Greenhouse Gas Emissions of Agriculture: A Comparative Analysis

*Dionisio Rodríguez*

## **Abstract**

Greenhouse gas emissions are accounted by greenhouse gases inventories, which must be produced by common accounting rules, called Guidelines, which are endorsed by the United Nations Framework Convention on Climate Change (UNFCCC). These inventories are fundamental to analyze the impact of agriculture on emissions, and as example of the difficulty and complexity of implementation of the guidelines, a comparative study is made on emissions from Agricultural Soil Management (CRF category 3D source) utilizing biological nitrogen fixation. The analysis carried out for the N2O emissions under this section of the agrarian sector of Spain, Europe, New Zealand, Canada and the USA, inventories and national communications from Argentina and Brazil permit to observe the wide spectrum of approaches and the importance of the management of the accounting rules to be used mainly if we need that the impact of mitigation policies are captured in a direct way by the inventory. New technologies could introduce changes in the rules and can be utilized for reducing emissions, and examples are also analyzed.

**Keywords:** inventory of agriculture greenhouse gas emissions, N2O emissions, biological nitrogen fixation, benchmark of countries, new technologies

## **1. Introduction**

Agriculture is one of the economic sectors that make up the economic structure of a country and, as such economic activity, contributes to generate part of greenhouse gas of the total emissions of each country and, therefore, is an activity co-responsible for climate change.

Emissions of greenhouse gas (GHG) are accounted by greenhouse gases inventories and allow us to characterize both the emitting sources and the amount emitted and must be made respecting common rules designed with high technical qualifications.

This accounting of emissions from the agricultural sector is particularly complex and should be a useful tool for the design of agricultural policies for emissions mitigation from this sector.

To be able to check the difficulty and complexity of application of accounting guidelines and, also, the wide spectrum of options that you can use, a comparative study of the treatment of emissions from a series of inventories or national communications from various countries is made in this chapter.

**20**

*Environmental Chemistry and Recent Pollution Control Approaches*

traitement des rejets fins et préservation de l'environnement. Annales Chimie Science Matériaux. 2001;**26**:S465-S470

[16] Rodier J, Bazin C, Broutin JP, Chambon P, Champsaur H, et Rodi L. L'analyse de l'eau, 8e édition. Dunod (Éditeur), Paris, France; 1996

édition. Paris, France; 1979

[17] AFNOR. Eau, Méthodes d'essais. 1er

[18] O.M.S. Fluor et santé. In: Série de monographie. Genève: Organisation mondiale de la Santé; 1972. p. 59

[19] Arafan A, Erraji M, Hassani E, Chik A. Traitement Thermique d'un Phosphate Très Riche En Matières Organiques Et Valorisation du Phosphate Calcine Y2. Rapport nom édité du Groupe OCP, Maroc, Maroc-Phosphore et Cerphos, 30 Juin; 1998

[20] Mountadar M, Garmes H, Bouraji M, Lhadi EK. Défluoruration d'une eau chargée en fluorures: cas du rejet de la laverie des phosphates (Khouribga-

[21] Moufti A, Annouar S, Mountadar S, Mountadar M. The regeneration

elimination fluorides ions from the underground waters. Journal of Materials and Environmental Science.

[22] Annouar S, Mountada M, Soufian A, Elmidaoui A, Menkouchi Sahli MA. Defluoridation of underground water by adsorption on the chitosan and by electrodialysis. Desalination.

[23] O.M.S. Directives de qualité de l'eau de boisson, v: 1, Recommendation. 2ème édition. Genève: Organisation mondiale

[24] Hassani EA, Znibar A, Bouhiaoui H. Traitement du phosphate noir de Youssoufia: Amélioration du système de

Maroc). In: Acte du colloque International : Gestion des Rejets Industriels pour un Développement

Durable (GRIDD). 1997

of the pre used ashes in the

2016;**7**(6):2069-2073

2004;**165**:437

de la Santé; 1994

It is undeniable that knowledge and the correct accounting of emissions we will strongly condition analyses and measures that specifically we design and pretend to implement to achieve lower emissions in this sector. It will enable us and also defines new technologies that may be incorporating gradually and its effect can be captured by each country GHG inventory.

## **2. Greenhouse inventories and agricultural sector**

From the year 2015, national GHG inventories have been developed following the Guidelines of the International Panel of Climate Change (IPCC) 2006 [4]. Until that year, 1996 IPCC Guidelines were used, and the introduction of these new guidelines meant significant changes in the accounting emissions of agricultural sector. In addition to new accounting rules that affect every economic sector, the potential of global warming greenhouse gases also changed, which meant to make updates of all the data series that are measured from the 1990 base year.

**Table 1** presents the global warming potential (GWP) of the three major gases that have been used and the new planned reform [1].

In addition to the changes made to the potential of global warming gases, two of which, (CH4 and N2O) particularly affect agriculture also changed certain accounting rules which generated significant changes in the volume of emissions in this economic sector. The changes that are made to inventories' rules will affect in proportion to each country's productive structure.

## **2.1 Emissions from agriculture**

Emissions from agriculture activity vary depending on the economic structure of countries and the extent of its territory because agriculture is mainly based on Earth's surface arable in each country. An idea of absolute importance (total emissions) and relative (percentage of agriculture with respect to the total emissions) can be seen in **Table 2** using data on inventories [2] and national communications of different countries [3].

We can see that the developed countries have a much less percentage of agrarian sector emissions (their emissions more importantly tend to belong to the energy sector), and the big countries such as Argentina and Brazil have a large amount of emissions in relative and absolute value. An exception is New Zealand that even being a developed country has a broad agricultural sector.


**23**

analysis.

exposure begins.

rules.

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

**Countries Agriculture** 

2016/AR4 EUR28 + Island 431,000 9.2 2016/AR4 USA 562,600 8.6 2016/AR4 Canada 72,000 10 2016/AR4 New Zealand 38,727 49.2 2016/AR4 Spain 34,405 12.1 2012/SAR Argentina 119,498 27.8 2010/SAR Brazil 407,067 32

**emissions Total kt CO2 eq.** **Agriculture emissions/total emissions (%)**

**3. A comparative analysis of methodologies**

of previous countries.

*Agriculture emissions by countries.*

biological fixation (NBF).

other countries could be used.

species in the IPCC guidelines.

**3.1 The treatment of legumes**

which explains very clearly this topic [5]:

In order to show the complexity and diversity of options that can be used, a comparative analysis of the methodologies used will be carried out to evaluate the emissions from agriculture's sector in the inventories and national communications

In a first epigraph, it will use a section of the inventory of emissions from agriculture, the emissions from managed agricultural soils: Agricultural Soil

Management by the analysis that the new guidelines have been given to the nitrogen

We will study methodologies, which not being developed in the United Nations Framework Convention on Climate Change (UNFCCC) guidelines are approved by the inspectors in different reviews carried out inventories and if it be known by

We will use, as demonstrative example, legumes' crops because they are widely cultivated throughout the world on a large part of the Earth's surface, and also they have the ability to fix atmospheric nitrogen to facilitate its growth; so it is very important to know the accounting treatment that has been given to these plant

New 2006 Guidelines introduced a very significant change in the treatment of legumes in GHG's inventories compared to previous 1996 guidelines [4]. This change meant a large reduction in emissions of the main producer countries, and this reduction is not due to an effective policy of mitigation emissions, but it is due to a simple change in accounting criteria, which is based on a technical scientist

To facilitate understanding of the problem, a brief theoretical nitrogen cycle

We will use the Centro Superior de Investigaciones Científicas (Spain) work,

A detailed analysis is performed, with respect to N2O emissions under this epigraph of the agrarian sector, of Spain, Europe, New Zealand, Canada and the USA inventories and national communications from Argentina and Brazil to see the broad spectrum of approaches and the importance of management of accounting

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

**Year/ methodology**

**Table 2.**

#### **Table 1.**

*Global warming potential (GWP) values relative to CO2.*

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*


**Table 2.**

*Environmental Chemistry and Recent Pollution Control Approaches*

**2. Greenhouse inventories and agricultural sector**

that have been used and the new planned reform [1].

proportion to each country's productive structure.

being a developed country has a broad agricultural sector.

**Second Assessment Report (SAR)**

Methane CH4 21 25 28

**2.1 Emissions from agriculture**

of different countries [3].

**Gases Chemical** 

Carbon dioxide

Nitrous oxide

**formula**

*Global warming potential (GWP) values relative to CO2.*

captured by each country GHG inventory.

base year.

It is undeniable that knowledge and the correct accounting of emissions we will strongly condition analyses and measures that specifically we design and pretend to implement to achieve lower emissions in this sector. It will enable us and also defines new technologies that may be incorporating gradually and its effect can be

From the year 2015, national GHG inventories have been developed following the Guidelines of the International Panel of Climate Change (IPCC) 2006 [4]. Until that year, 1996 IPCC Guidelines were used, and the introduction of these new guidelines meant significant changes in the accounting emissions of agricultural sector. In addition to new accounting rules that affect every economic sector, the potential of global warming greenhouse gases also changed, which meant to make updates of all the data series that are measured from the 1990

**Table 1** presents the global warming potential (GWP) of the three major gases

In addition to the changes made to the potential of global warming gases, two

Emissions from agriculture activity vary depending on the economic structure of countries and the extent of its territory because agriculture is mainly based on Earth's surface arable in each country. An idea of absolute importance (total emissions) and relative (percentage of agriculture with respect to the total emissions) can be seen in **Table 2** using data on inventories [2] and national communications

We can see that the developed countries have a much less percentage of agrarian sector emissions (their emissions more importantly tend to belong to the energy sector), and the big countries such as Argentina and Brazil have a large amount of emissions in relative and absolute value. An exception is New Zealand that even

**GWP values for 100-year time horizon**

**Fourth Assessment Report (AR4)**

CO2 1 1 1

N2O 310 298 265

**Fifth Assessment Report (AR5)**

of which, (CH4 and N2O) particularly affect agriculture also changed certain accounting rules which generated significant changes in the volume of emissions in this economic sector. The changes that are made to inventories' rules will affect in

**22**

**Table 1.**

*Agriculture emissions by countries.*

## **3. A comparative analysis of methodologies**

In order to show the complexity and diversity of options that can be used, a comparative analysis of the methodologies used will be carried out to evaluate the emissions from agriculture's sector in the inventories and national communications of previous countries.

In a first epigraph, it will use a section of the inventory of emissions from agriculture, the emissions from managed agricultural soils: Agricultural Soil Management by the analysis that the new guidelines have been given to the nitrogen biological fixation (NBF).

We will study methodologies, which not being developed in the United Nations Framework Convention on Climate Change (UNFCCC) guidelines are approved by the inspectors in different reviews carried out inventories and if it be known by other countries could be used.

A detailed analysis is performed, with respect to N2O emissions under this epigraph of the agrarian sector, of Spain, Europe, New Zealand, Canada and the USA inventories and national communications from Argentina and Brazil to see the broad spectrum of approaches and the importance of management of accounting rules.

We will use, as demonstrative example, legumes' crops because they are widely cultivated throughout the world on a large part of the Earth's surface, and also they have the ability to fix atmospheric nitrogen to facilitate its growth; so it is very important to know the accounting treatment that has been given to these plant species in the IPCC guidelines.

## **3.1 The treatment of legumes**

New 2006 Guidelines introduced a very significant change in the treatment of legumes in GHG's inventories compared to previous 1996 guidelines [4]. This change meant a large reduction in emissions of the main producer countries, and this reduction is not due to an effective policy of mitigation emissions, but it is due to a simple change in accounting criteria, which is based on a technical scientist analysis.

To facilitate understanding of the problem, a brief theoretical nitrogen cycle exposure begins.

We will use the Centro Superior de Investigaciones Científicas (Spain) work, which explains very clearly this topic [5]:

*"Nitrogen fixation may be purely abiotic or biological. In abiotic fixation, oxides are formed as a result of the combustion of organic compounds, electric shock, etc., which are dragged to the ground by rain, or ammonium by the industrial process Haber-Bosch. Biological nitrogen fixation process are carried out by prokaryotic organisms, N2 is reduced to ammonium and incorporated into the biosphere."*

"These bacteria from the soil that we could call fixation free, as those of the genus *Azotobacter*, require up to 100 units of glucose equivalents per unit of nitrogen fixed. For this reason, its agricultural significance is low, which increases considerably in the case of the symbiotic fixation, as the established between rhizobia and legumes, where the ratio decreases of 6–12 units of glucose consumed per unit of nitrogen fixed. In this case, moreover, the power source is carbon compounds supplied directly by the plant derived from photosynthesis, while free fixation has to take them from soil, where these carbon compounds (glucose) do not exist in the amount and form necessaries. So in fact, *Azotobacter* provides to the ground a few hundred grams of nitrogen per hectare/year, and on the other hand, this value goes up in the *Rhizobium* association with alfalfa, clover, peas or soybean, until a few hundred kilos. Despite these differences, free fixation alone represents, at global level, rather less than half of the total of N2 fixed per year [6], because symbiotic fixation, although was more high, is limited to a few plant species, including legumes." Therefore, the N2 is fixed not only by bacteria in the roots, mostly legumes, but also by the free bacteria (not symbiotic) in the soil.

Data show that 250 Mt. of N2 are fixed annually for bacteria and about 70 Mt. would be fixed by soil or free bacteria, which would represent 28% of N2 fixed and about 50% would be fixed for biological fixation.

It is very important to bear in mind this data because it will strongly affect the inventories and the ways of accounting for the whole issue of the fixation of atmospheric N2 as we will then develop.

On the one hand, it is very common that mitigation measures to tackle climate change are based on the property of legumes to fix atmospheric N2 by a series of bacteria (genus *Rhizobium* mainly).

Thus, for example, the road map of Spain for the reduction of diffuse emissions proposes, among others, the following course of action [7]: the introduction of legumes in managed grasslands with the aim of reducing the emissions from soils in meadows. The fixation of atmospheric nitrogen produced by legumes outweighs the need for mineral fertilizers.

On the other hand, the United States of America is the only country that counts in their inventory emissions of N2O due at atmospheric N2 fixed by the free soil bacteria.

#### **3.2 The measurement of the biological fixation by legumes**

With the 1996 IPCC Guidelines [8] to account for emissions of nitrous oxide (N2O) that occurs naturally in soils: "some agricultural activities bring nitrogen to the soil, increasing the amount of nitrogen (N) available for nitrification and denitrification and, ultimately, the amount of N2O emitted. Direct emissions of N2O from agricultural soils due to the application of N and other farming practices should reflect the contributions of anthropogenic (N) resulting from the use of synthetic fertilizers (NSF) and the animal manure applied (AMA), N of fixing varieties (NBF), the incorporation to the soils the crop residues, the nitrogen mineralization of the soil due to the cultivation of organic soils (i.e., histosols) (COS)."

The first conclusion we get is that those 1996 Guidelines address the plantation of legumes as an incorporation of N to the soil and, therefore, the producer of N2O

**25**

**Table 3.**

*The European Union greenhouse gas inventory 2014.*

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

2012 from the inventory of 15 European countries.

**3.3 New rules of measurement of biological fixation**

emissions. The captured N of the atmosphere as a sink is not considered, but it is a

To see how the emission due to the nitrogen biological fixation (NBF) is accounted, it can be seen from the following **Table 3** [9] that shows data of NBF in

We can see that because the property of N fixation of legumes Europe-15 have been issued 753,000 tons of nitrogen, which then result in N2O emissions.

As indicated above, from the year 2015, the 1996 IPCC guidelines are no longer used for inventory and entered into force the new 2006 Guidelines currently in the

These Guidelines say: "Biological nitrogen fixation has been removed as a direct source of N2O because of the lack of evidence of significant emissions arising from the fixation process itself [33]. These authors concluded that the N2O emissions induced by the growth of legume crops/forages may be estimated solely as a function of the above-ground and below-ground nitrogen inputs from crop/forage residue (the nitrogen residue from forages is only accounted for during pasture renewal). Conversely, the release of N by mineralization of soil organic matter as a result of change of land use or management is now included as an additional source. These are significant adjustments to the methodology previously described in the

This change means that they are accounted only for emissions from biological fixation of nitrogen for the purpose of the N2, which are produced from the crop

**Member states 2012 N-fixing crops (Gg N)**

Austria 23 Belgium 4 Denmark 42 Finland 0.7 France 224 Germany 78 Greece 0.8 Ireland 0.5 Italy 140 Luxemburg 0.1 The Netherlands 4 Portugal 10 Spain 172 Sweden 35 The United Kingdom 19 EU-15 753

Transformed into CO2 eq., they are equivalent to 4.575 Mt. of CO2 eq.

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

source of emission.

process of improvement.

1996 IPCC Guidelines."

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*

*Environmental Chemistry and Recent Pollution Control Approaches*

*"Nitrogen fixation may be purely abiotic or biological. In abiotic fixation, oxides are formed as a result of the combustion of organic compounds, electric shock, etc., which are dragged to the ground by rain, or ammonium by the industrial process Haber-Bosch. Biological nitrogen fixation process are carried out by prokaryotic organisms, N2 is reduced to ammonium and incorporated into the biosphere."*

"These bacteria from the soil that we could call fixation free, as those of the genus *Azotobacter*, require up to 100 units of glucose equivalents per unit of nitrogen fixed. For this reason, its agricultural significance is low, which increases considerably in the case of the symbiotic fixation, as the established between rhizobia and legumes, where the ratio decreases of 6–12 units of glucose consumed per unit of nitrogen fixed. In this case, moreover, the power source is carbon compounds supplied directly by the plant derived from photosynthesis, while free fixation has to take them from soil, where these carbon compounds (glucose) do not exist in the amount and form necessaries. So in fact, *Azotobacter* provides to the ground a few hundred grams of nitrogen per hectare/year, and on the other hand, this value goes up in the *Rhizobium* association with alfalfa, clover, peas or soybean, until a few hundred kilos. Despite these differences, free fixation alone represents, at global level, rather less than half of the total of N2 fixed per year [6], because symbiotic fixation, although was more high, is limited to a few plant species, including legumes." Therefore, the N2 is fixed not only by bacteria in the roots, mostly legumes, but also by the free bacteria (not

Data show that 250 Mt. of N2 are fixed annually for bacteria and about 70 Mt. would be fixed by soil or free bacteria, which would represent 28% of N2 fixed and

It is very important to bear in mind this data because it will strongly affect the inventories and the ways of accounting for the whole issue of the fixation of atmo-

On the one hand, it is very common that mitigation measures to tackle climate change are based on the property of legumes to fix atmospheric N2 by a series of

Thus, for example, the road map of Spain for the reduction of diffuse emissions proposes, among others, the following course of action [7]: the introduction of legumes in managed grasslands with the aim of reducing the emissions from soils in meadows. The fixation of atmospheric nitrogen produced by legumes outweighs the

On the other hand, the United States of America is the only country that counts in their inventory emissions of N2O due at atmospheric N2 fixed by the free soil bacteria.

