CDTA (1,2-cyclohexylenedinitrilotetraacetic acid) which acts as a
complexing ligand. Methods for preparation of different types of TISAB are given
in the appendix. Indications for use and sample volume, and the TISAB ratio for
different kinds of TISAB are given in Table 6.2.
6.5.12 Electrode Filling Solution
It connects the reference element to the sample and maximizes stability of the
potential developed at the reference element to the filling solution interface and
the filling solution to the sample junction. The electrode filling solution usually
contains 0.1 M sodium fluoride in 0.1 M sodium chloride (Khandpur 2007).
6.5.13 Storage of Ion Selective Electrode
Proper storage of the ISE is very important for its optimal performance and long
life. These steps should be followed during storage:
1. Storage between daily measurements and up to one week: Rinse the electrode
with deionized water and place in a solution containing 5 mg F/L and TISAB
in equal ratio. The filling solution inside the electrode should not be allowed to
evaporate as crystallization will occur. If the electrode filling solution is evapo-
rated to set as solid crystals, dip the electrode in warm (not hot) water for a few
minutes and gently remove the dry material present inside.
2. Storing for one week or longer (dry storage): Remove the electrode filling solu-
tion by pushing down the cap, flush and clean the electrode with deionized
water, mount the protective electrode cap, and store dry. Seal the filling hole
with parafilm and store at room temperature.
3. Disassembly (means unscrewing the cap and taking out the electrode): Should
not be done unless proper cleaning is essential.
4. Conditioning after dry storage: Rinse the electrode with deionized water, fill
the electrode filling solution, and soak in mixture consisting of 5.0 mg/L fluo-
ride and TISAB in ratio 1:1 for 30 min.
6.5.14 Fluoride in Aqueous Samples
Aqueous samples including water, urine, saliva, milk, and other body fluids usu-
ally do not require preanalysis processing. They can be analyzed directly by the
ISE method after mixing with TISAB in a suitable ratio. Care should be taken
when excess interfering ions are present, sample is turbid, or fluoride concentra-
tion is high in the sample. Direct calibration is a simple and rapid technique suit-
able for handling a large number of samples. Different steps can be summarized as
follows:
1. Prepare four standards that bracket the expected fluoride concentration range
and differ in concentration by a factor of 10.
2. Take a 50 ml standard solution with the lowest fluoride concentration and add
50 ml TISAB II or 5 ml of TISAB III into a 150 ml beaker and stir thoroughly.
3. Place the ISE into the beaker. Wait for a stable reading and then calibrate the
instrument.
4. Repeat the process for other standards. Rinse with distilled water and blot dry
the electrode before immersing it into the next solution.
5. Now take a 50 ml sample and 50 ml TISAB II and stir thoroughly.
6. Place the ISE into the sample mixture and wait for a stable reading.
Table 6.3 Methods for sample processing for fluoride assay in soft and calcified tissues
Sample Preanalysis processing/F extrac- Reference
tion method
Soft tissues (liver, kidney, Dry combustion/acid treatment Inkielewicz et al. (2003)
etc.)
Hair Alkali treatment Stolarska et al. (2000)
Obrink method Kokot and Drzewiecki
(2000)
Hair and fingernail Wash in diethylether, dry Schamschula et al. (1985)
and decompose in sodium
hydroxide
Bone Ashing, dissolve in perchloric Boivin et al. (1988)
acid
Bone Ashing, HMDS facilitated Buzalaf et al. (2005)
diffusion
Fingernail HMDS facilitated diffusion Whitford et al. (1999)
Tooth enamel Decalcify in perchloric acid Schamschula et al. (1982)
6.5.15 Fluoride in Soft and Calcified Tissues
Soft and calcified tissues require preanalysis processing to free the fluoride from the
organic or inorganic tissue matrix. Tissues can be prepared in different ways such as
simply dissolving in acids (perchloric acid), ashing and dissolving in p erchloric or
hydrochloric acid, or ashing and trapping of fluoride by a microdiffusion technique.
Ashing frequently results in refractory fluorides, hence the solubility of the ash
is ensured by fusing with alkali carbonate or hydroxide. After fusion, the melt is
dissolved and the fluoride is separated for analysis. Care should be taken d uring
sample processing to avoid contamination and loss of fluoride due to volatilization.
The percentage of fluoride recovery after processing by different techniques
is
variable; the highest recovery is achieved by an acid-hexamethyldisiloxane
(HMDS)—facilitated microdiffusion technique (Taves 1968).
A summary of the literature on different preanalysis sample processing
techniques applied for fluoride analysis in soft and calcified tissue is given in
Table 6.3. Readers can consult these references for a detailed methodology. A few
simple methods are also described in detail in the appendix.
6.5.16 Fluoride in Vegetation and Fodder Samples
Fluoride concentration in plants varies with the part of the plant, age of the
plant, type of soil, environmental conditions, and contamination with industrial
effluents. Industrial activities may emit particulate fluoride compounds that adhere
to leaves and stems. Hence do not wash the sample if overall intake of fluoride
through v egetation/fodder is to be estimated. However, washing is required before
processing if actual fluoride concentration in plant tissue is to be determined.
Sample preparation using one or more techniques such as distillation, acid
digestion/extraction, and alkali fusion is essential to free the fluorine compounds
from organic materials. Two simple methods for fluoride assay in vegetation/
fodder have been described in the appendix.
6.5.17 Fluoride in Soil, Feed, and Mineral Mixture
Collection of representative soil samples is essential for accurate results. Usually

multiple samples from different depths are collected, dried, pulverized, and
homogenized to make a composite sample. Organic matter present in the soil is
removed by ashing using fluoride-free calcium oxide as fixative (Horton 1962).
The soil contains labile fluoride and refractory fluoride in the form of iron,
aluminum, and silicon compounds. The labile fraction is of greater significance
than total soil fluoride, as this fraction is easily available for plant and animal
uptake (Murray 1982). This fraction can be estimated by simple acid extraction
(Madhavan and Subramanian 2002). Refractory fluoride compounds are converted
into readily soluble compounds by ashing, digestion, alkali fusion, and fluoride
trapping by microdiffusion techniques (Martínez-Mier et al. 2009). These steps are
required when estimation of total fluoride concentration in soil, feed, and mineral
mixture is required. Care should be taken during sample processing to avoid
contamination and loss of fluoride due to volatilization. One simple method for
fluoride analysis in a soil sample is given in the appendix.
References
Boivin G, Chapuy MC, Baud CA, Meunier PJ (1988) Fluoride content in human iliac bone:
results in controls, patients with fluorosis, and osteoporotic patients treated with fluoride.
J Bone Miner Res 3:497–502
Buck RP, Cosofret VV (1993) Recommended procedures for calibration of ion-selective
electrodes. Pure Appl Chem 65:1849–1858
Buzalaf MA, Caroselli EE, deCarvalho JG, Oliveira RC, da Silva Cardoso VE, Whitford GM
(2005) Bone surface and whole bone as biomarkers for acute fluoride exposure. J Anal
Toxicol 29:810–813
Campbell AD (1987) Determination of fluoride in various matrices. Pure Appl Chem 59:695–702
Horton CA (1962) Fluorine. In: Kolthoff JM, Elving PJ (eds) Treatise of analytical chemistry,
vol. 7, Part 2. Interscience Publishers, New York, pp. 207–334
Inkielewicz I, Czarnowski W, Krechniak J (2003) Determination of fluoride in soft tissues.
Fluoride 36:16–20
Jacobson JS, Weinstein LH (1977) Sampling and analysis of fluoride: methods for ambient air,
plant and animal tissues, water, soil and foods. J Occup Med 19:79–87
References 83
Khandpur RS (2007) Handbook of analytical instruments, 2nd edn. Tata McGraw-Hill

Education, Noida
Kokot Z, Drzewiecki D (2000) Fluoride levels in hair of exposed and unexposed populations in
Poland. Fluoride 33:196–204
Kovacs M, Magarini R, Halmos P (2009) Automated indirect fluoride determination by ICP-OES
using discontinuous-flow analysis. Toxicol Environ Chem 91:1217–1227
Madhavan N, Subramanian V (2002) Fluoride in fractionated soil samples of Ajmer district,
Rajasthan. J Environ Monitor 4:821–822
Martinez-Mier EM, Soto-Rojas AE, Buckley CM, Margineda J, Zero DT (2009) Evaluation
of the direct and diffusion methods for the determination of fluoride content in table salt.
Commun Dent Health 26:204–210
Murray F (l982) Fluoride retention by sandy soils. Water Air Soil Pollut 20:361–367
Schamschula RG, Sugar E, Un PS, Toth K, Barmes DE, Adkins BL (1985) Physiological
indicators of fluoride exposure and utilization: an epidemiological study. Commun Dent Oral
Epidemiol 13:104–107
Schamschula RG, Sugart E, Agus HM, Un PS, Toth K (1982) The fluoride content of human
tooth enamel in relation to environmental exposure to fluoride. Aust Dent J 27:243–247
Stolarska K, Czarnowski W, Urbanska B, Krechniak J (2000) Fluoride in hair as an indicator of
exposure to fluoride compounds. Fluoride 33:174–181
Taves DR (1968) Determination of submicromolar concentrations of fluoride in biological
samples. Talanta 15:1015–1023
Venkateswarlu P (1983) Overview of analytical methods for fluorine in air, water, soil vegetation,
body fluids and tissues. In: Shupe JL, Peterson H, Leone N (eds) Fluorides: effects on
vegetation, animals and humans. Paragon Press, Salt Lake City, UT, pp. 19–52
Wang CY, Xu J (1993) Developments in the analysis of fluoride 1980-1990. Fluoride 26:197–202
Wang CY, Zhou YM (1994) Developments in the analysis of fluoride 1991-1993. Fluoride
27:97–107
Wen ML, Li QC, Wang CY (1996) Developments in the analysis of fluoride 1993–1995. Fluoride
29:82–88
Whitford GM, Sampaio FC, Arneberg P, Von Der Fehr FR (1999) Fingernail fluoride: a method
for monitoring fluoride exposure. Caries Res 33:462–467
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Organization, Geneva
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Chapter 7
Mitigation and Prevention of Fluorosis
Abstract Nonavailability of a specific antidote and the irreversible nature of bony

and dental lesions poses a great challenge in management of fluoride toxicity;
hence prevention remains the best way to minimize suffering of animals. In
hydrofluorosis-endemic areas surface water, rain-harvested water, or defluoridated
groundwater should be offered to animals for drinking. Several defluoridation
techniques including the Nalgonda technique, use of activated alumina, KRASS
technology, reverse osmosis, distillation, and electrolysis are also available, but
the requirements of infrastructure, technical expertise, and high running cost are
major factors limiting their use in animal husbandry. Supplementation of certain
minerals, vitamins, antioxidants, and plant products can alleviate toxic symptoms
of fluorosis. Minimizing industrial fluoride emission by adapting advanced tech-
nology and imposition of stringent laws can help safeguard animal health from
industrial fluorosis. The generation of public awareness, particularly in rural areas,
is essential for effective use of various ameliorative and preventive measures avail-
able for fluorosis in animals.
In human beings, clinical symptoms, up to some extent, can be mitigated by pre-

venting high fluoride intake and long-term oral supplementation of certain min-
erals, vitamins, chemical compounds, antioxidants, and plant products (Susheela
and Bhatnagar 2002). Likewise, withdrawal of fluoride uptake and supplementa-
tion of certain therapeutic agents can alleviate animal suffering to a limited extent.
Nevertheless, dental and bony lesions of fluorosis cannot be fully reversed and
prevention remains the most effective way to minimize animal suffering. A brief
description of important preventive and therapeutic measures for fluorosis in ani-
mals is given in this chapter.

86 7 Mitigation and Prevention of Fluorosis
7.1 Minimizing/Withdrawing Excess Fluoride Intake
As described earlier, drinking groundwater containing excess fluoride, poor qual-

ity mineral mixture or feed supplement containing poorly defluoridated rock
phosphate, and industrial contamination of water, feed, and fodder are important
sources of excess fluoride intake for animals. In drinking water, fluoride is usually
present in soluble form which is rapidly and extensively absorbed in the gastroin-
testinal tract. Hence drinking water with high fluoride concentration is the most
common cause of fluorosis in animals as well as human beings. Therefore, reduc-
ing fluoride uptake through drinking water is the most important preventive meas-
ure that can be achieved in several ways as described below.
7.1.1 Search for Safe Groundwater Source
In almost all hydrofluorosis-endemic areas, a large variation in F concentration