With the 1996 IPCC Guidelines [8] to account for emissions of nitrous oxide (N2O) that occurs naturally in soils: "some agricultural activities bring nitrogen to the soil, increasing the amount of nitrogen (N) available for nitrification and denitrification and, ultimately, the amount of N2O emitted. Direct emissions of N2O from agricultural soils due to the application of N and other farming practices should reflect the contributions of anthropogenic (N) resulting from the use of synthetic fertilizers (NSF) and the animal manure applied (AMA), N of fixing varieties (NBF), the incorporation to the soils the crop residues, the nitrogen mineralization

The first conclusion we get is that those 1996 Guidelines address the plantation of legumes as an incorporation of N to the soil and, therefore, the producer of N2O

of the soil due to the cultivation of organic soils (i.e., histosols) (COS)."

**24**

symbiotic) in the soil.

about 50% would be fixed for biological fixation.

**3.2 The measurement of the biological fixation by legumes**

spheric N2 as we will then develop.

bacteria (genus *Rhizobium* mainly).

need for mineral fertilizers.

emissions. The captured N of the atmosphere as a sink is not considered, but it is a source of emission.

To see how the emission due to the nitrogen biological fixation (NBF) is accounted, it can be seen from the following **Table 3** [9] that shows data of NBF in 2012 from the inventory of 15 European countries.

We can see that because the property of N fixation of legumes Europe-15 have been issued 753,000 tons of nitrogen, which then result in N2O emissions. Transformed into CO2 eq., they are equivalent to 4.575 Mt. of CO2 eq.

### **3.3 New rules of measurement of biological fixation**

As indicated above, from the year 2015, the 1996 IPCC guidelines are no longer used for inventory and entered into force the new 2006 Guidelines currently in the process of improvement.

These Guidelines say: "Biological nitrogen fixation has been removed as a direct source of N2O because of the lack of evidence of significant emissions arising from the fixation process itself [33]. These authors concluded that the N2O emissions induced by the growth of legume crops/forages may be estimated solely as a function of the above-ground and below-ground nitrogen inputs from crop/forage residue (the nitrogen residue from forages is only accounted for during pasture renewal). Conversely, the release of N by mineralization of soil organic matter as a result of change of land use or management is now included as an additional source. These are significant adjustments to the methodology previously described in the 1996 IPCC Guidelines."

This change means that they are accounted only for emissions from biological fixation of nitrogen for the purpose of the N2, which are produced from the crop


#### **Table 3.**

*The European Union greenhouse gas inventory 2014.*

residue and mineralization and so the inventories will not reflect emissions that are previously counted as biological fixation.

The amounts saved because of this new methodology are significant because they can reduce emissions under this epigraph of the inventory by 50%.

As example, if we analyze successive inventories of Spain since the year 2012–2016, we obtain the following results (**Table 4**). The 1996 IPCC guidelines were used in the year 2012 and, therefore, included the biological fixation of nitrogen emission and also applied the N2O (SAR = 310) global warming potentials. That year was an emission result of the epigraph of agricultural soils 3D = 18,167 kt of CO2 eq.

Subsequently, this year 2012, inventories were calculated with the new warming potential of N2O (AR4 = 298) and began to gradually introduce the 2006 Guidelines, because as we have said is required to recalculate since 1990 with the new parameters. The result has been that the 2012 emissions calculated in the year 2018 and referred to the year 2016 have meant 9245 kt CO2 eq. for the year 2012.

This has meant that without changing the variables of activity of this section due to the recalculations, the year 2012 emissions were almost lower 50%. The inventory lowered emissions due to a change in accounting criteria, not the implementation of mitigation measures.

**Table 4** presents the evolution of Spain emissions for 2012, taking into account the recalculations marked in each annual inventory for the indicated methodological changes.

#### *3.3.1 The cultivation of soybean*

Then, we analyze the accounting treatment that different inventories of large producers of legumes make use of nitrogen biological fixation. We utilize, as an example, the crop of soybean, because it is the most widely legume cultivated worldwide and its high impact will allow us to better appreciate the distortions that occur in the accounting treatment of biological fixation. In **Table 5**, we can observe the increase of soybean crop between 2020 and 2016 surfaces mainly in Brazil and Argentina, and as in the United States, it has not changed, but remains as the maximum world producer of this crop.

These data provide us with an idea of the magnitude of the cultivation of these countries, some of which possess more hectares dedicated only to the soybean crop that the entire surface of Spain dedicated to all crops (Spain = 26.6 Million ha in the year 2016).

We can see that with such immense extensions dedicated to the cultivation of this legume, "accounting" treatment that Guidelines gives to the biological fixation will be a great importance for the inventory of emissions of these countries.

We will study two of the major producer countries (Argentina and Brazil) through their national communications and the inventories from the USA and Canada to observe how this phenomenon of biological fixation for the purposes of accounting has been treated.


**27**

**Table 6.**

*Argentina emissions.*

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

**Year 2000 2016**

Countries Million ha Million tonnes Million ha Million tonnes Argentina 8.6 20.1 19.5 58.8 Brazil 13.6 32.8 33.2 96.3 Canada 1.06 2.7 2.2 5.8 USA 29.3 75.1 33.5 117.2

To analyze how this item is addressed in Argentina, in your inventory, we will use data available from the second [11] and the third national communications [12]. These two communications are still made according to the 1996 IPCC guidelines

**Soybean crop**

The second national communication of Argentina says "the amount of nitrogen incorporated by NBF increased around 63% between 1990/91 and 2000/01 campaign. This fact was due to the strong increase in soybean production that went from 12 to nearly 20 million tons, making it the main crop of the country. The main increase in the amount of N was due, again, to the great increase of soybean produc-

The third communication data already indicate a rise of 5.354 Mt. CO2 eq., that

The data, in **Table 5**, show that soybean crop in Argentina increases until 19.5 Million ha and 58.8 Million tonnes in the year 2016. These data lead us to conclude that emissions from this crop and the NBF would grow strongly by applying the 1996 Guidelines. When they are finally implementing the 2006 Guidelines, the

As it is indicated above, Argentina will reduce its emissions simply by a change

The data show that the year 2012 Europe-15 saved with the accounting change of the NBF = 4.7 Mt. CO2 eq. front the 22.5 Mt. CO2 eq. that will save Argentina. That year 2012, Argentina only accounted 7.04 Mt. CO2 eq. (much lower than that of the NBF) due to the use of synthetic fertilizers that are usually the most important

We can see that the two large producers of soybean as Brazil (33.2 Mhas.) and the USA with (33.5 Mhas.) in 2016, compared the 17.6 Mhas. of Argentina in 2012, if they counted with this methodology that would have a great impact on their emissions.

**2000 2012**

**Argentina Years**

Surface of soybean (Mhas.) 8.6 17.6 Direct emissions from crops fixing (Mt. of CO2 eq.) 17.231 22.585

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

*3.3.1.1 Argentina*

*Data from FAOSTAT [10].*

**Table 5.**

since it closes its data in 2012.

tion, the main crop with contribution of the NBF."

is, an increase of 31% in emissions in those 12 years (**Table 6**).

emissions reduction, due to this item, will be very significant.

epigraph in agriculture emissions of developed countries.

in accounting criteria unless it really is because of a mitigation policy.

**Table 4.**

*Spain emissions N2O activity 3D.*


**Table 5.**

*Environmental Chemistry and Recent Pollution Control Approaches*

previously counted as biological fixation.

of CO2 eq.

mitigation measures.

*3.3.1 The cultivation of soybean*

accounting has been treated.

*Spain emissions N2O activity 3D.*

as the maximum world producer of this crop.

cal changes.

year 2016).

residue and mineralization and so the inventories will not reflect emissions that are

The amounts saved because of this new methodology are significant because

Subsequently, this year 2012, inventories were calculated with the new warming potential of N2O (AR4 = 298) and began to gradually introduce the 2006 Guidelines, because as we have said is required to recalculate since 1990 with the new parameters. The result has been that the 2012 emissions calculated in the year 2018 and

This has meant that without changing the variables of activity of this section due to the recalculations, the year 2012 emissions were almost lower 50%. The inventory lowered emissions due to a change in accounting criteria, not the implementation of

**Table 4** presents the evolution of Spain emissions for 2012, taking into account the recalculations marked in each annual inventory for the indicated methodologi-

Then, we analyze the accounting treatment that different inventories of large producers of legumes make use of nitrogen biological fixation. We utilize, as an example, the crop of soybean, because it is the most widely legume cultivated worldwide and its high impact will allow us to better appreciate the distortions that occur in the accounting treatment of biological fixation. In **Table 5**, we can observe the increase of soybean crop between 2020 and 2016 surfaces mainly in Brazil and Argentina, and as in the United States, it has not changed, but remains

These data provide us with an idea of the magnitude of the cultivation of these countries, some of which possess more hectares dedicated only to the soybean crop that the entire surface of Spain dedicated to all crops (Spain = 26.6 Million ha in the

We can see that with such immense extensions dedicated to the cultivation of this legume, "accounting" treatment that Guidelines gives to the biological fixation

**Emissions of Spain in 2012 in the successive inventories in kt. CO2** 2012 2013 2014 2015 2016 18,167 16,151 11,872 8823 9245

will be a great importance for the inventory of emissions of these countries. We will study two of the major producer countries (Argentina and Brazil) through their national communications and the inventories from the USA and Canada to observe how this phenomenon of biological fixation for the purposes of

they can reduce emissions under this epigraph of the inventory by 50%. As example, if we analyze successive inventories of Spain since the year 2012–2016, we obtain the following results (**Table 4**). The 1996 IPCC guidelines were used in the year 2012 and, therefore, included the biological fixation of nitrogen emission and also applied the N2O (SAR = 310) global warming potentials. That year was an emission result of the epigraph of agricultural soils 3D = 18,167 kt

referred to the year 2016 have meant 9245 kt CO2 eq. for the year 2012.

**26**

**Table 4.**

*Data from FAOSTAT [10].*

## *3.3.1.1 Argentina*

To analyze how this item is addressed in Argentina, in your inventory, we will use data available from the second [11] and the third national communications [12]. These two communications are still made according to the 1996 IPCC guidelines since it closes its data in 2012.

The second national communication of Argentina says "the amount of nitrogen incorporated by NBF increased around 63% between 1990/91 and 2000/01 campaign. This fact was due to the strong increase in soybean production that went from 12 to nearly 20 million tons, making it the main crop of the country. The main increase in the amount of N was due, again, to the great increase of soybean production, the main crop with contribution of the NBF."

The third communication data already indicate a rise of 5.354 Mt. CO2 eq., that is, an increase of 31% in emissions in those 12 years (**Table 6**).

The data, in **Table 5**, show that soybean crop in Argentina increases until 19.5 Million ha and 58.8 Million tonnes in the year 2016. These data lead us to conclude that emissions from this crop and the NBF would grow strongly by applying the 1996 Guidelines. When they are finally implementing the 2006 Guidelines, the emissions reduction, due to this item, will be very significant.

As it is indicated above, Argentina will reduce its emissions simply by a change in accounting criteria unless it really is because of a mitigation policy.

The data show that the year 2012 Europe-15 saved with the accounting change of the NBF = 4.7 Mt. CO2 eq. front the 22.5 Mt. CO2 eq. that will save Argentina. That year 2012, Argentina only accounted 7.04 Mt. CO2 eq. (much lower than that of the NBF) due to the use of synthetic fertilizers that are usually the most important epigraph in agriculture emissions of developed countries.

We can see that the two large producers of soybean as Brazil (33.2 Mhas.) and the USA with (33.5 Mhas.) in 2016, compared the 17.6 Mhas. of Argentina in 2012, if they counted with this methodology that would have a great impact on their emissions.


**Table 6.**

*Argentina emissions.*

The Argentine Government makes a [13] comparative study to analyze the impact that would have to apply the new accounting standards (1996 Guidelines against the 2006 Guidelines) and also apply the changes of global warming potentials from methane and nitrous oxide with a result of = −58.257 Mt. CO2 eq. if the new guidelines had been applied in the 2012 inventory (**Table 7**).

### *3.3.1.2 Brazil*

To analyze the accounting treatment of the emissions from this crop in Brazil, third national communication [14] sent to the UNFCCC in April 2016 will be used. In this document, Brazil already uses the IPCC 2006 Guidelines and, therefore, does not consider NBF as a source of N2O.

They also used a "study of Cardoso et al. (2008) that would demonstrate that don't exist any differences between the emissions of N2O measured in soils planted with inoculated varieties (in Brazil, soybean is inoculated with the specific bacteria for N2 fixation) and other varieties not inoculated." The authors of this national communication don't take into account, therefore the NBF, and they use the methodology of the 2006 Guidelines for analyses the N, which is incorporated into the soil by residue. To estimate these emissions of residues, annual productions and the amount of dry matter by crop type were used.

Brazil introduced an innovation by including a new measuring method and it explains their results also using the potential of global temperature that is proposed by the IPCC. To explain its emissions, results using three warming potential in addition to those specified in **Table 1** were used, the potential SAR and the AR5, and a new one is introduced: the global potential temperature (GTP) (**Table 8**). But, for the calculations, do not use the AR4 potential, which should be used according to the rules of implementation of the guidelines for 2006. Therefore, the results would be questionable and are not comparable.

Brazil with much more surface dedicated to the cultivation of soybean (33.2 Mhas. 2016) should produce much higher emissions than Argentina with much less soybean surface (in 2012 with 17.6 Mhas. produced 22.6 Mt. of CO2 eq.), but the use of 2006 Guidelines that do not account NBF and the use of different potential warming involve that this country has fewer emissions.

The gap would be much better if these divergent rules had been applied to the year 2016, in which both countries doubled their crop surfaces. The conclusion is that accounting rules are very important, because Brazil "save" important


**29**

**Table 9.**

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

Carbon dioxide CO2 1 Methane CH4 4 Nitrous oxide N2O 234

emissions, using the guidelines for 2006, which would be much greater with the

**Gases Chemical formula Global temperature potential: GTP/100years**

Knowing in depth how inventories are produced is, therefore, very important, and thus, we would conclude that the reduction of emissions from the agriculture sector was due to an "effective mitigation policy" when actually is due to a simple

Let us look at another example, the case of Canada [15–17], which also uses the 2006 Guidelines and it is another large producer of soybeans, although in smaller amounts (5, 8 Mt. in 2016). Canada, in its 2016 inventory, reports the emissions of leguminous crops in residues which are incorporated into the soil (6.5 Mt. CO2 eq.) [18]. As shown in **Table 9**, another novelty introduced in 2004 Canada's inventory is the appearance of new emissions due to summer fallow (0.43 Mt. CO2 eq.) and a sink effect (−0.63 Mt. CO2 eq.) due to the use of practices that do not till the soil,

These emissions and these sinks have no specific methodology in the 2006 Guidelines and their effects are calculated with methodologies developed by the country. According to the 2016 Canada's inventory, these emissions accounted for 0.22 Mt. CO2 eq. in the case of the fallow summer and −1.5 Mt. CO2 eq. as a sink due to conservation agriculture. Canada's inventory reports 20 Mt. CO2 eq. as direct sources of agricultural soils (N2O) and, therefore, conservation agriculture has been

Synthetic nitrogen fertilizers 8816.77 5800 11,000 Manure applied as fertilizers 3280.76 2100 2100 Biological nitrogen fixation 3779.12 — — Crop residue decomposition 6154.48 3800 6500 Cultivation of organic soils 61.01 60 60

Mineralization of soil organic carbon — — 800 Conservation tillage practices −630 −1500 Summer fallow 430 220 Irrigation 330

**Inventory 2003**

**Inventory 2004**

3272.71 4300 210

**Inventory 2016**

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

1996 Guidelines.

*Global temperature potential*

**Table 8.**

*3.3.2 Canada*

change of the accounting rules.

known as conservation agriculture.

a significant sink (7,5%).

**Agricultural soils (N2O) Direct sources. Kt. CO2 eq.**

*Evolution of Canada's inventories.*

manure)

Grazing animals (pasture, range and paddock

## **Table 7.**

*Evaluation guidelines effect.*

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*


#### **Table 8.**

*Environmental Chemistry and Recent Pollution Control Approaches*

*3.3.1.2 Brazil*

not consider NBF as a source of N2O.

amount of dry matter by crop type were used.

be questionable and are not comparable.

warming involve that this country has fewer emissions.

new guidelines had been applied in the 2012 inventory (**Table 7**).

The Argentine Government makes a [13] comparative study to analyze the impact that would have to apply the new accounting standards (1996 Guidelines against the 2006 Guidelines) and also apply the changes of global warming potentials from methane and nitrous oxide with a result of = −58.257 Mt. CO2 eq. if the

To analyze the accounting treatment of the emissions from this crop in Brazil, third national communication [14] sent to the UNFCCC in April 2016 will be used. In this document, Brazil already uses the IPCC 2006 Guidelines and, therefore, does

They also used a "study of Cardoso et al. (2008) that would demonstrate that don't exist any differences between the emissions of N2O measured in soils planted with inoculated varieties (in Brazil, soybean is inoculated with the specific bacteria for N2 fixation) and other varieties not inoculated." The authors of this national communication don't take into account, therefore the NBF, and they use the methodology of the 2006 Guidelines for analyses the N, which is incorporated into the soil by residue. To estimate these emissions of residues, annual productions and the

Brazil introduced an innovation by including a new measuring method and it explains their results also using the potential of global temperature that is proposed by the IPCC. To explain its emissions, results using three warming potential in addition to those specified in **Table 1** were used, the potential SAR and the AR5, and a new one is introduced: the global potential temperature (GTP) (**Table 8**). But, for the calculations, do not use the AR4 potential, which should be used according to the rules of implementation of the guidelines for 2006. Therefore, the results would

Brazil with much more surface dedicated to the cultivation of soybean (33.2 Mhas. 2016) should produce much higher emissions than Argentina with much less soybean surface (in 2012 with 17.6 Mhas. produced 22.6 Mt. of CO2 eq.), but the use of 2006 Guidelines that do not account NBF and the use of different potential

The gap would be much better if these divergent rules had been applied to the year 2016, in which both countries doubled their crop surfaces. The conclusion is that accounting rules are very important, because Brazil "save" important

4A. Livestock 52.900 49.372 3.528 +7% 4.B. Agriculture 35.242 70.130 −34.887 −50%

**guidelines. Mt. CO2 eq. Category IPCC 2006 IPCC 1996 Absolute** 

**Emissions from agriculture in Argentina in 2012 according to the different** 

63.616 90.515 −26.898 −30%

**difference**

−**58.257**

**Difference %**

**28**

**Table 7.**

5.A. Change of land use

*Evaluation guidelines effect.*

and forestry

*Global temperature potential*

emissions, using the guidelines for 2006, which would be much greater with the 1996 Guidelines.