in groundwater even coming from closely located sources is a common feature.
Hence, it is possible that one source may contain very high F concentration and
another may have a low or safe F level. Hence, water samples of all sources should
be analyzed for fluoride. Deep bore water sometimes contains less fluoride than
shallow bore water of the same area (Teotia et al. 1987). Only those sources (hand
pump or well) that contain a safe or minimum fluoride concentration should be used
by the community and others with high fluoride concentration should be closed.
7.1.2 Use of Surface Water
Surface water (from ponds, rivulets, etc.) usually has a lower fluoride concentra-
tion and can be used as drinking water for animals. But care should be taken as it
is often contaminated with other pollutants. Hence, purification using simple and
low-cost technologies, such as sand filtration, ultraviolet water disinfection, or
chlorine disinfection may be required before offering to animals.
7.1.3 Rainwater Harvesting
Rainwater harvesting is a technique used for collecting, storing, and using rain-
water for household, animal husbandry, and agricultural purposes. Rainwater
contains low F concentration and, therefore, can offer a low-cost source of safe
drinking water for animals even in hydrofluorosis-endemic areas. Rainwater
harvesting systems can be classified into two categories: (a) surface runoff

harvesting and (b) rooftop rainwater harvesting. In urban areas, rainwater always
7.1 Minimizing/Withdrawing Excess Fluoride Intake 87
flows as surface runoff which could be collected using suitable techniques for
recharging aquifers. In rooftop rainwater harvesting, the roof of the house serves
as catchments and the water collected flows though pipelines to a storage tank (an
enclosed rainwater harvesting tank is installed above the ground surface or may be
underground) or open catchment area (e.g., open tanks/ponds, etc.).
A rooftop rainwater harvesting system is more suitable for use in animal hus-
bandry. The system comprises several components including catchment, transpor-
tation, first flush, and filter. The catchment may consist of a terrace, courtyard, or
paved or unpaved open roof. Rainwater collected from the catchment is carried
through water pipes or drains to a storage/harvesting system. Wire mesh is fitted
between the catchment and transport system to prevent entry of coarse material.
First flush is a device used to flush off the water received in the first shower. The
first shower of rain often contains chemicals and particulate contaminants that
should be flushed off before storage of the next shower. It also helps in cleaning
the silt and other materials deposited on the roof during the dry season. Provisions
for the first-flush separator should be made at the outlet of each drainpipe. Filters
are used for removal of turbidity, color, and microorganisms that may enter in the
harvested rainwater as contaminants. Several types of homemade or commercial
rainwater filters can be used. In hydrofluorosis-endemic areas, each household
should prepare a rainwater harvesting tank for storage of rainwater for animal con-
sumption. Usually, the water collected in the rainy season may be used as drinking
water for animals year-round, although an occasional quality check is essential to
safeguard animal health. Sometimes, rejection of such water for drinking by some
animal species may be a problem.
It is important to note that in polluted areas where atmospheric fluoride concen-
tration is high, or sometimes even in those areas where atmospheric fluoride con-
centration is low, rainwater may have high fluoride concentration. For example, In
Poland, F concentration in atmospheric precipitation (rain and snow) was found as
high as 2 mg/L, whereas the F concentration in the atmosphere was within the nor-
mal range. In this case, high F concentration in rain was attributed to the flow of
polluted air from far-off western Poland and Germany (Walna et al. 2013). Hence,
fluoride analysis in rainwater is always advisable.
7.1.4 Water Defluoridation
The process of removal of excess fluoride using different physical or chemical

techniques is called defluoridation. There are several techniques available today
for water defluoridation (Table 7.1). Most of these were developed for water puri-
fication for human consumption. Their practical utility in animal husbandry is lim-
ited due to the large volume requirement and hence high operational cost. Efforts
have been made to develop low-cost techniques using indigenous materials such
as wheat bran, wood coat, charcoal, and so on for water defluoridation for large-
scale application in rural areas, but to date none appears to have high efficacy and
Table 7.1 Suitable defluoridation technique for different situations/conditions

Situation/condition Suitable technique
High fluoride concentration Reverse osmosis, activated alumina adsorption,
electrolysis
High total dissolved solvent (TDS)/brackish Reverse osmosis, electrolysis, solar
water evaporation
Low operational cost/rural areas Nalgonda technique, bone charcoal or calcined
clay adsorption, alum/lime precipitation
Large-scale use Nalgonda technique, reverse osmosis
Small-scale use Nalgonda technique, calcined clay adsorption,
activated alumina adsorption, reverse osmosis
wide public acceptability. Following are some essential qualities a defluoridation

technique should have for its large-scale use on a community basis in rural areas
(Mamatha and Rao 2005).
1. It should be user-friendly and easily used by common people without any need
for technical knowledge of handling or the requirement of electricity.
2. It should be effective in the treatment of water with different fluoride concen-
trations and water chemistry. It should be effective in the removal of fluoride
from water with variable pH and a high concentration of organic and inorganic
pollutants or sludge.
3. The taste of the treated water should not be unpleasant and no toxic residue
(such as aluminum compounds) should be left in the treated water.
4. It should be environmentally friendly and preferably the filter material should
be rechargeable or recyclable.
5. It should be portable and treatment time should be short. It should be suitable
for both domestic as well as community use.
6. It should be able to sustain different kinds of social, financial, environmental,
and technical constraints. Despite high efficacy and low cost, certain defluor-
idation techniques may not be widely accepted in the society. For examples,
bone-charcoal–based defluoridation techniques are not accepted by many reli-
gious cults in India, Thailand, and China.
The defluoridation may be performed as a continuous process or a batch pro-
cess. Defluoridation techniques/filters applied to the domestic water supply tap or
to hand-pump and reverse-osmosis-based instruments can defluoridate the water
continuously. However, many defluoridation techniques based on the precipita-
tion process can purify water in batches only; hence a given volume of water is
defluoridated at a time and is stored for household use.
Different water defluoridation techniques, based upon their applicability, can be
divided into two broad groups: (a) those that can be used for community water
defluoridation (large-scale use), and (b) those more suitable for household/individ-
ual use (small-scale use). These techniques can also be classified on the basis of
the defluoridation process principle as follows.
7.1.5 Precipitation-Based Techniques
These techniques require the addition of chemicals to form insoluble fluoride