Knowing in depth how inventories are produced is, therefore, very important, and thus, we would conclude that the reduction of emissions from the agriculture sector was due to an "effective mitigation policy" when actually is due to a simple change of the accounting rules.

### *3.3.2 Canada*

Let us look at another example, the case of Canada [15–17], which also uses the 2006 Guidelines and it is another large producer of soybeans, although in smaller amounts (5, 8 Mt. in 2016). Canada, in its 2016 inventory, reports the emissions of leguminous crops in residues which are incorporated into the soil (6.5 Mt. CO2 eq.) [18].

As shown in **Table 9**, another novelty introduced in 2004 Canada's inventory is the appearance of new emissions due to summer fallow (0.43 Mt. CO2 eq.) and a sink effect (−0.63 Mt. CO2 eq.) due to the use of practices that do not till the soil, known as conservation agriculture.

These emissions and these sinks have no specific methodology in the 2006 Guidelines and their effects are calculated with methodologies developed by the country. According to the 2016 Canada's inventory, these emissions accounted for 0.22 Mt. CO2 eq. in the case of the fallow summer and −1.5 Mt. CO2 eq. as a sink due to conservation agriculture. Canada's inventory reports 20 Mt. CO2 eq. as direct sources of agricultural soils (N2O) and, therefore, conservation agriculture has been a significant sink (7,5%).


#### **Table 9.**

*Evolution of Canada's inventories.*

They also incorporate emissions from irrigation practices by which we can conclude that a great evolution has suffered the emissions of this section of direct sources of agricultural soils.

## *3.3.3 The United States of America (USA)*

In addition to changes in the measurement of nitrogen biological fixation, the introduction of emissions' new categories, as we saw in Canada, "innovations" will continue to generate and, thus, the inventory of the United States [19], extends the computation of their emissions. This inventory includes in the accounting the nitrogen fixed, by what we have called-fixing bacteria N2 of the soil, which belong, for example, the genus *Azotobacter* and so-called biological fixation free. The USA in your inventory calls this fixation as asymbiotic fixation and, therefore, reports emissions. (**Figure 1**) shows the scheme how are calculated agricultural soils N2O emissions' in United States inventory.

The US inventory defines asymbiotic fixation as the fixation of atmospheric N2 by bacteria living in the soil and that do not have direct relationship with plants. This inventory says that although the nitrogen incorporated by asymbiotic N fixation is not specifically collected by the 2006 Guidelines, it is a

**31**

for longer.

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

of the synthetic fertilizer (64.5 Mt. CO2 eq.) to the soils.

Mhas. agricultural soils and Spain 26.6 Mhas. in 2016).

countries, it would have important effects on the emission amount.

component of the total emissions for managed soils and it should be included. It is calculated by a method of the high level developed to assess the source. To make the calculations, they use a combination of different methods using a

The result for the year 2016 was 95.1 Mt. CO2 eq., which includes the mineralization and asymbiotic fixation, and we can observe that it is a very significant amount. It is difficult to obtain which data belong to asymbiotic fixation and which

But what is striking is that it introduces a concept [20] that does not force the

We can observe in his inventory data that how meadows and crops emit more due to mineralization and asymbiotic fixation (95.1 Mt. of CO2 eq.) by the addition

We can conclude that the methodology used to measure the nitrogen biological fixation is very relevant for the emissions of a country and consideration of asymbiotic fixation (free or mineral) is an issue to consider. In the case of large agrarian

In this section, we will discuss, using New Zealand GHG inventories as example [21, 22], how we can make improvements in GHG inventories and we introduce measurement methodologies of new technologies applied in the agricultural sector.

The application of nitrogen fertilizers to the soil means the occurrence of biological and physicochemical reactions that leads to loss of nitrogen. The use of fertilizers with nitrification inhibitors has become a useful tool to reduce loss and improve the efficiency of the N. The use of nitrogen fertilizers stabilized become widespread and its are added, during the production process, with some substances, such as nitrification inhibitors, which can keep N applied as NH4

These products delayed the transformation of ammonia nitrogen (NH4

to nitrate nitrogen (NO3−) through temporary inhibition of various bacteria *Nitrosomonas* spp., and thus, the nitrogen is released in a progressive and gradual

Nitrification inhibitors degrade over time after being applied on the ground, and this degradation is influenced by temperature, moisture, pH and quantity of organic matter. There is already a long list of chemical compounds that have been tested as inhibitors of nitrification in the world (more than 64), but the most studied and used nitrification inhibitors are nitrapyrin, dicyandiamide (DCD) and

In the United States, nitrapyrin is being used in corn, sorghum, wheat, cotton and strawberries (in a manner restricted in these). However, more than 90% is used in corn. Nitrapyrin must be injected and immediately incorporated into the soil due to its volatility and therefore its use is limited in the regions where N is typically injected to the ground. In principle, only it is marketed in the United States.

way and, at that same rate, it is assimilated by the crop.

3,4-dimethylpyrazol phosphate (DMPP) [23].

+

+ )

Only these emissions be over all Canada emissions from agricultural soils (24 Mt. of CO2 eq.). Compared to Spain' emissions, only this item exceeds the emissions of all Spanish agriculture (34.4 Mt. CO2 eq. in 2016) (the United States has 405

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

specific model called Daycent.

are due to mineralization.

**4. New technologies**

**4.1 The nitrification inhibitors**

2006 Guidelines.

**Figure 1.** *Asymbiotic fixation [19].*

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*

component of the total emissions for managed soils and it should be included. It is calculated by a method of the high level developed to assess the source. To make the calculations, they use a combination of different methods using a specific model called Daycent.

The result for the year 2016 was 95.1 Mt. CO2 eq., which includes the mineralization and asymbiotic fixation, and we can observe that it is a very significant amount. It is difficult to obtain which data belong to asymbiotic fixation and which are due to mineralization.

But what is striking is that it introduces a concept [20] that does not force the 2006 Guidelines.

We can observe in his inventory data that how meadows and crops emit more due to mineralization and asymbiotic fixation (95.1 Mt. of CO2 eq.) by the addition of the synthetic fertilizer (64.5 Mt. CO2 eq.) to the soils.

Only these emissions be over all Canada emissions from agricultural soils (24 Mt. of CO2 eq.). Compared to Spain' emissions, only this item exceeds the emissions of all Spanish agriculture (34.4 Mt. CO2 eq. in 2016) (the United States has 405 Mhas. agricultural soils and Spain 26.6 Mhas. in 2016).

We can conclude that the methodology used to measure the nitrogen biological fixation is very relevant for the emissions of a country and consideration of asymbiotic fixation (free or mineral) is an issue to consider. In the case of large agrarian countries, it would have important effects on the emission amount.

## **4. New technologies**

*Environmental Chemistry and Recent Pollution Control Approaches*

sources of agricultural soils.

*3.3.3 The United States of America (USA)*

They also incorporate emissions from irrigation practices by which we can conclude that a great evolution has suffered the emissions of this section of direct

In addition to changes in the measurement of nitrogen biological fixation, the introduction of emissions' new categories, as we saw in Canada, "innovations" will continue to generate and, thus, the inventory of the United States [19], extends the computation of their emissions. This inventory includes in the accounting the nitrogen fixed, by what we have called-fixing bacteria N2 of the soil, which belong, for example, the genus *Azotobacter* and so-called biological fixation free. The USA in your inventory calls this fixation as asymbiotic fixation and, therefore, reports emissions. (**Figure 1**) shows the scheme how are calculated agricultural soils N2O emissions' in United States inventory. The US inventory defines asymbiotic fixation as the fixation of atmospheric N2 by bacteria living in the soil and that do not have direct relationship with plants. This inventory says that although the nitrogen incorporated by asymbiotic N fixation is not specifically collected by the 2006 Guidelines, it is a

**30**

**Figure 1.**

*Asymbiotic fixation [19].*

In this section, we will discuss, using New Zealand GHG inventories as example [21, 22], how we can make improvements in GHG inventories and we introduce measurement methodologies of new technologies applied in the agricultural sector.

### **4.1 The nitrification inhibitors**

The application of nitrogen fertilizers to the soil means the occurrence of biological and physicochemical reactions that leads to loss of nitrogen. The use of fertilizers with nitrification inhibitors has become a useful tool to reduce loss and improve the efficiency of the N. The use of nitrogen fertilizers stabilized become widespread and its are added, during the production process, with some substances, such as nitrification inhibitors, which can keep N applied as NH4 + for longer.

These products delayed the transformation of ammonia nitrogen (NH4 + ) to nitrate nitrogen (NO3−) through temporary inhibition of various bacteria *Nitrosomonas* spp., and thus, the nitrogen is released in a progressive and gradual way and, at that same rate, it is assimilated by the crop.

Nitrification inhibitors degrade over time after being applied on the ground, and this degradation is influenced by temperature, moisture, pH and quantity of organic matter. There is already a long list of chemical compounds that have been tested as inhibitors of nitrification in the world (more than 64), but the most studied and used nitrification inhibitors are nitrapyrin, dicyandiamide (DCD) and 3,4-dimethylpyrazol phosphate (DMPP) [23].

In the United States, nitrapyrin is being used in corn, sorghum, wheat, cotton and strawberries (in a manner restricted in these). However, more than 90% is used in corn. Nitrapyrin must be injected and immediately incorporated into the soil due to its volatility and therefore its use is limited in the regions where N is typically injected to the ground. In principle, only it is marketed in the United States.

The dicyandiamide (DCD) has a bacteriostatic effect on bacteria *Nitrosomonas* spp., which only has a depressive effect on those, without killing them (not bactericidal). The disadvantage is that it requires a large amount of DCD for to contribute with between a 10% to 15% of the N-NH4 + to the ground. (Applications are approximately 10 kg per hectare, twice a year, in spring and autumn).

It is a very soluble product that easily seeps with rainfall separating the fraction of ammonium. Another disadvantage is that this molecule can be absorbed by the plant, and in some cases, has generated toxicity. Currently, this product is not only used but also is formulated in combination with other molecules.

3,4-dimethylpyrazol phosphate (DMPP), equally to the DCD, has a bacteriostatic effect, not the bactericidal effect (does not kill bacteria but it inhibits its action for a certain period of time), and it is relatively immobile into the soil; so it does not occur losses by leaching. On the other hand, application rates are very low compared to other nitrification inhibitors (+ −1% of the N-NH4 + ). Their application rate is 16 times lower than the rate of application of the DCD. It has a high selectivity, because it effectively inhibits only the action of *Nitrosomonas* bacteria and it degrades completely into the soil without leaving any residue. To retard the passage of ammonium to nitrate, avoiding nitrogen losses by leaching, it also reduces the effect of soil acidification.

Used as an inhibitor of nitrification, 3,4- dimethylpyrazol phosphate is regulated in the European countries and also fertilizers are used with this product in Asia and Latin America. In contrast to the DCD, in which several authors have cited toxic effects, the 3,4-dimethylpyrazol phosphate has not been demonstrated, for the moment, toxic effects on the plants.

The GHG inventory from New Zealand in the year 2012 has incorporated an amendment to the IPCC methodology that consists in introducing the use of inhibitors of the nitrification for mitigation of emissions of N2O. They developed a methodology for incorporating the inhibitor of nitrification dicyandiamide (DCD) in the agriculture sector. N2O emissions in the agricultural soils category take into account the use of nitrification inhibitors on dairy farms.

Based on several investigations, they have produced a good management practice that consists of the incorporation of the DCD to pastures and maximize reductions of N2O emissions. The utilization of DCD has been reflected on the accounting and, so, incorporated in the inventory calculations and they modified parameter FracLEACH [24] and emission factor EF3PR & P [25] that are minor when using nitrification inhibitors. With these new emission factors, significant reductions of N2O emissions from soils in both direct (nitrate leaching) and indirect (volatilization of N2O) are achieved.

The emission factors are fixed by the Guidelines, but it is possible to modify the amount with scientific studies and this practice is done in New Zealand.

**Table 10**, [26] shows the differences between emission factors when DCD is not used (for example in 2012, EF3PR & P = 0.00994 front EF3PR & P = 0.01, that it is the amount fixed in the Guidelines, and FracLEACH = 0.06964 front FracLEACH = 0.07, that it is the amount fixed in the Guidelines) and as such these small differences of the emission factors meant, in total, that in 2102 its "save" 19.6 Mt. CO2 eq.

Currently, the dicyandiamide (DCD) retired voluntarily in New Zealand's market due to the concern of customers by the existence of certain residues in dairy products even though it is at a very low level. On this point, the inventory of Agriculture of New Zealand says: "there is no risk in dairy products for humans with low levels of inhibitor used." However, in the last inventory of 2018, they asserted that sales of this product have been suspended and they have not returned to use this discount since the year 2012.

**33**

fertilizers with DMPP.

urine while the animals are grazing.

**4.2 Urea inhibitors**

the urease inhibitor.

CO2 eq. of emissions.

0.025% rates.

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

Within the existing inhibitors, 3,4-dimethylpyrazol phosphate is the inhibitor which has major advantages over the rest of nitrification inhibitors existing, due to his effectiveness at low concentrations, its stability and movement on the ground. The DMPP is an inhibitor of nitrification considerate under different national regulations of fertilizers, including the Spanish. In particular, the Royal Decree 824/2005 on fertilizer products includes fertilizers with DMPP as suitable for marketing in Spain. Similarly, Portugal has authorized the commercialization of

*Emission factors, parameters and mitigation for New Zealand's DCD inhibitor calculations from 2007 to 2012.*

*Note: EF3(PRP) = 0.01 and FracLEACH = 0.07 when inhibitor is not applied. All other emission factors and parameters relating to animal excreta and fertilizer use (FracGASM, FracGASF, EF4 and EF5) remain unchanged* 

**2007 2008 2009 2010 2011 2012**

3.5 4.5 3.1 2.2 3.0 2.9

0.00992 0.00990 0.00993 0.00995 0.00993 0.00994

0.06957 0.06944 0.06962 0.06973 0.06963 0.06964

18.7 25.4 18.3 13.7 19.5 19.6

The New Zealand GHG inventory from agriculture has also developed a methodology for urea inhibitor called urease. Urea is the nitrogen fertilizer most used in the grasslands that are grazed in New Zealand and in addition to be excreted in the

Urea inhibitors suspend or delay, during a period of time, the transformation

the hydrolytic action of the urease enzyme. It reduces the speed at which urea is hydrolyzed in the soil and, therefore, losses of ammonium in the atmosphere by

The objective is to increase the efficiency of fertilizations with urea and to minimize the environmental impact of their use. For the purpose of the inclusion in the GHG inventory, they change the value of FracGASF [27] parameter when using

Field- and laboratory-based studies [28] have come to the conclusion that using these inhibitors could lower FracGASF = 0.1, which is the amount fixed in the Guidelines, to a new FracGASF = 0.055, when they apply the urea inhibitor at

As a result of these practices, we can see in **Table 11** [29] a strong increase in the use of inhibitor every year and this practice has meant that in 2016 will save 20.1 kt

+ by

of nitrogen in form of amide that exists in the urea to the ammonium NH4

volatilization or nitrate by runoff are reduced or avoided.

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

*when the inhibitor is used as an N2O mitigation technology.*

Percentage of dairy area applied with inhibitor

Final modified emission factor or parameter, EF3(PRP) (kg N2O-N/kg N)

Final modified emission factor or parameter, FracLEACH (kg N2O-N/kg N)

Mitigation (Gg CO2 eq.)

**Table 10.**

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*


*Note: EF3(PRP) = 0.01 and FracLEACH = 0.07 when inhibitor is not applied. All other emission factors and parameters relating to animal excreta and fertilizer use (FracGASM, FracGASF, EF4 and EF5) remain unchanged when the inhibitor is used as an N2O mitigation technology.*

#### **Table 10.**

*Environmental Chemistry and Recent Pollution Control Approaches*

mately 10 kg per hectare, twice a year, in spring and autumn).

used but also is formulated in combination with other molecules.

compared to other nitrification inhibitors (+ −1% of the N-NH4

account the use of nitrification inhibitors on dairy farms.

with between a 10% to 15% of the N-NH4

effect of soil acidification.

tion of N2O) are achieved.

to use this discount since the year 2012.

moment, toxic effects on the plants.

The dicyandiamide (DCD) has a bacteriostatic effect on bacteria *Nitrosomonas* spp., which only has a depressive effect on those, without killing them (not bactericidal). The disadvantage is that it requires a large amount of DCD for to contribute

+

3,4-dimethylpyrazol phosphate (DMPP), equally to the DCD, has a bacteriostatic effect, not the bactericidal effect (does not kill bacteria but it inhibits its action for a certain period of time), and it is relatively immobile into the soil; so it does not occur losses by leaching. On the other hand, application rates are very low

rate is 16 times lower than the rate of application of the DCD. It has a high selectivity, because it effectively inhibits only the action of *Nitrosomonas* bacteria and it degrades completely into the soil without leaving any residue. To retard the passage of ammonium to nitrate, avoiding nitrogen losses by leaching, it also reduces the

Used as an inhibitor of nitrification, 3,4- dimethylpyrazol phosphate is regulated in the European countries and also fertilizers are used with this product in Asia and Latin America. In contrast to the DCD, in which several authors have cited toxic effects, the 3,4-dimethylpyrazol phosphate has not been demonstrated, for the

The GHG inventory from New Zealand in the year 2012 has incorporated an amendment to the IPCC methodology that consists in introducing the use of inhibitors of the nitrification for mitigation of emissions of N2O. They developed a methodology for incorporating the inhibitor of nitrification dicyandiamide (DCD) in the agriculture sector. N2O emissions in the agricultural soils category take into

Based on several investigations, they have produced a good management practice that consists of the incorporation of the DCD to pastures and maximize reductions of N2O emissions. The utilization of DCD has been reflected on the accounting and, so, incorporated in the inventory calculations and they modified parameter FracLEACH [24] and emission factor EF3PR & P [25] that are minor when using nitrification inhibitors. With these new emission factors, significant reductions of N2O emissions from soils in both direct (nitrate leaching) and indirect (volatiliza-

The emission factors are fixed by the Guidelines, but it is possible to modify the

**Table 10**, [26] shows the differences between emission factors when DCD is not used (for example in 2012, EF3PR & P = 0.00994 front EF3PR & P = 0.01, that it is the amount fixed in the Guidelines, and FracLEACH = 0.06964 front FracLEACH = 0.07, that it is the amount fixed in the Guidelines) and as such these small differences of the

Currently, the dicyandiamide (DCD) retired voluntarily in New Zealand's market due to the concern of customers by the existence of certain residues in dairy products even though it is at a very low level. On this point, the inventory of Agriculture of New Zealand says: "there is no risk in dairy products for humans with low levels of inhibitor used." However, in the last inventory of 2018, they asserted that sales of this product have been suspended and they have not returned

amount with scientific studies and this practice is done in New Zealand.

emission factors meant, in total, that in 2102 its "save" 19.6 Mt. CO2 eq.

It is a very soluble product that easily seeps with rainfall separating the fraction of ammonium. Another disadvantage is that this molecule can be absorbed by the plant, and in some cases, has generated toxicity. Currently, this product is not only

to the ground. (Applications are approxi-

+

). Their application

**32**

*Emission factors, parameters and mitigation for New Zealand's DCD inhibitor calculations from 2007 to 2012.*

Within the existing inhibitors, 3,4-dimethylpyrazol phosphate is the inhibitor which has major advantages over the rest of nitrification inhibitors existing, due to his effectiveness at low concentrations, its stability and movement on the ground. The DMPP is an inhibitor of nitrification considerate under different national regulations of fertilizers, including the Spanish. In particular, the Royal Decree 824/2005 on fertilizer products includes fertilizers with DMPP as suitable for marketing in Spain. Similarly, Portugal has authorized the commercialization of fertilizers with DMPP.

#### **4.2 Urea inhibitors**

The New Zealand GHG inventory from agriculture has also developed a methodology for urea inhibitor called urease. Urea is the nitrogen fertilizer most used in the grasslands that are grazed in New Zealand and in addition to be excreted in the urine while the animals are grazing.

Urea inhibitors suspend or delay, during a period of time, the transformation of nitrogen in form of amide that exists in the urea to the ammonium NH4 + by the hydrolytic action of the urease enzyme. It reduces the speed at which urea is hydrolyzed in the soil and, therefore, losses of ammonium in the atmosphere by volatilization or nitrate by runoff are reduced or avoided.

The objective is to increase the efficiency of fertilizations with urea and to minimize the environmental impact of their use. For the purpose of the inclusion in the GHG inventory, they change the value of FracGASF [27] parameter when using the urease inhibitor.

Field- and laboratory-based studies [28] have come to the conclusion that using these inhibitors could lower FracGASF = 0.1, which is the amount fixed in the Guidelines, to a new FracGASF = 0.055, when they apply the urea inhibitor at 0.025% rates.

As a result of these practices, we can see in **Table 11** [29] a strong increase in the use of inhibitor every year and this practice has meant that in 2016 will save 20.1 kt CO2 eq. of emissions.


#### **Table 11.**

*Mitigation impact of urease inhibitors on nitrous oxide emissions from volatilization, from 2007 to 2016.*

We can conclude that nitrification inhibitors and urea inhibitors are chemical compounds whose use can be a valid methodology to reduce the accumulation of nitrates in the soil and prevent emissions both by leaching and by volatilization of N2O.

Different studies on mitigation policies propose the use of these practices with fertilizers or urea with inhibitors as, for example, France [30], that in a study of the INRA, proposes this mitigation measure in its roadmap for the agricultural sector. Similarly, the FAO [31], in his study of mitigation of emissions from production livestock, proposes the addiction of these inhibitors to the manure.

The use of inhibitors can be a useful tool to improve the efficiency of N in the soil, and for this reason, the use is being increased. However, this use needs still more securities, in particular, with regard to its possible effect on the food chain and in the environment and more research for the security on these products should be carried out.

Currently, these products accounted for GHG inventories do not appear and, the development of a methodology similar to the made by New Zealand could be used by other countries to reduce emissions. The 2018 USA inventory indicates that it will develop a methodology for use in next inventory due to the use of these products in the country.

#### **4.3 The inoculation of nitrogen-fixing bacteria**

The importance of legumes in the agricultural crops and its property of symbiotic fixation open the possibility of extend this property to other plant species of agricultural interest. The consequent descent of the need to use nitrogen fertilizers has made nitrogen biological fixation a subject of intense research over the years.

We will use other works of the Centro Superior de Investigaciones Científicas (Spain) for explaining this topic [32].

*"NBF is capable of providing between 25 and 84% of the nitrogen required for normal growth and development of the cultivation of soybean. Therefore, nitrogen fixation presents great economic and ecological interest. In fact, and as an example, the high productions of soybeans around the world are due to this process through the application of microbial inoculants."*

One of the new technologies to be applied in the agricultural field that could be used for reducing emissions, in addition to other benefits, is known as biofertilization, which continues influencing mutualistic symbiosis of nitrogen fixation.

**35**

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

jungle and soybeans should be inoculated for their cultivation.

tial. With the inoculation, up to 80% can be reached.

of the atmospheric N2 for themselves.

sponding symbiosis.

**5. Conclusions**

preparing mitigation policies.

Biofertilization is defined as the use of living organisms to improve the growth

of plants by two ways or increasing their nutrition making available available the required nutrients or acting on its development by the production of phytohormones. Also we can use biological control and biological remediation when with the inoculation of microorganisms we want to remove pathogens or increase the defensive response of the plants or remove xenobiotics compounds from the

The use of inoculants for legumes is essential if the vegetal species has not been grown in that soil and, therefore, there is no presence of the corresponding *Rhizobium* species. This is the case of soybean in Europe that has to be inoculated with the bacteria *Bradyrhizobium japonicum* or *Sinorhizobium fredii*. This practice is also made in Brazil because most of the farmland comes from deforestation of the

In a soil where is planted a new vegetal specie, if the natural infection's plant with these bacteria isn't possible, the crop efficiency is not superior to 40% poten-

For example, in Spain, there are, in our soil, bacteria appropriate for crops how, alfalfa, clover, pea, lentil, chickpea, etc., but are not always effective enough; these are nitrogen fixative, and there are cases in which the inoculation is necessary if we want to obtain satisfactory yields. The same occurs when characteristics of the soil, such as acidity, drought, etc., influence the persistence of *Rhizobium*

New knowledge is being developed in this field of biological nitrogen fixation and investigators start to get the extension of the fixing capacity to other nonleguminous plants of interest. So, they are trying to achieve that corn, wheat or rice be infected efficiently by *Rhizobium*, and begin to glimpse the possibility of transferring to these plants the fixing capacity. So its plants will be able to take advantage

In the short term, the selection of strains and their appropriate genetic manipulation are underway, to prepare the most suitable inoculants and, also, to improve the plant so that there are no limiting factors in the establishment of the corre-

*"Researchers point that not everything is optimal in obtaining self-sufficient plants for nitrogen, because although the crops should not be fertilized, it would be less productive. The energy cost involved in fixing becomes up to three times higher than the utilization of nitrate and the plants would grow less, the performance would be lower and may even were reduced the area of cultivation. But this independence of nitrogen fertilization would possibly more profitable crop, more suitable for* 

In short, as we have seen above, the 2006 Guidelines do not consider the contribution of nitrogen by biological fixation, which involves a direct emission of N2O to the atmosphere and, therefore, this technology should be taken into account.

The GHG inventory is a great source of information, not only for its environmental aspect, but also by the possibility of using their data for relevant technical and economic analyses. Other quality is its role for serving us a guidance when

This comparative study of different inventories show the wide spectrum of approaches and the importance of the management of the accounting rules.

*economically weak areas and environmentally cleaner" [32].*

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

environment.

bacteria.

#### *Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*

*Environmental Chemistry and Recent Pollution Control Approaches*

**urease inhibitor (urea treated/total urea)**

2007 5.0 3.0 2008 5.2 3.0 2009 9.4 4.7 2010 6.9 4.1 2011 5.3 3.5 2012 7.0 4.6 2013 8.6 5.9 2014 20.2 13.6 2015 16.2 13.1 2016 26.5 20.1

**Year Percentage of urea applied that included** 

We can conclude that nitrification inhibitors and urea inhibitors are chemical compounds whose use can be a valid methodology to reduce the accumulation of nitrates in the soil and prevent emissions both by leaching and by volatilization of N2O.

*Mitigation impact of urease inhibitors on nitrous oxide emissions from volatilization, from 2007 to 2016.*

**Estimated greenhouse gas mitigation from using urease inhibitor kt. CO2 eq.**

Different studies on mitigation policies propose the use of these practices with fertilizers or urea with inhibitors as, for example, France [30], that in a study of the INRA, proposes this mitigation measure in its roadmap for the agricultural sector. Similarly, the FAO [31], in his study of mitigation of emissions from production

The use of inhibitors can be a useful tool to improve the efficiency of N in the soil, and for this reason, the use is being increased. However, this use needs still more securities, in particular, with regard to its possible effect on the food chain and in the environment and more research for the security on these products should

Currently, these products accounted for GHG inventories do not appear and, the development of a methodology similar to the made by New Zealand could be used by other countries to reduce emissions. The 2018 USA inventory indicates that it will develop a methodology for use in next inventory due to the use of these products in

The importance of legumes in the agricultural crops and its property of symbiotic fixation open the possibility of extend this property to other plant species of agricultural interest. The consequent descent of the need to use nitrogen fertilizers has made nitrogen biological fixation a subject of intense research over the years. We will use other works of the Centro Superior de Investigaciones Científicas

*"NBF is capable of providing between 25 and 84% of the nitrogen required for normal growth and development of the cultivation of soybean. Therefore, nitrogen fixation presents great economic and ecological interest. In fact, and as an example, the high productions of soybeans around the world are due to this process through* 

One of the new technologies to be applied in the agricultural field that could be used for reducing emissions, in addition to other benefits, is known as biofertilization, which continues influencing mutualistic symbiosis of nitrogen fixation.

livestock, proposes the addiction of these inhibitors to the manure.

**4.3 The inoculation of nitrogen-fixing bacteria**

*the application of microbial inoculants."*

(Spain) for explaining this topic [32].

**34**

be carried out.

**Table 11.**

the country.

Biofertilization is defined as the use of living organisms to improve the growth of plants by two ways or increasing their nutrition making available available the required nutrients or acting on its development by the production of phytohormones. Also we can use biological control and biological remediation when with the inoculation of microorganisms we want to remove pathogens or increase the defensive response of the plants or remove xenobiotics compounds from the environment.

The use of inoculants for legumes is essential if the vegetal species has not been grown in that soil and, therefore, there is no presence of the corresponding *Rhizobium* species. This is the case of soybean in Europe that has to be inoculated with the bacteria *Bradyrhizobium japonicum* or *Sinorhizobium fredii*. This practice is also made in Brazil because most of the farmland comes from deforestation of the jungle and soybeans should be inoculated for their cultivation.

In a soil where is planted a new vegetal specie, if the natural infection's plant with these bacteria isn't possible, the crop efficiency is not superior to 40% potential. With the inoculation, up to 80% can be reached.

For example, in Spain, there are, in our soil, bacteria appropriate for crops how, alfalfa, clover, pea, lentil, chickpea, etc., but are not always effective enough; these are nitrogen fixative, and there are cases in which the inoculation is necessary if we want to obtain satisfactory yields. The same occurs when characteristics of the soil, such as acidity, drought, etc., influence the persistence of *Rhizobium* bacteria.

New knowledge is being developed in this field of biological nitrogen fixation and investigators start to get the extension of the fixing capacity to other nonleguminous plants of interest. So, they are trying to achieve that corn, wheat or rice be infected efficiently by *Rhizobium*, and begin to glimpse the possibility of transferring to these plants the fixing capacity. So its plants will be able to take advantage of the atmospheric N2 for themselves.

In the short term, the selection of strains and their appropriate genetic manipulation are underway, to prepare the most suitable inoculants and, also, to improve the plant so that there are no limiting factors in the establishment of the corresponding symbiosis.

*"Researchers point that not everything is optimal in obtaining self-sufficient plants for nitrogen, because although the crops should not be fertilized, it would be less productive. The energy cost involved in fixing becomes up to three times higher than the utilization of nitrate and the plants would grow less, the performance would be lower and may even were reduced the area of cultivation. But this independence of nitrogen fertilization would possibly more profitable crop, more suitable for economically weak areas and environmentally cleaner" [32].*

In short, as we have seen above, the 2006 Guidelines do not consider the contribution of nitrogen by biological fixation, which involves a direct emission of N2O to the atmosphere and, therefore, this technology should be taken into account.

## **5. Conclusions**

The GHG inventory is a great source of information, not only for its environmental aspect, but also by the possibility of using their data for relevant technical and economic analyses. Other quality is its role for serving us a guidance when preparing mitigation policies.

This comparative study of different inventories show the wide spectrum of approaches and the importance of the management of the accounting rules.

A detailed analysis of the nitrogen biological fixation and, particularly, the cultivation of soybeans, allow us to appreciate the importance of the follow-up to the guidelines that govern the preparation of inventories.

This article also shows significant differences in the volume of emissions due to the use of the 1996 Guidelines front the 2006 Guidelines, and both change the rules and the changes of global warming potentials. Using the emissions from the cultivation of an important legume such as Argentina and Brazil soybeans, we can observe in a practical way the importance of methodological changes in "accounting standards."

The case of the USA includes emissions that would not be bound by these guidelines, such as those due to asymbiotic fixation and the case of Canada incorporates the non-tillage (conservation agriculture) sink effect as the emissions due to fallow and irrigation systems.

On the other hand, examples of introduction of new technologies are exposed that are not included in the Guidelines, which require the development of specific methodologies. The case of the inventory of New Zealand regarding the nitrification inhibitors and urea inhibitor is a relevant example. Nitrogen Biological Fixation should be one of the fields to research in more depth because the specificity of some bacteria to capture atmospheric N2 could provide large reductions in the use of synthetic nitrogen fertilizers.

Although the guidelines seek to unify criteria, we have exposed the full spectrum of options that have all these inventories to the same heading of emission from agricultural soils. We must bear in mind that correct accounting will condition strongly the analyses and measures that specifically we design and try to implement in the agricultural sector.

The objective should be that the effectiveness of a mitigation policy was validated for the concrete results of the GHG inventory and, of course, that this policy could be applied at the farm level. Deep knowledge of accounting rules is a necessary premise. It is very important that all persons participating in the measurement of emissions (technicians, researchers, public, professional managers of agriculture, etc.) are aware of the use of the same rules.

A country greenhouse gas measurement methodology is a science, not well known among professionals, which requires, in addition to a large sectoral specialization, to be addressed, by large multidisciplinary teams, and should be given in college and, perhaps, the most difficult, to be conducted at the level of agricultural farm in an understandable way. We must bear in mind that farmers will be responsible for putting into practice any measure of mitigation or generation of sinks that intends to.

## **Author details**

Dionisio Rodríguez Xunta de Galicia, Santiago de Compostela, Spain

Address all correspondence to: dionisio.rodriguez.alvarez@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**37**

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

1996. Available from: https://www.ipccnggip.iges.or.jp/public/gl/invs1.html

[9] EEA, European Environment Agency. Annual European Union Greenhouse Gas Inventory 1990-2012 and Inventory Report 2014. European Environment Agency. 2014. Available from: http://www.eea.europa.eu/ publications/european-uniongreenhouse-gas-inventory-2014

[10] FAOSTAT. Available from: http:// www.fao.org/faostat/en/#data/QC

[11] Segunda comunicación nacional de la República Argentina a la convención

[12] Tercera comunicación nacional de la República Argentina a la convención marco de las Naciones Unidas sobre el cambio climático.2015. Available from: https://unfccc.int/documents/67499

[13] Asociación Argentina de Consorcios Regionales de Experimentación Agrícola (AACREA), Fundación Torcuato Di Tella (FTDT), Price Waterhouse & Co. Asesores de Empresas SRL. Informe comparativo de las Directrices IPCC 1996 vs. 2006 Agricultura, Ganadería, y Cambio de Uso del Suelo y Silvicultura. 2015. Available from: http://ambiente.gob.ar/archivos/ web/ProyTerceraCNCC/file/5\_%20 Comparativa%20Gu%c3%adas%20 IPCC%201996%20y%202006%20-%20 Agricultura,%20Ganaderia%20y%20

[14] Third National Communication of Brazil to the United Nations Framework

Convention on Climate Change. Available from: https://unfccc.int/

[15] Canada – Initial Submission 2005 GHG Inventory 1990-2003.

marco de las Naciones Unidas sobre el cambio climático. 2008. Available from: https://unfccc.int/

documents/67498

CUSS.pdf

documents/66129

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

[1] IPCC Assessment Reports. Available from: http://www.ipcc.ch/publications\_ and\_data/publications\_and\_data\_

[2] National Inventories Submisions. Available from: https://unfccc.int/ process-and-meetings/transparencyand-reporting/reporting-andreview-under-the-convention/ greenhouse-gas-inventories-annexi-parties/national-inventory-

**References**

reports.shtml

submissions-2018

[3] National Communication Submisions. Available from: https:// unfccc.int/process#:0c4d2d14-7742- 48fd-982e-d52b41b85bb0:f666393f-34f5- 45d6-a44e-8d03be236927:cc852874- 8331-492c-a332-cc6313dec434

[4] International Panel of Climate Change. UNEP.WMO (2006) Directrices del IPCC de 2006 para los inventarios nacionales de gases de efecto invernadero. IPCC. Available from: http://www.ipcc-nggip.iges.or.jp/ public/2006gl/spanish/pdf/4\_Volume4/

V4\_11\_Ch11\_N2O&CO2.pdf

ciencia/fijacion/

fijacion/

[5] Pascual JO. Fijación biológica de Nitrógeno. (Última Act. 08-02-2008). CSIC, Granada: Estación Experimental del Zaidín; 2008. Available from: https://www2.eez.csic.es/olivares/

[6] Pascual JO. Fijación biológica de Nitrógeno. (Última Act. 08-02-2008). CSIC, Granada: Estación Experimental del Zaidín; 2008. Available from: http:// www.eez.csic.es/~olivares/ciencia/

[7] Oficina Española de Cambio Climático. Hoja de ruta de los sectores difusos a 2020. MAGRAMA. Oficina Española de Cambio Climático; 2014

[8] Revised 1996 IPCC guidelines for National Greenhouse Gas Inventories. *Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*

## **References**

*Environmental Chemistry and Recent Pollution Control Approaches*

the guidelines that govern the preparation of inventories.