compounds that precipitate and settle down. Calcium and aluminum salts are
commonly used in these techniques. Here the pH of the water plays an important
role in completion of the precipitation process. The amount of chemical required
depends upon the water fluoride concentration. These techniques generate sludge
which requires careful disposal to avoid pollution and chances of toxicity by acci-
dental exposure to animals and human beings.
7.1.5.1 Nalgonda Technique
The Nalgonda technique was developed in India in year 1975 by the National
Environmental Engineering Research Institute (NEERI), Nagpur, India and named
after the village where it was pioneered. It is an aluminum-sulfate-based technique
where coagulation–flocculation and sedimentation are applied. A simple model of
a treatment system that was developed for use in African countries for domestic
use is described below (Fawel et al. 2006).
Two 20-L-capacity plastic buckets are taken and fitted with taps 5 cm above the
bottom of the buckets in order to enable trapping of the sludge below the draw-
off point. The upper bucket tap is fitted with a tea sieve on which a piece of cot-
ton cloth is placed allowing the water to flow directly into the second clean water
bucket. In the first bucket, about 18 L of raw water are filled, and aluminum sul-
fate and lime are added simultaneously and dissolved/suspended by stirring with a
wooden paddle. Stirring is done fast for the first minute and then slowly for next
5 min. Water pH is checked in between; preferably it should remain between 6.2
and 7.6. Aluminum sulfate makes the water acidic and lime is added to adjust
the pH towards neutral. The amount of aluminum sulfate and lime required thus
depends upon the quality of the untreated water. Usually lime is added at 5 % of
the aluminum sulfate added for the treatment. The flocs formed are left to settle for
about one hour. The treated water is then run from the tap through the cloth into
the treated water bucket from where it is stored for daily drinking and cooking. It
is desirable to remove the treated water within a couple of hours after initiating the
flocculation process.
Advantages
1. Low-cost technique, ideal for household and community use.
2. Applicable in batch as well as in continuous operation.
3. Simple to construct, operate, and maintain. Materials required are easily available.
4. Simultaneously removes color, odor, turbidity, bacteria, and organic compounds.
5. Wastage of water is minimal.
Limitations/Disadvantages
1. Only water having fluoride concentration <10 ppm and turbidity <1500 ppm
can be treated.
2. Concentration of residual aluminum in treated water may vary from 2.01 to

6.80 ppm. The WHO-permitted maximum aluminum concentration in drinking
water is 0.2 ppm.
7.1.6 Adsorption and Ion-Exchange-Based Techniques
These techniques utilize different adsorbents including activated alumina, carbon,

bone charcoal, and synthetic ion exchange resins. When water is passed through
a contact bed or column made up of these adsorbents, fluoride is removed by ion
exchange or surface chemical reaction. These contact beds or columns saturate
after a period of operation. However, they can be regenerated or recharged sev-
eral times by chemical treatment. For example, activated alumina can be recharged
about 30 times. The operational cost of adsorption and ion-exchange-based tech-
niques is usually higher than precipitation-based techniques.
7.1.6.1 Activated Alumina-Based Technique
In this process water is passed through a filter packed with activated alumina. Activated
alumina (Al2O3) is made from aluminum hydroxide by dehydroxylation in a way that
produces a highly porous material that can adsorb fluoride, arsenic, and selenium from
water. For household purposes, activated alumina may be filled in ordinary water filter-
ing candles and used for fluoride removal (Srimurali and Karthikeyan 2008). The acti-
vated alumina (filter material) requires regeneration after 3–4 months’ use.
Limitations/Disadvantages
1. Expensive process; reactivation of filter material is cumbersome.
2. Trained personnel are required for reactivation of the filter.
3. The treated water contains high residual aluminum ranging from 0.16 to 0.45 ppm.
7.1.6.2 KRASS Technology
KRASS technology applies a direct flow-through type column for defluoridation

that can be easily recharged after its exhaustion (Agarwal et al. 1999). The fluo-
ride from water can be removed for an influent pH range of 4.3–9.0 and influent
fluoride concentration up to 24 mg/L. Residues of aluminum in treated water are
minimal in this process. The exhausted media bed is claimed to be rechargeable up
to 35 cycles (Singh and Yadava 2003).
7.1.7 Reverse-Osmosis-Based Techniques
In reverse osmosis, water is forced through a semipermeable membrane having a

pore size of 0.0001 μ, which rejects all undissolved and dissolved contaminants
of the water including fluoride up to 98 %. In addition, it also removes arsenic and
some other pollutants. Reverse osmosis is, perhaps, the most efficient and popu-
lar technology for drinking water purification. It can convert raw water into pota-
ble water as per the WHO and European Union specifications. However, the high
operational cost seems to limit its practical utility in water purification for animals.
7.1.8 Distillation-Based Techniques
Distillation units can also be used for removal of fluoride from drinking water.
This is particularly useful for conversion of brackish water into drinking water.
Electrical, coal, or other kinds of heat sources such as solar heat can be utilized for
water distillation.
7.1.9 Electrocoagulation/Electrolysis-Based Techniques
Instruments based on the principle of electrolysis have aluminum plate electrodes

that are placed in the raw water containing excess fluoride. During electrolysis, the
anode is ionized and fluoride is removed by complex formation, adsorption, pre-
cipitation, and coagulation.
Domestic animals consume a large quantity of drinking water. Hence, low-cost
techniques are more suitable for water defluoridation. Readers are advised to con-
sult the WHO monograph, “Fluoride in Drinking Water” (Fawel et al. 2006), for
details about some other water defluoridation techniques.
7.2 Preventive and Therapeutic Measures
Although a specific antidote for fluoride is not available, several minerals, vita-
mins, drugs, hormones, and plant products have been claimed effective in mitigat-
ing fluoride toxicity in animals (NRC 1974; Wheeler and Fell 1983).
7.2.1 Minerals, Drugs, and Other Chemicals
Gastric absorption of fluoride is significantly modulated by dietary factors includ-