A detailed analysis of the nitrogen biological fixation and, particularly, the cultivation of soybeans, allow us to appreciate the importance of the follow-up to

This article also shows significant differences in the volume of emissions due to the use of the 1996 Guidelines front the 2006 Guidelines, and both change the rules and the changes of global warming potentials. Using the emissions from the cultivation of an important legume such as Argentina and Brazil soybeans, we can observe in a practical way the importance of methodological changes in "accounting standards." The case of the USA includes emissions that would not be bound by these guidelines, such as those due to asymbiotic fixation and the case of Canada incorporates the non-tillage (conservation agriculture) sink effect as the emissions due to fallow

On the other hand, examples of introduction of new technologies are exposed that are not included in the Guidelines, which require the development of specific methodologies. The case of the inventory of New Zealand regarding the nitrification inhibitors and urea inhibitor is a relevant example. Nitrogen Biological Fixation should be one of the fields to research in more depth because the specificity of some bacteria to capture atmospheric N2 could provide large reductions in the use of

Although the guidelines seek to unify criteria, we have exposed the full spectrum of options that have all these inventories to the same heading of emission from agricultural soils. We must bear in mind that correct accounting will condition strongly the analyses and measures that specifically we design and try to implement

The objective should be that the effectiveness of a mitigation policy was validated for the concrete results of the GHG inventory and, of course, that this policy could be applied at the farm level. Deep knowledge of accounting rules is a necessary premise. It is very important that all persons participating in the measurement of emissions (technicians, researchers, public, professional managers of agricul-

A country greenhouse gas measurement methodology is a science, not well known among professionals, which requires, in addition to a large sectoral specialization, to be addressed, by large multidisciplinary teams, and should be given in college and, perhaps, the most difficult, to be conducted at the level of agricultural farm in an understandable way. We must bear in mind that farmers will be responsible for putting into practice any measure of mitigation or generation of sinks that intends to.

**36**

**Author details**

Dionisio Rodríguez

and irrigation systems.

synthetic nitrogen fertilizers.

in the agricultural sector.

Xunta de Galicia, Santiago de Compostela, Spain

provided the original work is properly cited.

ture, etc.) are aware of the use of the same rules.

Address all correspondence to: dionisio.rodriguez.alvarez@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] IPCC Assessment Reports. Available from: http://www.ipcc.ch/publications\_ and\_data/publications\_and\_data\_ reports.shtml

[2] National Inventories Submisions. Available from: https://unfccc.int/ process-and-meetings/transparencyand-reporting/reporting-andreview-under-the-convention/ greenhouse-gas-inventories-annexi-parties/national-inventorysubmissions-2018

[3] National Communication Submisions. Available from: https:// unfccc.int/process#:0c4d2d14-7742- 48fd-982e-d52b41b85bb0:f666393f-34f5- 45d6-a44e-8d03be236927:cc852874- 8331-492c-a332-cc6313dec434

[4] International Panel of Climate Change. UNEP.WMO (2006) Directrices del IPCC de 2006 para los inventarios nacionales de gases de efecto invernadero. IPCC. Available from: http://www.ipcc-nggip.iges.or.jp/ public/2006gl/spanish/pdf/4\_Volume4/ V4\_11\_Ch11\_N2O&CO2.pdf

[5] Pascual JO. Fijación biológica de Nitrógeno. (Última Act. 08-02-2008). CSIC, Granada: Estación Experimental del Zaidín; 2008. Available from: https://www2.eez.csic.es/olivares/ ciencia/fijacion/

[6] Pascual JO. Fijación biológica de Nitrógeno. (Última Act. 08-02-2008). CSIC, Granada: Estación Experimental del Zaidín; 2008. Available from: http:// www.eez.csic.es/~olivares/ciencia/ fijacion/

[7] Oficina Española de Cambio Climático. Hoja de ruta de los sectores difusos a 2020. MAGRAMA. Oficina Española de Cambio Climático; 2014

[8] Revised 1996 IPCC guidelines for National Greenhouse Gas Inventories. 1996. Available from: https://www.ipccnggip.iges.or.jp/public/gl/invs1.html

[9] EEA, European Environment Agency. Annual European Union Greenhouse Gas Inventory 1990-2012 and Inventory Report 2014. European Environment Agency. 2014. Available from: http://www.eea.europa.eu/ publications/european-uniongreenhouse-gas-inventory-2014

[10] FAOSTAT. Available from: http:// www.fao.org/faostat/en/#data/QC

[11] Segunda comunicación nacional de la República Argentina a la convención marco de las Naciones Unidas sobre el cambio climático. 2008. Available from: https://unfccc.int/ documents/67498

[12] Tercera comunicación nacional de la República Argentina a la convención marco de las Naciones Unidas sobre el cambio climático.2015. Available from: https://unfccc.int/documents/67499

[13] Asociación Argentina de Consorcios Regionales de Experimentación Agrícola (AACREA), Fundación Torcuato Di Tella (FTDT), Price Waterhouse & Co. Asesores de Empresas SRL. Informe comparativo de las Directrices IPCC 1996 vs. 2006 Agricultura, Ganadería, y Cambio de Uso del Suelo y Silvicultura. 2015. Available from: http://ambiente.gob.ar/archivos/ web/ProyTerceraCNCC/file/5\_%20 Comparativa%20Gu%c3%adas%20 IPCC%201996%20y%202006%20-%20 Agricultura,%20Ganaderia%20y%20 CUSS.pdf

[14] Third National Communication of Brazil to the United Nations Framework Convention on Climate Change. Available from: https://unfccc.int/ documents/66129

[15] Canada – Initial Submission 2005 GHG Inventory 1990-2003. Available from: https://unfccc.int/ process/transparency-and-reporting/ reporting-and-review-under-theconvention/greenhouse-gas-inventories/ submissions-of-annual-greenhouse-gasinventories-for-2017/submissions-ofannual-ghg-inventories-2005

[16] National Inventory Report 1990- 2004. Greenhouse Gas Sources and Sinks in Canada — Final Submission. Available from: https://unfccc.int/ process/transparency-and-reporting/ reporting-and-review-under-theconvention/greenhouse-gas-inventories/ submissions-of-annual-greenhouse-gasinventories-for-2017/submissions-ofannual-ghg-inventories-2006

[17] National Inventory Report 1990- 2016. Greenhouse Gas Sources and Sinks in Canada. Available from: https:// unfccc.int/documents/65715

[18] "There is no evidence that measurable amounts of N2O are produced in Canadian agricultural soils during the N fixation process itself. Therefore, Canada decided to report this source as "not occurring." However, the contribution of legume N to N2O emissions is included as a source of N2O emissions from crop residue decomposition on agricultural soils (NRES)".The Canadian Government. (2014). National Inventory report. 1990-2012. Green house gases and sources in Canada. The Canadian Government's Submission to the UN Framework Convention on Climate Change. 2014. Available from: https:// unfccc.int/process-and-meetings/ transparency-and-reporting/reportingand-review-under-the-convention/ greenhouse-gas-inventories-annexi-parties/national-inventorysubmissions-2018

[19] Inventory of U.S greenhouse gas emissions and sinks 1990-2016. EPA 2018. Available from: http:// www.epa.gov/ghgemissions/

inventory-us-greenhouse-gasemissionsand-sinks-1990-2016

[20] Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990-2012. U.S. Environmental Protection Agency. U.S.A; 2014. Available from: http:// unfccc.int/files/national\_reports/ annex\_i\_ghg\_inventories/national\_ inventories\_submissions/application/ zip/usa-2014-nir-15apr.zip

[21] "N inputs from asymbiotic N fixation are not directly addressed in *2006 IPCC Guidelines*, but are a component of the total emissions from managed lands and are included in the Tier 3 approach developed for this source". U.S. Environmental Protection Agency. (2014). Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990- 2012. Ministry for the Environment. 2014. Available from: http:// unfccc.int/files/national\_reports/ annex\_i\_ghg\_inventories/national\_ inventories\_submissions/application/ zip/nzl-2014-nir-14apr.zip

[22] Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990-2016. Ministry for the Environment. 2018. Available from: https://unfccc.int/documents/65588

[23] Trenkel ME. Slow- and Controlled-Release and Stabilized Fertilizers: An Option for Enhancing Nutrient Use Efficiency in Agriculture. Paris, France: International Fertilizer Industry Association (IFA); 2010. Available from: https://www.google.es/?gws\_ rd=ssl#q=Slow-+and+Controlled-Releas e+and+Stabilized+Fertilizers:+An+Opti on+for+Enhancing+Nutrient+Use+Effic iency+in+Agriculture

[24] FracLEACH-(H) = fraction of all N added to/mineralised in managed soils in regions where leaching/runoff occurs that is lost through leaching and runoff, kg N (kg of N additions)-1

**39**

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis*

nes+de+gases+de+efecto+invernadero+ en+la+producci%C3%B3n+ganadera.+U na+revisi%C3%B3n+de+las+opciones+t %C3%A9cnicas+para+la+reducci%C3% B3n+de+las+emisiones+de+gases+difere

[32] Pascual JO. Tiene futuro la fijación biológica de nitrógeno? CSIC, Granada: Estación Experimental del Zaidín; 2008. Available from: http://www.eez.csic. es/~olivares/ciencia/futuro2/index.html

[33] Rochette P, Janzen H. Towards a revised coefficient for estimating N2O emissions from legumes. Nutrient Cycling in Agroecosystems.

ntes+al+CO2.+FAO+2013

2005;**73**(2):171-179

*DOI: http://dx.doi.org/10.5772/intechopen.84208*

[25] EF3PRP = emission factor for N2O emissions from urine and dung N deposited on pasture, range and paddock by grazing animals, kg N2O–N

[26] Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990-2016. Ministry for the Environment; 2018. p. 198. Available from: https://unfccc.int/

[27] FracGASF = fraction of synthetic fertiliser N that volatilises as NH3 and NOx, kg N volatilised (kg of N

[28] "Saggar et al. (2013) showed that the presently recommended country-specific value of FracGASF of 0.1 be reduced to 0.055. This finding was based on field and laboratory studies conducted both in New Zealand and worldwide." Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990 – 2016. Ministry for the Environment; 2018.

[29] Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990-2016. Ministry for the

[30] European Parliament. Measures at farm level to reduce greenhouse gas emissions from EU agriculture. Directorate General for Internal Policies. Policy Department Structural and Cohesion Policies. Agriculture and Environmental Development. European

[31] Gerber PJ, et al. Mitigación de las emisiones de gases de efecto invernadero

en la producción ganadera. Una revisión de las opciones técnicas para la reducción de las emisiones de gases diferentes al CO2. FAO; 2013. Available from: https://www.google.es/?gws\_rd=s sl#q=Mitigaci%C3%B3n+de+las+emisio

(kg N input)-1

documents/65588

applied)-1

p. 196

Environment; 2018

Parliament; 2014

*Greenhouse Gas Emissions of Agriculture: A Comparative Analysis DOI: http://dx.doi.org/10.5772/intechopen.84208*

[25] EF3PRP = emission factor for N2O emissions from urine and dung N deposited on pasture, range and paddock by grazing animals, kg N2O–N (kg N input)-1

*Environmental Chemistry and Recent Pollution Control Approaches*

inventory-us-greenhouse-gasemissions-

[20] Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990-2012. U.S. Environmental Protection Agency. U.S.A; 2014. Available from: http:// unfccc.int/files/national\_reports/ annex\_i\_ghg\_inventories/national\_ inventories\_submissions/application/

zip/usa-2014-nir-15apr.zip

2014. Available from: http:// unfccc.int/files/national\_reports/ annex\_i\_ghg\_inventories/national\_ inventories\_submissions/application/

zip/nzl-2014-nir-14apr.zip

iency+in+Agriculture

kg N (kg of N additions)-1

[22] Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990-2016. Ministry for the Environment. 2018. Available from: https://unfccc.int/documents/65588

[23] Trenkel ME. Slow- and Controlled-Release and Stabilized Fertilizers: An Option for Enhancing Nutrient Use Efficiency in Agriculture. Paris, France: International Fertilizer Industry Association (IFA); 2010. Available from: https://www.google.es/?gws\_ rd=ssl#q=Slow-+and+Controlled-Releas e+and+Stabilized+Fertilizers:+An+Opti on+for+Enhancing+Nutrient+Use+Effic

[24] FracLEACH-(H) = fraction of all N added to/mineralised in managed soils in regions where leaching/runoff occurs that is lost through leaching and runoff,

[21] "N inputs from asymbiotic N fixation are not directly addressed in *2006 IPCC Guidelines*, but are a component of the total emissions from managed lands and are included in the Tier 3 approach developed for this source". U.S. Environmental Protection Agency. (2014). Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990- 2012. Ministry for the Environment.

and-sinks-1990-2016

Available from: https://unfccc.int/ process/transparency-and-reporting/ reporting-and-review-under-theconvention/greenhouse-gas-inventories/ submissions-of-annual-greenhouse-gasinventories-for-2017/submissions-of-

annual-ghg-inventories-2005

annual-ghg-inventories-2006

unfccc.int/documents/65715

[18] "There is no evidence that measurable amounts of N2O are produced in Canadian agricultural soils during the N fixation process itself. Therefore, Canada decided to report this source as "not occurring." However, the contribution of legume N to N2O emissions is included as a source of N2O emissions from crop residue decomposition on agricultural soils (NRES)".The Canadian Government. (2014). National Inventory report. 1990-2012. Green house gases and sources in Canada. The Canadian Government's Submission to the UN Framework Convention on Climate Change. 2014. Available from: https:// unfccc.int/process-and-meetings/ transparency-and-reporting/reportingand-review-under-the-convention/ greenhouse-gas-inventories-annexi-parties/national-inventory-

[17] National Inventory Report 1990- 2016. Greenhouse Gas Sources and Sinks in Canada. Available from: https://

[16] National Inventory Report 1990- 2004. Greenhouse Gas Sources and Sinks in Canada — Final Submission. Available from: https://unfccc.int/ process/transparency-and-reporting/ reporting-and-review-under-theconvention/greenhouse-gas-inventories/ submissions-of-annual-greenhouse-gasinventories-for-2017/submissions-of-

**38**

submissions-2018

[19] Inventory of U.S greenhouse gas emissions and sinks 1990-2016. EPA 2018. Available from: http:// www.epa.gov/ghgemissions/

[26] Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990-2016. Ministry for the Environment; 2018. p. 198. Available from: https://unfccc.int/ documents/65588

[27] FracGASF = fraction of synthetic fertiliser N that volatilises as NH3 and NOx, kg N volatilised (kg of N applied)-1

[28] "Saggar et al. (2013) showed that the presently recommended country-specific value of FracGASF of 0.1 be reduced to 0.055. This finding was based on field and laboratory studies conducted both in New Zealand and worldwide." Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990 – 2016. Ministry for the Environment; 2018. p. 196

[29] Ministry for the Environment. New Zealand's Greenhouse Gas Inventory 1990-2016. Ministry for the Environment; 2018

[30] European Parliament. Measures at farm level to reduce greenhouse gas emissions from EU agriculture. Directorate General for Internal Policies. Policy Department Structural and Cohesion Policies. Agriculture and Environmental Development. European Parliament; 2014

[31] Gerber PJ, et al. Mitigación de las emisiones de gases de efecto invernadero en la producción ganadera. Una revisión de las opciones técnicas para la reducción de las emisiones de gases diferentes al CO2. FAO; 2013. Available from: https://www.google.es/?gws\_rd=s sl#q=Mitigaci%C3%B3n+de+las+emisio

nes+de+gases+de+efecto+invernadero+ en+la+producci%C3%B3n+ganadera.+U na+revisi%C3%B3n+de+las+opciones+t %C3%A9cnicas+para+la+reducci%C3% B3n+de+las+emisiones+de+gases+difere ntes+al+CO2.+FAO+2013

[32] Pascual JO. Tiene futuro la fijación biológica de nitrógeno? CSIC, Granada: Estación Experimental del Zaidín; 2008. Available from: http://www.eez.csic. es/~olivares/ciencia/futuro2/index.html

[33] Rochette P, Janzen H. Towards a revised coefficient for estimating N2O emissions from legumes. Nutrient Cycling in Agroecosystems. 2005;**73**(2):171-179

**41**

**Chapter 3**

Fluorosis

*and Huajie Zhang*

pounds are important.

skeletal fluorosis

**1. Introduction**

**Abstract**

Progressive Research in the

*Liming Shen, Chengyun Feng, Sijian Xia, Yan Wei,* 

Molecular Mechanisms of Chronic

*Hua Zhang, Danqing Zhao, Fang Yao, Xukun Liu, Yuxi Zhao* 

Long-term excessive intake of fluoride (F) leads to chronic fluorosis, resulting in dental fluorosis and skeletal fluorosis. Chronic exposure to high doses of fluoride can also cause damage to soft tissues, especially when it passes through the blood-brain, blood-testis, and blood-placenta barrier, causing damage to the corresponding tissues. Fluorosis has become a public health problem in some countries or regions around the world. Understanding the pathogenesis of fluorosis is very important. Although the exact mechanism of fluorosis has not been fully elucidated, various mechanisms of fluoride-induced toxicity have been proposed. In this chapter, we will introduce the research progress of the mechanism of fluorosis, focusing on dental fluorosis, skeletal fluorosis, nervous and reproductive system toxicity, and influential factors related to fluoride toxicity (i.e., genetic background, co-exposure with other element). In addition, the application of proteomics and metabolomics in the study of the pathogenesis of fluorosis is also introduced. Currently, there is still no specific treatment for fluorosis. However, since fluorosis is caused by excessive intake of fluoride, avoiding excessive fluoride intake is the critical measure to prevent the disease. In endemic regions, health education and supplement diet with vitamins C, D and E, and calcium and antioxidant com-

**Keywords:** chronic fluorosis, fluoride, influential factor, mechanisms, proteomics,

Fluorine is a highly active gaseous element found widely in nature. Fluoride in small doses is beneficial for preventing dental caries and is commonly used in the prevention of dental caries [1, 2]. However, long-term excessive fluoride intake will affect human health, causing chronic fluorosis. Chronic fluorosis is a systemic disease, high doses of fluoride leads to bioaccumulation in the body, especially hard tissues such as bones and teeth, and primarily harms bones and teeth [3–5]. Besides skeletal and dental damage, excessive exposure to fluoride can also cause other non-phrenological hazards, such as metabolic, structural, and functional damage to

## **Chapter 3**

## Progressive Research in the Molecular Mechanisms of Chronic Fluorosis

*Liming Shen, Chengyun Feng, Sijian Xia, Yan Wei, Hua Zhang, Danqing Zhao, Fang Yao, Xukun Liu, Yuxi Zhao and Huajie Zhang*

## **Abstract**

Long-term excessive intake of fluoride (F) leads to chronic fluorosis, resulting in dental fluorosis and skeletal fluorosis. Chronic exposure to high doses of fluoride can also cause damage to soft tissues, especially when it passes through the blood-brain, blood-testis, and blood-placenta barrier, causing damage to the corresponding tissues. Fluorosis has become a public health problem in some countries or regions around the world. Understanding the pathogenesis of fluorosis is very important. Although the exact mechanism of fluorosis has not been fully elucidated, various mechanisms of fluoride-induced toxicity have been proposed. In this chapter, we will introduce the research progress of the mechanism of fluorosis, focusing on dental fluorosis, skeletal fluorosis, nervous and reproductive system toxicity, and influential factors related to fluoride toxicity (i.e., genetic background, co-exposure with other element). In addition, the application of proteomics and metabolomics in the study of the pathogenesis of fluorosis is also introduced. Currently, there is still no specific treatment for fluorosis. However, since fluorosis is caused by excessive intake of fluoride, avoiding excessive fluoride intake is the critical measure to prevent the disease. In endemic regions, health education and supplement diet with vitamins C, D and E, and calcium and antioxidant compounds are important.