ing the presence of certain divalent or trivalent cations (Rao 1984; Cerklewski
and Ridlington 1987). Supplementation of chemical compounds containing these
cations can reduce fluoride absorption, hence delay progression of the disease or
help alleviate clinical symptoms.
Different aluminum salts (viz. hydroxide, chloride, and sulphate) have been
tried with varying degrees of success. Aluminum ions form an insoluble c omplex
with fluoride and reduce absorption of fluoride from the gastrointestinal tract
(Spencer et al. 1985). Feed containing 3 % aluminum sulphate and chloride in
equal ratio reduced fluoride absorption by 15 % (Grunder 1972). In another study,
a 33–45 % decrease in fluoride absorption and lower serum, urine, bone, and teeth
fluoride concentrations were observed in sheep after administration of a luminum
sulphate (Kessabi et al. 1988). Aluminum chloride has also been reported effec-
tive in alleviation of experimental fluorosis in calves and sheep (Said et al. 1977;
Mehra 1981). Dietary supplementation of small doses of aluminum hydroxide
with 15 mg of fluoride as sodium fluoride greatly reduced the plasma fluoride
levels (Spencer et al. 1981). Severity of clinical signs in sheep experimentally
intoxicated with fluoride decreased when aluminum hydroxide was administered
simultaneously (Zhai et al. 1987).
Calcium compounds are also effective in reducing the toxic effects and fluoride
burden in animals. Lower retention of fluoride in the femur of rats was observed
when calcium chloride was added to the diet along with sodium fluoride (Weddle
and Muhler 1954). Calcium carbonate supplementation in rats is helpful in main-
taining a serum fluoride concentration at a less toxic level (Ekambaram and Paul
2002). Addition of 200 g calcium carbonate/day in the diet can reduce the sever-
ity of clinical symptoms in lactating animals given a high dose of fluoride (Suttie
et al. 1957). As with aluminum, it also reduces the intestinal absorption of fluoride
(Harrison et al. 1984). Lower dietary calcium intake is considered a predisposing
factor for fluorosis in humans (Mithal et al. 1993; Moudgil et al. 1996).
Boron supplementation reduces the severity of osteofluorosis (Elsair et al.
1980). In a study on water buffalo calves (Bubalus bubalis) exposed to 60 ppm
elemental fluoride (as sodium fluoride) on dry matter basis in concentrate ration
for 3 months, supplementation of borax (140 ppm elemental boron on dry
matter basis) in concentrate ration helped minimize fluoride-induced changes

in the hemogram and urinary biochemical profile (Bharti et al. 2007). Likewise,
magnesium and selenium compounds are also reported to alleviate animal

fluorosis (Han et al. 2006; Khandare et al. 2011).
Practical use of these chemical compounds for a long period is, however, not
advisable due to their deleterious side effects. Feeding of aluminum sulphate
(1 %) depressed the milk yield of cows (Burns and Allcortt 1964). The addition
of aluminum salts can reduce the palatability of the feed (Boddie 1960; Burns and
Allcortt 1964). Moreover, aluminum has been found to enhance the toxic effects
of fluoride in in vitro studies (Van-der-voet et al. 1999). Fluoride induces release
of IL-6 and IL-8, which are mediators of inflammation. Addition of Al3+ ions can
enhance fluoride-induced IL-6 and IL-8 synthesis in human epithelial lung cells
(Refsnes et al. 1999). Long-term boron and selenium supplementation for prophy-
lactic and therapeutic purposes also has limitations including low efficacy, high
cost, and undesirable health effects.
7.2 Preventive and Therapeutic Measures 93
7.2.2 Vitamins and Antioxidants
In humans, the toxic effects of fluoride are minimal when the diet is rich in
different nutrients, particularly protein, calcium, antioxidant minerals, and

vitamins such as A, D, E, and C. Clinical symptoms of fluorosis subside after a
decrease in fluoride intake and consumption of a diet rich in essential nutrients and
antioxidants (Susheela and Bhatnagar 2002). Vitamins A, C, and D are claimed
to mitigate the symptoms of fluorosis in humans and retard the development of
fluoride toxicosis with vitamin C possessing the greatest effect (Suttie and Phillips
1959). Therapeutic and preventive efficacy of vitamin C has been demonstrated
in several experimental studies (Chinoy et al. 1993; Chinoy and Memon 2001).
Choubisa and his coworkers opined that high levels of calcium and vitamin C pre-
sent naturally in plants, grasses, and forage consumed by sheep, goats, and camels
are responsible for the lower prevalence of dental and bony lesions in these ani-
mal species in comparison to cattle and water buffaloes reared in the same hydro-
fluorotic areas of Rajasthan, India (Choubisa 2010, 2013; Choubisa et al. 2011).
However, this hypothesis does not appear plausible as after oral intake, vitamin
C is almost completely destroyed by rumen microorganisms and is not utilized in
ruminants (Ranjan et al. 2012). Hence, vitamin C may have some role in fluorosis
prevention in simple-stomach animals and humans, but further study is required
to elucidate its role in ruminants. Contrary to this, some laboratory animal stud-
ies even indicated that blood fluoride concentration (Chan et al. 1992) and fluo-
ride accumulation in bone and soft tissues (Muhler 1958) increase after vitamin C
administration during high fluoride intake. In a study in fluorotic human patients,
daily oral supplementation of 2 g vitamin C failed to have any effect on urinary
fluoride excretion (Krishnamachari and Laxmaiah 1975).
Copper supplementation has been reported to reduce bone fluoride accumula-
tion in rabbits exposed to high dietary fluoride intake (Khandare et al. 2005). In a
study, vitamin E and methionine supplementation had a hepatoprotective effect in
rats intoxicated with sodium fluoride (Stawiarska-Pieta et al. 2011).
7.2.3 Plant Products/Herbal Medicines
Herbal medicines and plant products can be helpful in the management of vari-
ous ailments in animals including wounds, fever, anorexia, diarrhea, and snake
bite, among others. Due to their low cost, easy availability, and less/no potential
toxicity or side effects even at high doses, the quest for effective herbal medicine
appears as the rational solution to fluorosis in humans and animals. Several plant
products have been tested by various workers from time to time with encouraging
results. However, the requirement of large doses and oral administration for a long
time seems to limit their practical utility. Details about important plant products
found effective in animal fluorosis are summarized in Table 7.2.
Table 7.2 Plant products found effective in mitigation of fluorosis