**Keywords:** chronic fluorosis, fluoride, influential factor, mechanisms, proteomics, skeletal fluorosis

## **1. Introduction**

Fluorine is a highly active gaseous element found widely in nature. Fluoride in small doses is beneficial for preventing dental caries and is commonly used in the prevention of dental caries [1, 2]. However, long-term excessive fluoride intake will affect human health, causing chronic fluorosis. Chronic fluorosis is a systemic disease, high doses of fluoride leads to bioaccumulation in the body, especially hard tissues such as bones and teeth, and primarily harms bones and teeth [3–5]. Besides skeletal and dental damage, excessive exposure to fluoride can also cause other non-phrenological hazards, such as metabolic, structural, and functional damage to the nervous system [6–12], kidneys [13–16], liver [14, 16–19], cardiovascular system [20–23], and reproductive system [24–26].

Chronic fluorosis is an endemic disease; it is endemic in at least 25 countries across the globe, China and India being the worst affected among them [27]. Most cases of fluorosis were caused by drinking fluorous water. In China, fluorosis is caused by drinking water as well as inhaling combustion fumes of coal being used as an indoor fuel source [28–31]. Guizhou is one of the most severely afflicted areas of endemic fluorosis in China and this occurrence is due to indoor coal burning [30]. Another type of fluorosis is brick tea-type fluorosis, due to fluoride accumulation in brick tea. It is more prevalent in Tibet than in other regions of China [32]. It is also worth noting that chronic exposure to volcanic environments may lead to the exposure of excessive amounts of fluoride [33]. It is estimated that more than 10% of the worldwide population live within the potential exposure range of some active or historically active volcano, either erupting or in a post-eruption phase [34].

In recent years, numerous studies focused on the molecular mechanisms associated with fluoride toxicity [35–39]. Although the underlying mechanisms of chronic fluorosis is still not well understood, the results of the previous studies indicated that fluoride can induce oxidative stress; regulate intracellular redox homeostasis; and lead to mitochondrial damage, endoplasmic reticulum stress, and alteration of gene expression [35–39]. Other mechanisms include enzyme inhibition, induction of apoptosis, cell cycle arrest, etc. [35–39]. This chapter reviews the present research on the potential adverse effects of overdose fluoride on various organisms, summarizes the molecular mechanism of fluorosis, and aims to improve our understanding of fluoride toxicity.

## **2. Mechanisms of skeletal fluorosis**

Fluoride is a cumulative toxin, which accumulates in mineralized tissues, notably in the lattice of bone and tooth crystals. The bones and teeth are recognized as the target organs of fluoride, and bone tends to accumulate this element with age. The main features of the disease are dental fluorosis and skeletal fluorosis. Dental fluorosis is the first visible toxic effect of F exposure, which manifests as pitting of tooth enamel and yellow cracked teeth in adults and in children [37]. Skeletal fluorosis is a metabolic bone disease with osteosclerosis as the major clinical sign, mostly involving bone joints [40]. It results in ligament calcifications, accompanied by osteopenia, osteoporosis, and osteomalacia to varying degrees [40, 41]. Fluorine is a trace element that is incorporated into bone mineral during bone formation [42]. Fluoride substitutes for the hydroxyl group in hydroxyapatite, forming fluorapatite. Bone metabolism includes the process of osteoblasts forming bone and the osteoclasts degrading bone. Fluoride has an effect on bone mineral, bone cells, and bone architecture [42]. Fluoride at physiological levels promotes osteoblast proliferation, increases bone mass, as well as increases osteoblast activity via the up-regulation of markers such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP), and bone gla protein (BGP) [43]. The levels of ALP and BGP were higher in patients with skeletal fluorosis than the control group [44]. However, fluoride may stimulate osteoblastic activity and delay mineralization of new bone [42]. On the other hand, osteoclasts are derived from hematopoietic progenitors in bone marrow and are only responsible for bone resorption. The mechanism associated with the osteoclasts is complicated; some studies showed that high fluoride concentrations may promote the formation of osteoclasts [45], or reduce the number of osteoclasts and decrease their bone resorption ability [46, 47]. Others suggested that fluoride had little effect on the number of osteoclasts and no effect on the osteoclast formation

**43**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

[48]. Indeed, excessive fluoride intake can destroy the processes of bone formation and resorption, which may lead to bone turnover disorders and result in skeletal fluorosis. Bone turnover is a dynamic balance regulated by osteoblasts and osteoclasts. Excessive fluoride disrupts this balance, influencing the differentiation of osteoblasts and osteoclasts and resulting in the development of bone lesions [49]. This may be related to certain signaling pathways and mechanisms. Fluoride influences bone turnover by regulating certain factors, such as runt-related transcription factor 2 (Runx2) and receptor activator for nuclear factor-κB ligand (RANKL), which act as markers of osteoblasts and osteoclasts [48, 50]. Through the mitogenactivated protein kinases (MAPK) pathway, fluoride mediates gene expression and cell viability. In ameloblasts, fluoride activates the Rho/ROCK pathway. Fluoride can also induce endoplasmic reticulum (ER) stress, leading to protein misfolding [51]. In addition, TGFβ-SMAD signaling regulates expression of essential genes (MMP13, Collagen Type I, Collagen Type VII, Aggrecan, and Biglycans) involved in the formation of extracellular matrix (ECM). Fluoride exposure affects the expression of these genes through TGFβ-SMAD signaling [52]. Additionally, excessive fluoride exposure leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation, suggesting that c-Fos might negatively regulate osteoprotegerin (OPG) expression

Furthermore, collagen and noncollagenous proteins are of significant importance for maintaining the biomechanical integrity of the bone and many bone matrix proteins play important roles in mineralization [42]. Excessive intake of fluoride affects the bone matrix proteins, that is, collagen and noncollagenous proteins, which may be another possible mechanism of skeletal fluorosis [42, 54]. For example, it has been shown that fluoride could inhibit the synthesis of type I collagen and decrease the degree of collagen cross-linking [54–59] or affect other collagen proteins [60–62], and affect the synthesis of proteoglycan [63], and

expression of matrix metalloproteinases (MMPs) [54, 64, 65]. Taken together, these studies suggested that exposure to fluoride alters growth, ECM formation, bone mineralization, and skeletal development and induced bone formation and bone

Excessive fluoride may cross the blood-brain barrier and accumulate in the brain, causing dysfunction of the central nervous system (CNS). In recent years, many studies have focused on fluorine neurotoxicity. The central nervous system during development is highly sensitive to the influence of fluorine due to its weakened protective mechanisms [66]. Studies showed that children in high fluoride areas had significantly lower IQ (intelligence quotient) scores than those who lived in low fluoride areas [67, 68]. The results of meta-analyses supported the possibility of adverse effects of fluoride exposures on children's neurodevelopment [67, 69]. In the animal experiments, as exposed to high levels of fluorine, the content of fluorides in the rats' brains was even 220 and 300 times higher than in the control group [70], and fluoride exposure affects the behavior, memory, cognitive and learning ability [71–73]. Dendritic thickening and disappearance, mitochondrial swelling, neuronal endoplasmic reticulum dilation, and impaired hippocampus synaptic interface structure can be observed in the brain of fluoride exposed rats [73]. The numbers of Nissl bodies in neurons in the hippocampus and cortex of brains from both adult rats and their pups with fluorosis were reduced, suggesting an injury of neurons [10]. These data indicate that excessive exposure to fluoride

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

induced by fluoride in osteoblastic cells [53].

resorption, thus leading to the development of fluorosis.

**3. Nervous system toxicity**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

*Environmental Chemistry and Recent Pollution Control Approaches*

[20–23], and reproductive system [24–26].

understanding of fluoride toxicity.

**2. Mechanisms of skeletal fluorosis**

the nervous system [6–12], kidneys [13–16], liver [14, 16–19], cardiovascular system

Chronic fluorosis is an endemic disease; it is endemic in at least 25 countries across the globe, China and India being the worst affected among them [27]. Most cases of fluorosis were caused by drinking fluorous water. In China, fluorosis is caused by drinking water as well as inhaling combustion fumes of coal being used as an indoor fuel source [28–31]. Guizhou is one of the most severely afflicted areas of endemic fluorosis in China and this occurrence is due to indoor coal burning [30]. Another type of fluorosis is brick tea-type fluorosis, due to fluoride accumulation in brick tea. It is more prevalent in Tibet than in other regions of China [32]. It is also worth noting that chronic exposure to volcanic environments may lead to the exposure of excessive amounts of fluoride [33]. It is estimated that more than 10% of the worldwide population live within the potential exposure range of some active or historically active volcano, either erupting or in a post-eruption phase [34].

In recent years, numerous studies focused on the molecular mechanisms associated with fluoride toxicity [35–39]. Although the underlying mechanisms of chronic fluorosis is still not well understood, the results of the previous studies indicated that fluoride can induce oxidative stress; regulate intracellular redox homeostasis; and lead to mitochondrial damage, endoplasmic reticulum stress, and alteration of gene expression [35–39]. Other mechanisms include enzyme inhibition, induction of apoptosis, cell cycle arrest, etc. [35–39]. This chapter reviews the present research on the potential adverse effects of overdose fluoride on various organisms, summarizes the molecular mechanism of fluorosis, and aims to improve our

Fluoride is a cumulative toxin, which accumulates in mineralized tissues, notably in the lattice of bone and tooth crystals. The bones and teeth are recognized as the target organs of fluoride, and bone tends to accumulate this element with age. The main features of the disease are dental fluorosis and skeletal fluorosis. Dental fluorosis is the first visible toxic effect of F exposure, which manifests as pitting of tooth enamel and yellow cracked teeth in adults and in children [37]. Skeletal fluorosis is a metabolic bone disease with osteosclerosis as the major clinical sign, mostly involving bone joints [40]. It results in ligament calcifications, accompanied by osteopenia, osteoporosis, and osteomalacia to varying degrees [40, 41]. Fluorine is a trace element that is incorporated into bone mineral during bone formation [42]. Fluoride substitutes for the hydroxyl group in hydroxyapatite, forming fluorapatite. Bone metabolism includes the process of osteoblasts forming bone and the osteoclasts degrading bone. Fluoride has an effect on bone mineral, bone cells, and bone architecture [42]. Fluoride at physiological levels promotes osteoblast proliferation, increases bone mass, as well as increases osteoblast activity via the up-regulation of markers such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP), and bone gla protein (BGP) [43]. The levels of ALP and BGP were higher in patients with skeletal fluorosis than the control group [44]. However, fluoride may stimulate osteoblastic activity and delay mineralization of new bone [42]. On the other hand, osteoclasts are derived from hematopoietic progenitors in bone marrow and are only responsible for bone resorption. The mechanism associated with the osteoclasts is complicated; some studies showed that high fluoride concentrations may promote the formation of osteoclasts [45], or reduce the number of osteoclasts and decrease their bone resorption ability [46, 47]. Others suggested that fluoride had little effect on the number of osteoclasts and no effect on the osteoclast formation

**42**

[48]. Indeed, excessive fluoride intake can destroy the processes of bone formation and resorption, which may lead to bone turnover disorders and result in skeletal fluorosis. Bone turnover is a dynamic balance regulated by osteoblasts and osteoclasts. Excessive fluoride disrupts this balance, influencing the differentiation of osteoblasts and osteoclasts and resulting in the development of bone lesions [49]. This may be related to certain signaling pathways and mechanisms. Fluoride influences bone turnover by regulating certain factors, such as runt-related transcription factor 2 (Runx2) and receptor activator for nuclear factor-κB ligand (RANKL), which act as markers of osteoblasts and osteoclasts [48, 50]. Through the mitogenactivated protein kinases (MAPK) pathway, fluoride mediates gene expression and cell viability. In ameloblasts, fluoride activates the Rho/ROCK pathway. Fluoride can also induce endoplasmic reticulum (ER) stress, leading to protein misfolding [51]. In addition, TGFβ-SMAD signaling regulates expression of essential genes (MMP13, Collagen Type I, Collagen Type VII, Aggrecan, and Biglycans) involved in the formation of extracellular matrix (ECM). Fluoride exposure affects the expression of these genes through TGFβ-SMAD signaling [52]. Additionally, excessive fluoride exposure leads to disturbances of bone homeostasis. c-Fos is known to be essential in bone development by affecting osteoblast and osteoclast differentiation, suggesting that c-Fos might negatively regulate osteoprotegerin (OPG) expression induced by fluoride in osteoblastic cells [53].

Furthermore, collagen and noncollagenous proteins are of significant importance for maintaining the biomechanical integrity of the bone and many bone matrix proteins play important roles in mineralization [42]. Excessive intake of fluoride affects the bone matrix proteins, that is, collagen and noncollagenous proteins, which may be another possible mechanism of skeletal fluorosis [42, 54]. For example, it has been shown that fluoride could inhibit the synthesis of type I collagen and decrease the degree of collagen cross-linking [54–59] or affect other collagen proteins [60–62], and affect the synthesis of proteoglycan [63], and expression of matrix metalloproteinases (MMPs) [54, 64, 65]. Taken together, these studies suggested that exposure to fluoride alters growth, ECM formation, bone mineralization, and skeletal development and induced bone formation and bone resorption, thus leading to the development of fluorosis.

## **3. Nervous system toxicity**

Excessive fluoride may cross the blood-brain barrier and accumulate in the brain, causing dysfunction of the central nervous system (CNS). In recent years, many studies have focused on fluorine neurotoxicity. The central nervous system during development is highly sensitive to the influence of fluorine due to its weakened protective mechanisms [66]. Studies showed that children in high fluoride areas had significantly lower IQ (intelligence quotient) scores than those who lived in low fluoride areas [67, 68]. The results of meta-analyses supported the possibility of adverse effects of fluoride exposures on children's neurodevelopment [67, 69]. In the animal experiments, as exposed to high levels of fluorine, the content of fluorides in the rats' brains was even 220 and 300 times higher than in the control group [70], and fluoride exposure affects the behavior, memory, cognitive and learning ability [71–73]. Dendritic thickening and disappearance, mitochondrial swelling, neuronal endoplasmic reticulum dilation, and impaired hippocampus synaptic interface structure can be observed in the brain of fluoride exposed rats [73]. The numbers of Nissl bodies in neurons in the hippocampus and cortex of brains from both adult rats and their pups with fluorosis were reduced, suggesting an injury of neurons [10]. These data indicate that excessive exposure to fluoride

results in structural and functional damages to the central nervous system, and may significantly hinder the neurodevelopment.

Fluorine neurotoxicity may be associated with oxidative stress, neuroinflammatory and neurotransmitter alterations. Fluorine induces increase in ROS (reactive oxygen species) and lipid peroxidation and decrease in anti-oxidative enzyme activity in neurons and glia, resulting in oxidative stress, which in turn causes cell damage and metabolism disorders [12, 74]. Fluorine causes glial cell activation which is involved in inflammation through producing proinflammatory cytokines. Chronic inflammation in the brain appears to cause neuronal damage [66, 75]. Moreover, fluorine influences the synthesis of neurotransmitters, the activity of enzymes, the expression of receptors, and the plasticity of neurons [76–78]. Therefore, excessive exposure to fluoride results in structural and functional damages to the central nervous system.

Of note, because fluoride can not only cross the blood-brain barrier, but also penetrate through the placenta, fluorine exposure in the prenatal and neonatal periods is dangerous [66, 79]. A recent study showed that during pregnancy and lactation, even at very low concentrations, F exposure may alter parameters of the central nervous system functionality, producing a delay in eye-opening development in the offspring as well as hypoactivity in adult offspring [80]. Further studies will be crucial to elucidate the molecular mechanisms through which F exposure during gestation and lactation trigger neurobehavioral changes [80].

## **4. Reproductive system toxicity**

Research on the effects of fluoride on the reproductive system has been carried out for many years. As early as 1925, Schulz and Lamb reported the reproductive toxicity of fluoride [81]. Fluoride shows adverse effects on the male reproductive system, including spermatogenesis defect, sperm count loss, sperm differentiation, and maturation impairment [82], and increase in chromosomal aberrations in primary testicular cells and the rate of sperm deformity [83]. Interestingly, recent studies showed that exposure to fluoride can alter the BTB (blood-testis-barrier) [84, 85]; fluoride induced structural and functional alterations in the BTB by increasing the expression levels of Arp3 protein with a concomitant increase in the expression levels of IL-1ɑ (interleukin-1ɑ) that led to the reorganization of the highly branching F-actin and the decreased expression of F-actin [25]. A significant increase in the fluoride concentration in the testes of mice that were exposed to sodium fluoride (NaF) has been observed [85]. In addition, ovaries of albino rats treated with high doses of NaF exhibited abnormal ovarian follicles, dilated blood vessels, stromal congestion, and necrotic granulose cells [86].

Cell apoptosis is one early sign of genotoxic damage in mature testis, and plays critical roles in spermatozoa output. Fluoride may induce oxidative stress through the activation of MAPK cascade and Jun N-terminal kinase (JNK, c-Jun) and extracellular signal-regulated protein kinase (ERK) signaling pathway lead to cell apoptosis that includes both intrinsic and extrinsic apoptotic pathways [82]. Fluoride could also cause leakage of potassium ions, thereby reducing sodium and potassium levels in spermatozoa [87]. In addition, higher levels of inflammatory factor such as IL-1ɑ were detected in the testes of NaF-treated rats [25], suggesting that inflammation was involved in the in the toxicity of fluoride to the reproductive system [25, 88]. More recently, a proteomics study analyzed the proteome characteristics of sperm from fluoride-exposed mice, and identified 15 differentially expressed proteins between fluoride-exposed and control groups. Most of them are associated with sperm functions such as sperm motility, maturation, capacitation

**45**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

and acrosome reaction, lipid peroxidation, detoxification, inflammation, and stability of membrane structure [89]. Another study reported altered MicroRNA (miRNA) expression profiling in sperm of mice induced by fluoride. Sixteen altered miRNAs were identified and they mainly were involved in protease inhibitor activity, apoptosis, ubiquitin-mediated proteolysis, and signaling pathways of calcium, JAK-STAT, MAPK, p53, and Wnt [90]. These findings provide new insights into the mechanism underlying fluoride reproductive toxicity. However, the toxicity mecha-

nism of fluoride on the reproductive system still needs further exploration.