Name of the plant Part used/ Biological effects Subject Reference
type of extract (animal/
human)
Tamarindus indicia Fruit pulp Enhances urinary Humans and Khandare et al.
fluoride excretion dogs (2000, 2002)
Tamarindus indicia Aqueous extract Protection from Rabbits Ranjan et al. (2009)
of fruit pulp bony changes and
hepatic and renal
damage
Tamarindus indicia Hydromethanolic Reduces fluoride Rats Dey et al. (2011)
fruit pulp extract accumulation in
bone, enhanced
urinary fluoride
excretion
Tamarindus indicia Dried powder Reduces serum F Cattle Gupta et al. (2013)
of fruit pulp concentration
and urinary
hydroxyproline
concentration,
increases serum
Ca level
Morus nigra (black Juice Reduces Rats Hassan and Yousef
berry) hepatotoxicity (2009)
and oxidative
stress
Terminalia arjuna Ethanolic extract Protects mouse Rats Sinha et al. (2008)
of bark heart against
oxidative stress
Terminalia arjuna Aqueous extract Protects hepatic Mice Sinha et al. (2007)
of bark and renal tissues
from oxidative
damage
Moringa oleifera Aqueous extract Protection from Rabbits Ranjan et al.
of seed bony changes (2009)
and hepatic and
renal damage
Mangifera Fruit powder Protects hepatic Rats Amaravadi and
indica and renal tissues Vasant (2011)
from oxidative
damage
Curcuma Curcumin Protects human Human Tiwari and Rao
longa (diferuloylmethane) lymphocytes from peripheral (2010)
genotoxic effects lymphocytes
(in vitro
study)
Curcuma Curcumin Protects Rats Nabavi et al. (2012)
longa erythrocytes
from oxidative
damage
(continued)
7.2 Preventive and Therapeutic Measures 95
Table 7.2 (continued)
Name of the plant Part used/ Biological effects Subject Reference
type of extract (animal/
human)
Spirulina platensis Spray-dried Ameliorates Rats Banji et al. (2013)
(blue-green alga) powder behavioral
changes, neuronal
damage, and
thyroid
dysfunction
Emblica officinalis Dry fruit powder Protects against Rats Vasant and
hyperlipidemia Narasimhacharya
and oxidative (2013)
stress
Ocimum sanctum Aqueous extract of Protects CNS from Rats Madhusudhan
leaves free radicals et al. (2010)
Aloe vera Whole leaf juice Protects bone Rats Navathej et al.
microarchitecture (2014)
Limonia acidissima Fruit pulp Protects against Rats Vasant and
hyperglycemia Narasimhacharya
and hyperlipidemia (2012)
The fruit pulp of Tamarindus indica contains L-ascorbic acid, alpha-tocopherol,

carotenes, essential minerals, sugars, phytosterols, and triterpenes (Wilson 1974)
and its extract has the capacity to bind fluoride ions (Maruthamuthu and Reddy
1987). In addition, oral supplementation of fruit pulp of Tamarindus indica has
been shown to increase urinary excretion of fluoride in children (Khandare et al.
2002) and dogs (Khandare et al. 2000). The beneficial effects of its fruit pulp sup-
plementation have also been reported in natural cases of bovine fluorosis (Gupta
et al. 2013).
Seeds of the Moringa oleifera plant are an excellent coagulant that can be used
for wastewater treatment (Ndabigengeser and Narasiah 1998). The leaf contains
a high concentration of vitamin A, vitamin C, zinc, and calcium (Barminas et al.
1998; Nambiar and Seshadri 2001). The beneficial effects of the seed extract of the
plant in fluoride toxicity have been reported in several laboratory animal species
including rats (Stanley et al. 2002) and rabbits (Ranjan et al. 2009).
Camellia sinensis, commonly known as black tea, was found to counter-
act fluoride-induced alterations in DNA, RNA, and protein concentration in the
brain and other tissues of mice (Trivedi et al. 2006; Verma et al. 2007). Sodium-
fluoride–induced hemolysis in humans was prevented by black tea extract (Verma
et al. 2006). However, the high fluoride content in tea plants was suggested as a
drawback for its use in fluorosis (Hudaykuliyev et al. 2005; Mahvi et al. 2006).
Amaranth oil is reported to reduce oxidative-stress-induced damage in the liver
and heart in fluoride-intoxicated rats (Kyonk et al. 2002). Some Chinese herbs
have also been claimed to have beneficial effects in fluorosis, although more stud-
ies are required for their scientific validation.
7.3 Minimizing Industrial Fluoride Emissions
Major contributors to industrial fluoride emissions include aluminum smelters, steel

production plants, superphosphate plants, coal-burning power plants, ceramic industry,
glassworks, and oil refineries. Among these, aluminum smelters are the highest con-
tributors (about 10 %) to worldwide annual industrial fluoride emission (WHO 2000).
That is why monitoring fluoride emission by aluminum smelters is a legal issue in
many countries. The regulated airborne fluoride emission limits vary from one coun-
try to another, but usually lie between 0.2 and 1.5 kg fluoride per ton of aluminum
produced. The Light Metals Research Centre (LMRC), the University of Auckland in
collaboration with the Australian Aluminum Council (AAC), has compiled a docu-
ment, “Fluoride Emissions Management Guide (FEMG),” which describes various
operational and maintenance practices for aluminum smelters and techniques that can
help reduce fluoride emissions into the environment (LMRC 2011). Industrial fluoride
emissions can be minimized by strict adherence to these guidelines.
7.4 Generating Public Awareness
Chronic fluorosis in animals remains mostly insidious and fails to draw the
attention of farmers as well as veterinarians as overt signs of illness are minimal
or even absent. Nevertheless, as with subclinical mastitis, it may be responsible
for huge economic losses, largely due to suboptimal reproductive and p roductive
performances of dairy animals. Extension workers should discuss the various aspects
of the fluorosis problem with farmers, highlighting the ill effects on health, produc-
tion, and reproduction in livestock. Various sources of excess fluoride intake and
their relative significance in a particular geographical area should also be studied
and discussed. Animal husbandry practices including the source of drinking water
for animals, grazing pattern, and nutritional aspects must be taken into considera-
tion while evaluating the overall impact. Farmers should be encouraged to submit
samples including water, blood, fodder, concentrate ration, mineral mixture, and so
on for fluoride analysis. The feed phosphates having a phosphorus and fluorine ratio
100:1 or more should only be allowed for livestock consumption (Thompson 1980).
Various water defluoridation techniques should be demonstrated and people should
be encouraged to offer defluoridated or rain-harvested water to animals for drinking.
Knowledge regarding industrial fluoride pollution and related legal issues and emis-
sion norms should be disseminated to the people living in industrial areas.
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Appendix
Abbreviations
Abbreviation Full form

ALD Approximate lethal dose
BW Body weight
CDTA 1,2-cyclohexylenedinitrilotetraacetic acid
CSF Cerebrospinal fluid
F Fluoride
g Gram
h Hour
HF Hydrogen fluoride
ISE Ion selective electrode
kg Kilogram
km Kilometer
L Liter
LC50 Median lethal concentration
LD50 Median lethal dose
LOAEL Lowest observed adverse effect level
m Milli
µg Microgram
mg Milligram
ml Milliliter
mV Millivolt
NaF Sodium fluoride
NOAEL No observed adverse effect level
NOEC No observed effect concentration (also called as NOEL: No observed effect
level)
NRC National Research Council
ppm Parts per million

SpringerBriefs in Animal Sciences, DOI 10.1007/978-3-319-17512-6
102 Appendix
Abbreviation Full form

PMNs Polymorphonuclear cells
SPADNS Trisodium 2-(parasulfophenylazo)-1,8-dihydroxy-3,6-napthalenedisulfonate salt
TISAB Total ionic strength adjustment buffer
UNICEF United Nations Children’s Fund
USEPA United States Environmental Protection Agency
WHO World Health Organization
Preparation of Different Types of TISAB
Methods for preparation of different types of TISAB are given below. It is impor-
tant to note that composition of commercially available TISABs may differ from
those described herein.
1. TISAB I (Low-Level TISAB)

(Reference: Frant MS, Ross JW (1968) Use of a total ionic strength adjustment
buffer for electrode determination of fluoride in water supplies. Anal Chem 40:
1169–1174)
Chemicals Required
1. Sodium chloride
2. Sodium citrate dihydrate
3. Glacial acetic acid
4. Sodium hydroxide
Take a beaker (one-liter capacity) and add 500 ml distilled water. Add 57 ml gla-
cial acetic acid, 58 g sodium chloride, and 0.3 g sodium citrate dihydrate. Mix
well by stirring. Adjust the pH of this mixture between 5.0 and 5.5 by dropwise
adding 5 M NaOH. Cool the solution to room temperature and transfer the con-
tents into a one-liter volumetric flask. Adjust the final volume to one liter by add-
ing distilled water.
2. TISAB II
(Reference: Liberti A, Mascini M (1969) Anion determination with ion selective
electrodes using Gran’s plot. Anal Chem. 41: 676–680)
Chemicals Required
1. Sodium nitrate
2. Sodium acetate trihydrate
Appendix 103
Take a beaker (one-liter capacity) and add 500 ml distilled water. Add 170 g
sodium nitrate, 68 gm sodium acetate trihydrate, and 92.4 g sodium citrate dihy-
drate. Mix well by stirring. Adjust the pH of this mixture between 5.0 and 5.5 by
dropwise adding 5 M NaOH. Cool the solution to room temperature and transfer
the contents into a one-liter volumetric flask. Adjust the final volume to one liter
by adding distilled water.
3. TISAB III
(Reference: Peters MA, Ladd DM (1971) Determination of fluoride in oxides with
fluoride-ion activity electrode. Talanta 18: 655–664)
Chemicals Required
1. CDTA
2. Sodium hydroxide
4. Sodium chloride
Take a beaker (one-liter capacity) and add 500 ml distilled water. Add 17.65 g
CDTA and 40 % sodium hydroxide drop by drop until the salt is dissolved. Add
300 g sodium citrate dehydrate and 60 g sodium chloride and mix well using a
magnetic stirrer and rotator. Transfer the content into a one-liter volumetric flask.
Adjust the final volume to one liter by adding distilled water.
4. TISAB IV
Chemicals Required
1. Tris (hydroxymethyl) aminomethane
2. Sodium tartarate dihydrate (Na2C4H406-2H2O)
3. Hydrochloric acid (36–38 %)
Take a beaker (one-liter capacity) and add 500 ml distilled water. Add 84 ml of
concentrated hydrochloric acid (36–38 %) and 242 g tris (hydroxymethyl) amino-
methane and 230 g sodium tartrate dihydrate. Dissolve the solids by stirring and
cool the solution to room temperature. Transfer the mixture into a one-liter volu-
metric flask and adjust the final volume to one liter by adding distilled water.
Analytical Methods for Fluoride Analysis
Given below are some simple methods for fluoride analysis developed/adopted by
different workers. The authors of this book are not responsible for the accuracy
and efficacy of these methods.
104 Appendix
1. Fluoride Assay in Bone

(Reference: Mikaelian I, QuallsJr CW, Guise S De, Whaley MW, Martineau D
(1999) Fluoride concentrations in Beluga Whales from Canada. J Wildlife Dis 35:
356–360)
The bone sample is cleaned by boiling in water for 15 min and adhering tissue
is removed with a razor blade. Care should be taken to avoid removal of any
bony material because it may affect the actual fluoride concentration in the bone.
Thereafter, the sample is placed in petroleum ether for 72 h and the ether is changed
twice daily. The clean bone sample is then dried overnight in an oven, weighed, and
ashed at 550 °C. The ash obtained is weighed and then pulverized into fine powder.
The ash powder is dissolved in 2 ml of 0.25 M HCL to which 8 ml of TISAB IV
buffer is added and fluoride concentration is measured using ISE.
2. Fluoride Assay in Soft Tissues

(Reference: Inkielewicz I, Czarnowski W, Krechniak J (2003) Determination
of fluoride in soft tissues. Fluoride 36: 16–20)
The soft tissue sample is cut into pieces with the help of a scalpel. A platinum
crucible is taken and 0.5 to 1.0 g of the tissue sample is transferred into it. The
appropriate NaF solution is then added and then 1.0 ml of 5 % magnesium acetate
solution is poured over the tissue sample. The mixture is dried at 105 °C for 12 h.
The dried tissue sample is pyrolized in an oven at 500 °C for 24 h. The cooled
sample is then transferred into a polyethylene measuring vessel by rinsing the
crucible successively with 1 ml perchloric acid solution, add 1.5 ml of 1 M sodium
citrate solution, and 1.5 ml TISAB buffer, measure the fluoride concentration
using ISE.
3. Fluoride Assay in Vegetation and Fodder Samples

Method 1
The fodder sample is cut into small pieces of approximately one inch length. Ten
grams of the sample are dried at 110 °C for 24 h in a hot air oven and weighed
again after drying. Another 5 to 10 g of the fodder sample is taken into a 50-ml
nickel crucible and 10 ml (6.7 g) of NaOH is added. The mixture is dried for 2 h at
150 °C. Thereafter, the sample is ashed at 550 °C in a muffle furnace for 2 h. The
sample is cooled and the ash is dissolved into distilled water and the final volume
is adjusted to 50 ml. The solution obtained is filtered through a Whatman No. 41
paper into a plastic beaker and the pH is adjusted to 5.2 with acetic acid. The fluo-
ride concentration in the final solution is measured using ISE.
Method 2
The fodder is washed with distilled water and dried at 800 °C for 24 h. Then dried
material is ground to pass through a 60-mesh sieve. The powered material (2 g) is
taken into a 150-ml plastic beaker and 100 ml of 0.1 M perchloric acid is added.
The mixture is stirred continuously on a magnetic stirrer for at least 20 min. The
suspension obtained is divided into two parts for duplicate analysis. The fluoride
concentration in the suspension is measured using ISE.
Appendix 105
4. Fluoride Assay in Soil, Feed, and Mineral Mixture