As mentioned above, excess fluoride uptake affects other organs including liver and kidneys, and cardiovascular system. Liver is the most important detoxification organ in the body. The effect of fluoride on the liver has been widely studied and it has been demonstrated that excessive intake of fluoride causes serious liver damage

The kidneys are the main route of F removal from the body, and approximately 60% of the total daily F absorbed is filtered and excreted in urine [91]. The link between fluoride and kidney disease has been known and confirmed for many years [13–16], the toxicity or damage of fluoride to the kidney has been observed in population and experimental animals, including the kidneys of the fetus and suckling mammal [92]. Of note, people on kidney dialysis, patients with reduced glomerular filtration rates, and diabetic mammals are particularly susceptible to

A rising number of research studies have been carried out on the toxic effect of F in cardiovascular system [20–23, 93]. Fluoride can accumulate in the cardiovascular system, resulting in arterial calcifications, elastic properties of ascending aorta

Clearly, toxic effects in humans due to chronic fluoride ingestion mainly depend on the total dosage and duration of exposure. However, dose and time alone are not the only factor affecting fluorosis. Some studies have shown the existence of nonresponder populations to fluorine [94], while others have shown that some people seem to be very sensitive to fluorine [95, 96]. Animal experiments have observed that three inbred strains of mice (A/J, SWR/J, 129P3/J) displayed variations in the onset and severity of dental/enamel fluorosis with equivalent fluoride exposure [97]. The bone mechanical properties were reduced in the "susceptible strain" (A/J), moderately altered in the "intermediate strain" (SWR/J), and unaffected in the "resistant strain" (129P3/J), suggesting a genetic contribution to the variation in bone response to fluoride content [97]. Fluoride effects on bone formation and mineralization are influenced by genetics [42]. Another study showed that exposure to the same dosage and time, as compared with Wistar rats, the urine fluoride of SD rats was higher while bone and teeth fluoride levels were lower. Meanwhile, dental

Interestingly, the association between genetic polymorphisms in candidate genes and the susceptibility in the development of fluorosis has been well reviewed [27]. Candidate genes associated with human dental fluorosis and skeletal fluorosis are

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

**5. Other systems**

fluoride exposure [15].

**6. Influence factor**

**6.1 Genetic susceptibility to fluorosis**

fluorosis susceptibility of SD rats is higher [98].

disruption, and ventricular diastolic dysfunction [93].

[14, 16–19].

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

and acrosome reaction, lipid peroxidation, detoxification, inflammation, and stability of membrane structure [89]. Another study reported altered MicroRNA (miRNA) expression profiling in sperm of mice induced by fluoride. Sixteen altered miRNAs were identified and they mainly were involved in protease inhibitor activity, apoptosis, ubiquitin-mediated proteolysis, and signaling pathways of calcium, JAK-STAT, MAPK, p53, and Wnt [90]. These findings provide new insights into the mechanism underlying fluoride reproductive toxicity. However, the toxicity mechanism of fluoride on the reproductive system still needs further exploration.

## **5. Other systems**

*Environmental Chemistry and Recent Pollution Control Approaches*

significantly hinder the neurodevelopment.

**4. Reproductive system toxicity**

nervous system.

results in structural and functional damages to the central nervous system, and may

Fluorine neurotoxicity may be associated with oxidative stress, neuroinflammatory and neurotransmitter alterations. Fluorine induces increase in ROS (reactive oxygen species) and lipid peroxidation and decrease in anti-oxidative enzyme activity in neurons and glia, resulting in oxidative stress, which in turn causes cell damage and metabolism disorders [12, 74]. Fluorine causes glial cell activation which is involved in inflammation through producing proinflammatory cytokines. Chronic inflammation in the brain appears to cause neuronal damage [66, 75]. Moreover, fluorine influences the synthesis of neurotransmitters, the activity of enzymes, the expression of receptors, and the plasticity of neurons [76–78]. Therefore, excessive exposure to fluoride results in structural and functional damages to the central

Of note, because fluoride can not only cross the blood-brain barrier, but also penetrate through the placenta, fluorine exposure in the prenatal and neonatal periods is dangerous [66, 79]. A recent study showed that during pregnancy and lactation, even at very low concentrations, F exposure may alter parameters of the central nervous system functionality, producing a delay in eye-opening development in the offspring as well as hypoactivity in adult offspring [80]. Further studies will be crucial to elucidate the molecular mechanisms through which F exposure

Research on the effects of fluoride on the reproductive system has been carried out for many years. As early as 1925, Schulz and Lamb reported the reproductive toxicity of fluoride [81]. Fluoride shows adverse effects on the male reproductive system, including spermatogenesis defect, sperm count loss, sperm differentiation, and maturation impairment [82], and increase in chromosomal aberrations in primary testicular cells and the rate of sperm deformity [83]. Interestingly, recent studies showed that exposure to fluoride can alter the BTB (blood-testis-barrier) [84, 85]; fluoride induced structural and functional alterations in the BTB by increasing the expression levels of Arp3 protein with a concomitant increase in the expression levels of IL-1ɑ (interleukin-1ɑ) that led to the reorganization of the highly branching F-actin and the decreased expression of F-actin [25]. A significant increase in the fluoride concentration in the testes of mice that were exposed to sodium fluoride (NaF) has been observed [85]. In addition, ovaries of albino rats treated with high doses of NaF exhibited abnormal ovarian follicles, dilated blood

during gestation and lactation trigger neurobehavioral changes [80].

vessels, stromal congestion, and necrotic granulose cells [86].

Cell apoptosis is one early sign of genotoxic damage in mature testis, and plays critical roles in spermatozoa output. Fluoride may induce oxidative stress through the activation of MAPK cascade and Jun N-terminal kinase (JNK, c-Jun) and extracellular signal-regulated protein kinase (ERK) signaling pathway lead to cell apoptosis that includes both intrinsic and extrinsic apoptotic pathways [82]. Fluoride could also cause leakage of potassium ions, thereby reducing sodium and potassium levels in spermatozoa [87]. In addition, higher levels of inflammatory factor such as IL-1ɑ were detected in the testes of NaF-treated rats [25], suggesting that inflammation was involved in the in the toxicity of fluoride to the reproductive system [25, 88]. More recently, a proteomics study analyzed the proteome characteristics of sperm from fluoride-exposed mice, and identified 15 differentially expressed proteins between fluoride-exposed and control groups. Most of them are associated with sperm functions such as sperm motility, maturation, capacitation

**44**

As mentioned above, excess fluoride uptake affects other organs including liver and kidneys, and cardiovascular system. Liver is the most important detoxification organ in the body. The effect of fluoride on the liver has been widely studied and it has been demonstrated that excessive intake of fluoride causes serious liver damage [14, 16–19].

The kidneys are the main route of F removal from the body, and approximately 60% of the total daily F absorbed is filtered and excreted in urine [91]. The link between fluoride and kidney disease has been known and confirmed for many years [13–16], the toxicity or damage of fluoride to the kidney has been observed in population and experimental animals, including the kidneys of the fetus and suckling mammal [92]. Of note, people on kidney dialysis, patients with reduced glomerular filtration rates, and diabetic mammals are particularly susceptible to fluoride exposure [15].

A rising number of research studies have been carried out on the toxic effect of F in cardiovascular system [20–23, 93]. Fluoride can accumulate in the cardiovascular system, resulting in arterial calcifications, elastic properties of ascending aorta disruption, and ventricular diastolic dysfunction [93].

## **6. Influence factor**

#### **6.1 Genetic susceptibility to fluorosis**

Clearly, toxic effects in humans due to chronic fluoride ingestion mainly depend on the total dosage and duration of exposure. However, dose and time alone are not the only factor affecting fluorosis. Some studies have shown the existence of nonresponder populations to fluorine [94], while others have shown that some people seem to be very sensitive to fluorine [95, 96]. Animal experiments have observed that three inbred strains of mice (A/J, SWR/J, 129P3/J) displayed variations in the onset and severity of dental/enamel fluorosis with equivalent fluoride exposure [97]. The bone mechanical properties were reduced in the "susceptible strain" (A/J), moderately altered in the "intermediate strain" (SWR/J), and unaffected in the "resistant strain" (129P3/J), suggesting a genetic contribution to the variation in bone response to fluoride content [97]. Fluoride effects on bone formation and mineralization are influenced by genetics [42]. Another study showed that exposure to the same dosage and time, as compared with Wistar rats, the urine fluoride of SD rats was higher while bone and teeth fluoride levels were lower. Meanwhile, dental fluorosis susceptibility of SD rats is higher [98].

Interestingly, the association between genetic polymorphisms in candidate genes and the susceptibility in the development of fluorosis has been well reviewed [27]. Candidate genes associated with human dental fluorosis and skeletal fluorosis are

listed in **Table 1**. Candidate genes in dental fluorosis include BGLAP (Osteocalcin), COL1A2 (Collagen type 1 alpha 2), CTR/CALCR (Calcitonin Receptor), ESR (Estrogen Receptor), and VDR (Vitamin D Receptor). Candidate genes in skeletal fluorosis include MMP-2 (Matrix metallopeptidase2), MPO (Myeloperoxidase), GSTP1 (Glutathione S-transferase pi 1), PRL (Prolactin), and VDR (Vitamin D Receptor). These genes are involved in different functions—BGLAP, ESR, and COL1A2 are related to bone formation and development; VDR and CTR are related to bone formation and metabolism; and PTH and PRL are related to hormones' secretion. GSTP1, MMP, COMT, and MPO are related to detoxifying enzymes, extracellular matrix, cognitive and immune responses, respectively [27]. These results suggest that an individual's genetic background plays a major role in influencing the risk to fluorosis.

## **6.2 Co-exposure with other element**

Co-exposure to other elements is another major factor affecting fluorosis. All of these could complicate the overall toxic response. For example, in geothermal areas, volcanic activity includes CO2-rich hot springs, steaming vents, hot ground and boiling mud pools that normally contain unusually high concentrations of Li, Rb, Cs, Si, B, As, and F [33]. Thus, chronic exposure to volcanic environments may lead to the exposure of excessive amounts of fluoride and other elements. Interestingly, a recent study indicated that an increase or decrease in various elements (including F, Al, Se, Zn, Cu, Fe, Mo, Mn, B, V, Ca, Mg, and P) in the environment is related to the abnormal levels of the corresponding elements in a fluoride-exposed population [28]. High levels of F, Al, As, Pb, and Cr were a risk factor for dental fluorosis, but not Se, Zn, Cu, B, Ca, and P, which was a protective factor for dental fluorosis [28].


**47**

reduction in the toxicity of F [135].

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

combined, can decrease learning and memory ability in rats [118].

Combined exposure to fluoride and aluminum is another noteworthy problem related to fluorosis. It mainly occurs through indoor combustion of coal, especially kaolin mixed with coal [119], and high Al content in tea such as brick tea [120]. The interaction mechanism of F and Al is also complicated, may be independent, synergistic, or antagonistic. Aluminum exposure impairs bone formation; inhibition of bone formation by aluminum through different signal transduction pathways has been reported [121]. Exposure to Al is associated with low bone mineral density (BMD) and an increased risk of osteoporosis [121–124]. Fluoride enhances the uptake of aluminum; the simultaneous administration of fluorine and aluminum increased plasma [125], and bone [126] concentrations of aluminum in rats, whereas aluminum suppresses the uptake of fluoride [127]. Decreased bone mineral density was observed in fluorine and aluminum-treated rats [126]. Patients with co-exposure to fluoride and aluminum display with osteomalacia or osteoporosis may be due to fluoride promoting aluminum accumulation in bone, while aluminum inhibits bone formation. However, the vitro study showed that there was a synergistic effect of fluoride and aluminum on the expression of Runx2 and Osterix mRNA in osteoblastic MC3T3-E1 cells, thereby enhance MC3T3-E1 cells proliferation and differentiation [128], and contribute to osteosclerosis. This may explain the different clinical features of skeletal fluorosis, that is, osteosclerosis accompanied with osteomalacia, and osteopenia.

It is worth mentioning that Al is a well-known neurotoxic agent, and it has long since been implicated in the etiopathology of AD [129]. Fluorine and aluminum are able to cross the blood-brain barrier and the placental barrier [66, 130]. They can accumulate in the brain, and fluoride did not affect the accumulation of aluminum in the CNS [131]. It has been reported that increases of microglia activation and inflammatory response were seen in aluminum, fluoride, and a combination of aluminum-fluoride-treated rat brain [132]. Excessive fluoride and aluminum intake induces the progression of cell death which inhibits acetylcholinesterase (AChE) activities and triggers the release of lysosomal and cell cycle proteins in the brain of rats [133]. More recently, Xie et al. found that continuous exposure to fluorine and/ or aluminum of mother rats impaired the neurobehavioral reflexes, spatial learning, and memory of offspring rats [134, 135]. The effects of F were obvious, but the effects of Al were slight. There were antagonistic effects between F and Al, with Al

In addition, Chinoy et al. reported that simultaneous exposure of the animals to NaF and AlCl3 was associated with an increased toxic effect on gonadal

At present, some studies have been reported on the co-exposure of fluorine and arsenic (As) or fluorine and aluminum (Al). Both arsenic and fluoride are ubiquitous in the environment. The co-exposure of fluorine and arsenic is mainly through drinking water [109–111] or burning coal [112, 113]. The latter is a unique type in China, which was attributed to exposure to high levels of As and F in food and breathing As-laden air, caused by polluted food and air due to indoor combustion of coal [112, 113]. The interaction mechanism of these two elements is complicated, which may be independent, synergistic, or antagonistic [114]. A recent study indicated that arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway [115]. Arsenic affects the expression of c-Fos, thereby affecting the expression of transcription factor AP1, indirectly involved in fluoride-induced bone toxicity [115]. Another study showed that the joint effect of fluoride and arsenate on the gene expression of ODF (osteoclast differentiation factor) is antagonistic, while the combined effect on the gene expression of OPG is synergistic [116]. Ma et al. reported that As and F can induce the expression of adhesion molecules, chemokines and pro-inflammatory cytokines in rabbit aorta separately, and antagonistic effects were observed on inflammatory response [117]. Fluoride and arsenic, either alone or

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

## **Table 1.**

*Candidate genes in dental fluorosis and skeletal fluorosis.*

#### *Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

*Environmental Chemistry and Recent Pollution Control Approaches*

encing the risk to fluorosis.

**Dental fluorosis**

**Skeletal fluorosis**

*Candidate genes in dental fluorosis and skeletal fluorosis.*

**6.2 Co-exposure with other element**

listed in **Table 1**. Candidate genes in dental fluorosis include BGLAP (Osteocalcin), COL1A2 (Collagen type 1 alpha 2), CTR/CALCR (Calcitonin Receptor), ESR (Estrogen Receptor), and VDR (Vitamin D Receptor). Candidate genes in skeletal fluorosis include MMP-2 (Matrix metallopeptidase2), MPO (Myeloperoxidase), GSTP1 (Glutathione S-transferase pi 1), PRL (Prolactin), and VDR (Vitamin D Receptor). These genes are involved in different functions—BGLAP, ESR, and COL1A2 are related to bone formation and development; VDR and CTR are related to bone formation and metabolism; and PTH and PRL are related to hormones' secretion. GSTP1, MMP, COMT, and MPO are related to detoxifying enzymes, extracellular matrix, cognitive and immune responses, respectively [27]. These results suggest that an individual's genetic background plays a major role in influ-

Co-exposure to other elements is another major factor affecting fluorosis. All of these could complicate the overall toxic response. For example, in geothermal areas, volcanic activity includes CO2-rich hot springs, steaming vents, hot ground and boiling mud pools that normally contain unusually high concentrations of Li, Rb, Cs, Si, B, As, and F [33]. Thus, chronic exposure to volcanic environments may lead to the exposure of excessive amounts of fluoride and other elements. Interestingly, a recent study indicated that an increase or decrease in various elements (including F, Al, Se, Zn, Cu, Fe, Mo, Mn, B, V, Ca, Mg, and P) in the environment is related to the abnormal levels of the corresponding elements in a fluoride-exposed population [28]. High levels of F, Al, As, Pb, and Cr were a risk factor for dental fluorosis, but not Se, Zn, Cu, B, Ca, and P, which was a protective factor for dental fluorosis [28].

**mutational bases)**

rs412777 (A/C)

rs2234693 (A > C, XbaI)

**References**

[99] [100]

[101]

**Candidate genes Polymorphism site (restriction sites or** 

*AMBN* (Ameloblastin) rs4694075 (C/T) [102] *TFIP11* (Tuftelin interacting protein 11) rs5997096 (C/T) [102] *TUFT1* (Tuftelin) rs4970957 (A/G) [102] *DLX1* (Homeobox protein DLX-1) rs788173 (A/G) [103] *DLX2* (Homeobox protein DLX-2) rs743605 (A/G) [103] *TIMP1* (Metalloproteinase inhibitor 1) rs4898 (C/T) [103]

*FRZB1* (frizzled-related protein 1) rs2242070 (A/G) [104] *VDR* (Vitamin D receptor) rs2228570 (Fok I) [105] *GSTP1* (Glutathione S-transferase pi 1) rs1695 (A/G) [106] *PRL* (Prolactin) rs1341239 [107] *MMP-2* (Matrix metallopeptidase 2) rs2287074 (G/A) rs243865 [108]

*COL1A2* (Collagen type1 alpha 2) rs414408 (PvuII)

*ESR* (Estrogen receptor) rs1256049 (G > A, RsaI)

**46**

**Table 1.**

At present, some studies have been reported on the co-exposure of fluorine and arsenic (As) or fluorine and aluminum (Al). Both arsenic and fluoride are ubiquitous in the environment. The co-exposure of fluorine and arsenic is mainly through drinking water [109–111] or burning coal [112, 113]. The latter is a unique type in China, which was attributed to exposure to high levels of As and F in food and breathing As-laden air, caused by polluted food and air due to indoor combustion of coal [112, 113]. The interaction mechanism of these two elements is complicated, which may be independent, synergistic, or antagonistic [114]. A recent study indicated that arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway [115]. Arsenic affects the expression of c-Fos, thereby affecting the expression of transcription factor AP1, indirectly involved in fluoride-induced bone toxicity [115]. Another study showed that the joint effect of fluoride and arsenate on the gene expression of ODF (osteoclast differentiation factor) is antagonistic, while the combined effect on the gene expression of OPG is synergistic [116]. Ma et al. reported that As and F can induce the expression of adhesion molecules, chemokines and pro-inflammatory cytokines in rabbit aorta separately, and antagonistic effects were observed on inflammatory response [117]. Fluoride and arsenic, either alone or combined, can decrease learning and memory ability in rats [118].