(Reference: Madhavan N, Subramanian V (2002) Fluoride in fractionated soil
samples of Ajmer district, Rajasthan. J Environ Monit 4: 821–822)
The soil sample is air dried and 1 gm of the sample is mixed with 10 ml of
aqua regia and digested in a Teflon bomb at 60°C for 90 min. The volume of the
digested sample is adjusted to 100 ml with distilled water in a polypropylene volu-
metric flask. Then 2 ml of the sample is mixed with 8 ml of 45 % sodium acetate
solution to make the pH of the final solution neutral. Then the sample is diluted
with TISAB and the fluoride concentration is determined using ISE.
Some Other Analytical Techniques
Baker RL (1972) Determination of fluoride in vegetation using the specific ion
electrode. Anal Chem 44: 1326–1327
McQuaker NR and Gurney M (1977) Determination of total fluoride in soil and
vegetation using an alkali fusion-selective ion electrode technique. Anal Chem 49:
53–56
Patterson MM, Reddy KJ and Jackson R (2003) Comparison of two methods
for analysis of fluoride in vegetation standard reference material. Commun Soil
Sci Plant Anal 34: 1077–1082
Vijan PN and Alder B (1984) Determination of fluoride in vegetation. Am Lab
83: 16–24
Villa EV (1979) Rapid method for determining fluoride in vegetation using an
ion-selective electrode. Analyst 104: 545–551

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SPRINGER BRIEFS IN ANIMAL SCIENCES

ISSN 2211-7504 ISSN 2211-7512 (electronic)

Library of Congress Control Number: 2015936363

Springer Cham Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

2 Sources of Fluoride Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3 Fluoride Kinetics and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3.2 Distribution and Retention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6.3 Gas Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

7 Mitigation and Prevention of Fluorosis. . . . . . . . . . . . . . . . . . . . . . . . . . 85

Fluorine is a highly electronegative and reactive halogen element belonging to

© The Author(s) 2015 1

Before discussing the role of fluorine in the maintenance of health or ­production

1.1.1 Physical and Chemical Properties

At room temperature elemental fluorine is a pale yellow-green gas with a char-

Table 1.1 Chemical formula Mineral Chemical formula Fluorine concentration (%)

1.2 Is Fluoride Essential for Health?

Whether fluoride is essential for maintenance of health is an issue of much

1.2.1 Fluoride and Human Health

Fluoride is required for mineralization of bone and teeth, maintenance of fertil-

acid and alkaline phosphatases, and isocitrate dehydrogenase (Kirck 1991). In

1.2.2 Fluoride and Animal Health

Many biologists doubt the essentiality of fluoride for maintenance of a­nimal

documented the beneficial effects of fluoride supplementation on animal health

1.3.1 Fluorosis in the Human Population

Naturally occurring high fluoride concentration in drinking water is the most

Alberts B (1998) Discussion section: is fluoride a nutrient? Fluoride 31:153–157

© The Author(s) 2015 11

Feed supplements Vegetation

Volcanic ash and

Fig. 2.1 Sources of fluoride toxicity in animals

2.1.1 Forage, Grasses, and Grains

thereafter F is carried to the shoot by transpiration. Most of the plants do not

The fluoride concentration of natural groundwater depends upon geological factors,

2.2.1 Mineral Mixture and Other Feed Supplements

There are several reports documenting mineral supplements as a major source of

The mean F concentration in ambient air in unpolluted or nonindustrial areas is

2.2.4 Agrochemicals and Household Products

USEPA (1980) Reviews of the environmental effects of pollutants: IX Fluoride. US Environmental

Abstract Fluoride, after absorption from the gastrointestinal tract, respiratory

Fluoride absorption, distribution, deposition in body tissues, and excretion are

© The Author(s) 2015 21

Excretion Sweat & Saliva

Fig. 3.1 Fluoride metabolism in animal body after its oral intake

The respiratory tract appears to be an important route of fluoride uptake in

3.1.3 Dermal and Other Routes

3.2 Distribution and Retention

3.2.3 Skeleton and Other Calcified Tissues

Approximately 99 % of the body F burden is associated with the calcified tissues.

3.2.6 Hair and Fingernails

Some studies on human and laboratory animals suggested that F concentration in

3.3 Elimination and Excretion

Unabsorbed fluoride from the gastrointestinal tract is eliminated through feces,

The urinary excretion of fluoride is a pH-dependent process. The renal clearance

A fraction of the ingested fluoride not absorbed in the gastrointestinal tract is

In humans, average salivary fluoride concentration is about 75 % of the plasma

Fluoride concentration in perspiration in humans is approximately 20 % of plasma

Shupe JL (1980) Clinicopathologic features of fluoride toxicosis in cattle. J Anim Sci

Abstract Clinical manifestations of acute fluoride toxicity include hypersaliva-

Before discussing the role of fluorine in the maintenance of health or production

1.1.1 Physical and Chemical Properties

1.2 Is Fluoride Essential for Health?

1.2.1 Fluoride and Human Health

1.2.2 Fluoride and Animal Health

Many biologists doubt the essentiality of fluoride for maintenance of animal

1.3.1 Fluorosis in the Human Population

2.1.1 Forage, Grasses, and Grains

2.2.1 Mineral Mixture and Other Feed Supplements

2.2.4 Agrochemicals and Household Products

3.1.3 Dermal and Other Routes

3.2 Distribution and Retention

3.2.3 Skeleton and Other Calcified Tissues

3.2.6 Hair and Fingernails

3.3 Elimination and Excretion

4.2.1 General Health Effects

4.2.2 Effects on Calcified Tissues

4.2.3 Effects on Soft Tissues (Nonskeletal,

4.2.3.2 Renal and Hepatic Toxicity

4.2.3.6 Genotoxicity and Carcinogenicity

4.3 Molecular Mechanism of Toxicity

5.1 Fluoride Tolerance in Different Animal Species

5.1.4 Poultry and Other Birds

5.1.5 Insects and Other Invertebrates

5.1.6 Fish and Other Aquatic Animals

5.2 Factors Affecting Fluoride Tolerance

5.2.2 Dietary and Nutritional Factors

5.2.3 Chemical Form of Fluoride

5.2.4 Dose, Duration, and Continuity of Fluoride Intake

5.2.5 Environmental and Other Factors

6.4 Neutron or Proton Activation Technique

6.5 Potentiometric Analysis/Ion Selective

6.5.1 Working of Ion Selective Electrode

6.5.2 Factors Affecting Performance of ISE

6.5.2.3 Presence of Fluoride Complexing Ion

6.5.3 Sample Collection and Preservation

6.5.5 Checking Electrode Operation