Combined exposure to fluoride and aluminum is another noteworthy problem related to fluorosis. It mainly occurs through indoor combustion of coal, especially kaolin mixed with coal [119], and high Al content in tea such as brick tea [120]. The interaction mechanism of F and Al is also complicated, may be independent, synergistic, or antagonistic. Aluminum exposure impairs bone formation; inhibition of bone formation by aluminum through different signal transduction pathways has been reported [121]. Exposure to Al is associated with low bone mineral density (BMD) and an increased risk of osteoporosis [121–124]. Fluoride enhances the uptake of aluminum; the simultaneous administration of fluorine and aluminum increased plasma [125], and bone [126] concentrations of aluminum in rats, whereas aluminum suppresses the uptake of fluoride [127]. Decreased bone mineral density was observed in fluorine and aluminum-treated rats [126]. Patients with co-exposure to fluoride and aluminum display with osteomalacia or osteoporosis may be due to fluoride promoting aluminum accumulation in bone, while aluminum inhibits bone formation. However, the vitro study showed that there was a synergistic effect of fluoride and aluminum on the expression of Runx2 and Osterix mRNA in osteoblastic MC3T3-E1 cells, thereby enhance MC3T3-E1 cells proliferation and differentiation [128], and contribute to osteosclerosis. This may explain the different clinical features of skeletal fluorosis, that is, osteosclerosis accompanied with osteomalacia, and osteopenia.

It is worth mentioning that Al is a well-known neurotoxic agent, and it has long since been implicated in the etiopathology of AD [129]. Fluorine and aluminum are able to cross the blood-brain barrier and the placental barrier [66, 130]. They can accumulate in the brain, and fluoride did not affect the accumulation of aluminum in the CNS [131]. It has been reported that increases of microglia activation and inflammatory response were seen in aluminum, fluoride, and a combination of aluminum-fluoride-treated rat brain [132]. Excessive fluoride and aluminum intake induces the progression of cell death which inhibits acetylcholinesterase (AChE) activities and triggers the release of lysosomal and cell cycle proteins in the brain of rats [133]. More recently, Xie et al. found that continuous exposure to fluorine and/ or aluminum of mother rats impaired the neurobehavioral reflexes, spatial learning, and memory of offspring rats [134, 135]. The effects of F were obvious, but the effects of Al were slight. There were antagonistic effects between F and Al, with Al reduction in the toxicity of F [135].

In addition, Chinoy et al. reported that simultaneous exposure of the animals to NaF and AlCl3 was associated with an increased toxic effect on gonadal steroidogenesis, uterine metabolism of carbohydrates, and hypercholesterolemia, as compared with each compound administered separately [136]. Recently, Dong et al. reported that F induced the reduction in testosterone and sperm amount; however, Al had antagonism effects on F and weakened the toxicity of F to some extent [137]. Moreover, fluoride interacts with aluminum to form a fluoro-aluminum complex AlFx (e.g., AlF3 and AlF4−), which can interact with the G protein (guanine nucleotide-binding proteins) and activated effect or enzymes, providing false information, and amplify the processes of signal transmission [138]. Together, further investigation is needed on the underlying mechanisms by which fluorine and arsenic or fluorine and aluminum induce toxicity.

## **7. Proteomics and metabolomics applications**

Proteomics and metabolomics are useful and powerful tools for clarifying toxicological mechanisms associated with diseases. Proteomics offers the possibility to map the entire proteome of an organism or cells and detect toxic effects at significantly lower doses, as well as faster screening for potential adaptive mechanisms by the use of high-throughput technologies [139]. In particular, during the last 10 years, apart from the gel-based techniques (e.g., 2D-PAGE and 2D-DIGE), gel-free techniques (e.g., stable-isotope labeling or using label-free methods) have been dominating the field of MS-based quantitation in proteomics [140]. This enhances the ability of proteomics to explore disease mechanisms. As mentioned above, proteomics analysis has been used to investigate the toxicity mechanism of fluorine on sperm [89]. Proteomics analysis associated with F-toxicity has also been studied in other tissues including gastrocnemius muscle, kidney, liver, midgut, bone, cells, serum, and urine [18, 54, 62, 68, 141–147]. All of these studies are listed in **Table 2**. As shown in **Table 2**, proteomic techniques 2D-PAGE, LC-MS/MS (liquid chromatography-tandem mass spectrometry), and iTRAQ (isobaric tags for relative and absolute quantification) labeling coupled with LC-MS/MS analysis were employed in these studies. The proteins associated with fluoride exposure were found involved in oxidative stress, ER stress, cell proliferation and apoptosis, mitochondrial-metabolism, tricarboxylic acid (TCA) cycle, unfolded protein response, inflammatory response, etc. These pathways or biological processes have previously been linked to the pathophysiology of fluorosis. The results support the current views on the molecular mechanism of F-toxicity.

Interestingly, Khan et al. evaluated the effects of F on the liver proteome of mice susceptible (A/J) or resistant (129P3/J) to the effects of F. As compared with 129P3/J mice, most of the proteins with fold change upon treatment with lower F concentrate (15 ppm) were increased in the A/J mice, suggesting an attempt of the former to fight the deleterious effects of F. However, upon treatment with 50 ppm F, most proteins with fold change were decreased in the A/J mice, especially proteins related to oxidative stress and protein folding, which might be related to the higher susceptibility of the A/J animals to the deleterious effects of F [18]. These results add light to the mechanisms underlying genetic susceptibility to fluorosis [18].

It is worth mentioning that in our previous comparative proteomic analysis of fluoride treated rat bone [54], 13, 35, and 34 differentially expressed proteins were identified in low-, medium-, and high-dose NaF exposure group. The medium- and high-dose groups shared a more similar protein expression pattern. Most of these proteins belong to collagen proteins, matrix metalloproteinases, proteoglycans (PGs), proteolytic protein, osteoclast-related protein, and myosin proteins, involved in collagen metabolism, bone mass change, mineralization process, dysfunction of the motor cell, and affected osteoblasts and/or osteoclasts, finally, contributing to the pathophysiology of skeletal and chronic fluorosis [54].

**49**

**Models/materials**

Calvarial osteoblasts

Cells were treated with NaF

2-DE MALDI-TOF MS

**Toxic effects**

**Method**

**Critical proteins**

ATP synthase, Dihydropyrimidinase-like 2, Heat shock 70-kDa protein(HSP70), Nucleoside diphosphate kinase, Glutamate oxaloacetate transaminase, Phosphatidylethanolamine binding prote in Proteasome 26S ATPase, Nucleoside diphosphate kinase, Protein disulfide isomerase, Ras-GTPase-activating protein, Thioredoxin, Tubulin, beta

Androgen regulated 20 kDa protein, Aflatoxin B1 aldehyde reductase, alpha-2-μ-globulin

Twenty five (25), 30 and 32 differentially expressed proteins were successfully identified between different doses of NaF treatment groups and their respective controls.

Urine from the fluoride-treated Wistar rat Kidney (A/J and 129P3/J mice)

Dental fluorosis

 Kidney impairment

2D-PAGE LC-MS/MS

Genetic susceptibility

Gastrocnemius

Alter glucose

2D-PAGE

78 kDa glucose-regulated protein, Alpha-enolase, Beta-enolase,

Gamma-enolase, Gelsolin, Glyceraldehyde-3-phosphate

dehydrogenase, Glycerol-3-phosphate dehydrogenase [NAD(+)],

Heat shock cognate 71 kDa protein, L-lactate dehydrogenase A chain,

L-lactate dehydrogenase B chain, L-lactate dehydrogenase C chain,

Malate dehydrogenase, Myosin-3, Myosin-6, Myosin-7, Myosin-8,

Myosin-binding protein C\_ slow-type, Myosin-binding protein H,

Pyruvate kinase isozymes M1/M2

LC-MS/MS

homeostasis and

lead to insulin

resistance

muscle

Streptozotocininduced diabetes exposed to fluorides

Femurs, tibiae, and

Bone architecture

LC-ESI-MS/MS

129P3/J vs A/J mice:

Bone morphogenetic protein 1, Bone sialoprotein 2, Collagen alpha-1(I) chain, Collagen alpha-2(I) chain, Exportin-2, NADPH oxidase 4,

Protocadherin beta 15, Secreted frizzled-related sequence protein 4

129P3/J vs F-treated 129P3/J mice:

Aflatoxin B1 aldehyde reductase member 2, Carbonyl reductase

[NADPH] 2, Catenin alpha-2, Chromodomain-helicase-DNA-binding

protein

Mineral apposition

lumbar vertebrae

(129P3/J and A/J

rate

Genetic

susceptibility

mice)

Dental fluorosis

2D-PAGE MALDI-TOF-

TOF MS/MS

**Pathways/biology processes**

Cell proliferation

 Nucleotide metabolism

Xu et al. [141]

Signal transduction

 Protein oxidative folding

Hydrophobic ligands

Cell motility Detoxification Hormone regulation

Metabolic and cellular processes Response to stimuli

Development Regulation of cellular

processes

Muscle contraction

Leite et al.

[144]

Carbohydrate catabolic

processes

Generation of precursor

Metabolites and energy

NAD metabolic processes

Gluconeogenesis

Osteogenesis

Kobayashi

et al. [62]

Osteoclastogenesis

Kobayashi et al [142]

**References**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

Carvalho

et al. [143]

*DOI: http://dx.doi.org/10.5772/intechopen.84548*


### *Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

*Environmental Chemistry and Recent Pollution Control Approaches*

and arsenic or fluorine and aluminum induce toxicity.

**7. Proteomics and metabolomics applications**

steroidogenesis, uterine metabolism of carbohydrates, and hypercholesterolemia, as compared with each compound administered separately [136]. Recently, Dong et al. reported that F induced the reduction in testosterone and sperm amount; however, Al had antagonism effects on F and weakened the toxicity of F to some extent [137]. Moreover, fluoride interacts with aluminum to form a fluoro-aluminum complex AlFx (e.g., AlF3 and AlF4−), which can interact with the G protein (guanine nucleotide-binding proteins) and activated effect or enzymes, providing false information, and amplify the processes of signal transmission [138]. Together, further investigation is needed on the underlying mechanisms by which fluorine

Proteomics and metabolomics are useful and powerful tools for clarifying toxicological mechanisms associated with diseases. Proteomics offers the possibility to map the entire proteome of an organism or cells and detect toxic effects at significantly lower doses, as well as faster screening for potential adaptive mechanisms by the use of high-throughput technologies [139]. In particular, during the last 10 years, apart from the gel-based techniques (e.g., 2D-PAGE and 2D-DIGE), gel-free techniques (e.g., stable-isotope labeling or using label-free methods) have been dominating the field of MS-based quantitation in proteomics [140]. This enhances the ability of proteomics to explore disease mechanisms. As mentioned above, proteomics analysis has been used to investigate the toxicity mechanism of fluorine on sperm [89]. Proteomics analysis associated with F-toxicity has also been studied in other tissues including gastrocnemius muscle, kidney, liver, midgut, bone, cells, serum, and urine [18, 54, 62, 68, 141–147]. All of these studies are listed in **Table 2**. As shown in **Table 2**, proteomic techniques 2D-PAGE, LC-MS/MS (liquid chromatography-tandem mass spectrometry), and iTRAQ (isobaric tags for relative and absolute quantification) labeling coupled with LC-MS/MS analysis were employed in these studies. The proteins associated with fluoride exposure were found involved in oxidative stress, ER stress, cell proliferation and apoptosis, mitochondrial-metabolism, tricarboxylic acid (TCA) cycle, unfolded protein response, inflammatory response, etc. These pathways or biological processes have previously been linked to the pathophysiology of fluorosis. The results support the current views on the molecular mechanism of F-toxicity.

Interestingly, Khan et al. evaluated the effects of F on the liver proteome of mice susceptible (A/J) or resistant (129P3/J) to the effects of F. As compared with 129P3/J mice, most of the proteins with fold change upon treatment with lower F concentrate (15 ppm) were increased in the A/J mice, suggesting an attempt of the former to fight the deleterious effects of F. However, upon treatment with 50 ppm F, most proteins with fold change were decreased in the A/J mice, especially proteins related to oxidative stress and protein folding, which might be related to the higher susceptibility of the A/J animals to the deleterious effects of F [18]. These results add light

It is worth mentioning that in our previous comparative proteomic analysis of fluoride treated rat bone [54], 13, 35, and 34 differentially expressed proteins were identified in low-, medium-, and high-dose NaF exposure group. The medium- and high-dose groups shared a more similar protein expression pattern. Most of these proteins belong to collagen proteins, matrix metalloproteinases, proteoglycans (PGs), proteolytic protein, osteoclast-related protein, and myosin proteins, involved in collagen metabolism, bone mass change, mineralization process, dysfunction of the motor cell, and affected osteoblasts and/or osteoclasts, finally, contributing to

to the mechanisms underlying genetic susceptibility to fluorosis [18].

the pathophysiology of skeletal and chronic fluorosis [54].

**48**


**51**

**Models/materials**

Sperm samples from Kunming mice

Bone samples (Sprague-Dawley rats ) Liver (A/J, 129P3/J

Disturbances in

Nano-LC-ESI-MS/

soft tissues

Genetic

MS

susceptibility

Plasma from

Children

2-DE,

Alpha-1-B glycoprotein, Apolipoprotein E precursor, Complement

C1s subcomponent precursor, Hemopexin, Immunoglobulin light

MALDI-TOF/

TOF-MS

chain variable region

intelligence

Gene

polymorphism

**Table 2.**

*Fluorosis-related proteomics studies reported in the literatures.*

Children

mice)

Chronic fluorosis

iTRAQ labeling, NanoLC-MS/

Thirteen (13), 35, and 34 differentially expressed proteins were identified in low-, medium-, and high-dose NaF-treated group, respectively.

These proteins belong to collagen proteins, matrix metalloproteinases (MPPs), proteoglycans( PGs),

proteolytic protein, osteoclast related protein, and myosin proteins

Eighty one (81) differentially expressed proteins were identified in

the liver of A/J and 129P3/J mice treated with 15 ppm F.

One hundred one (101) differentially expressed proteins were

identified in the liver of A/J and 129P3/J mice treated with 50 ppm F

MS

Sperm damage

2D-PAGE, MALDI- TOF-MS

Adenylate kinase isoenzyme 1 isoform 2, Aldose reductase-related protein 1 Annexin A13, Annexin A4, Dihydrolipoyllysine-

residue acetyltransferase component of pyruvate dehydrogenase complex, Gamma-actin, Inhibitor alpha1 Phosphoglycerate kinase 2, Proteasome (prosome, macropain) subunit, alpha type 3, Serotransferrin precursor, Triosephosphate isomerase

**Toxic effects**

**Method**

**Critical proteins**

**Pathways/biology processes**

Sperm motility

Sun et al. [89]

> Maturation Capacitation and acrosome reaction

 Lipid peroxidation

Detoxification Inflammation Stability of membrane structure

**References**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

Wei et al. [54]

Bone mass change

 Mineralization process

Dysfunction of the motor cell

Affected on osteoblasts and/or osteoclasts Carboxylic acid metabolic

Khan et al.

[18]

process

Cellular amino acid

metabolic

Process

Oxidative stress and protein

folding might be related

to the susceptibility to the

deleterious effects of F

Cell immunity

Zhang et al.

[68]

Metabolism

*DOI: http://dx.doi.org/10.5772/intechopen.84548*


### *Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

*Environmental Chemistry and Recent Pollution Control Approaches*

**50**

**Models/materials**

**Toxic effects**

**Method**

**Critical proteins**

4, Chromodomain-helicase-DNA-binding protein 7, NADPH oxidase 4,

Phosphatidylinositol 3,4,5-trisphosphate

5-phosphatase 2, Protocadherin beta 9

A/J vs F-treated A/J mice:

Eukaryotic translation initiation factor 2 alpha kinase 3, Exportin-2,

Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1

Kidney (Wistar rats)

Hippocampus from

Leaning ability

2D-PAGE,

MALDI-TOF-MS

Memory

rats

Alteration of renal

2D-PAGE

Control vs 10 ppm F:

Aldo–keto reductase, Adnylate kinase 3-like 1, Enoyl coenzyme A

Detoxification

Kobayashi

et al [145]

Metabolism

Housekeeping

hydratase, Pyruvate carboxylase,

Control vs 5 ppm F:

Aldolase B, Endoplasmic reticulum protein 29

5'-AMP-activated protein kinase, Aconitate hydratase, Actin,

Biosynthesis of amino acids

Pan et al.

[146]

Carbon metabolism

Insulin signaling pathway

Phagosome

Oxytocin signaling pathway

cytoplasmic2-like isoform 3, Actr2 protein, Beta-actin, Cytosolic

aspartate, dehydrogenase [NADP+], and fructose-bisphosphate

aldolase C, Dynamin, Fascin, Fructose-bisphosphate aldolase C-B,

Gln synthetase, Glycogen phosphorylase, glycoprotein 1 precursor,

Lysosome-associated membrane, MHC class I antigen, Mitogenactivated protein kinase 1, Mixture 1: alcohol, N-ethylmaleimide

sensitive, Otub1 protein, PDZ and LIM domain prptein 3,

Phosphatase 1E isoform 1, Pyruvate carboxylase, Serum albumin,

Tropomyosin 1, Ulip2 protein, Voltage-dependent anion-selective

channel protein 1, WD repeat-containing protein 1

A total of 37 differentially expressed proteins were identified in

Complement and

Wei et al.

[147]

coagulation cascade

Inflammatory response

Complement activation

Defense response

Wound response

different doses of the NaF treatment group

Serum from Wistar

Dental fluorosis

iTRAQ labeling,

NanoLC-MS/

MS

rats treated by NaF

MALDI-TOF

MS

metabolism

**Pathways/biology** 

**References**

**processes**

**Table 2.**

Metabolomics can capture low-molecular weight metabolites that are the closest to the phenotype, which is believed to be one of the most powerful techniques to study the metabolic alteration associated with the treatment of environmental toxicants. However, the study on metabolic profile response to fluoride exposure is limited. A recent study carried out a metabonomics study on NaF treated human oral squamous cell carcinoma cells. The results showed that inhibition of the enolase reaction in glycolysis pathway was observed in the early stages of fluoride treatment. In the later stages, gradual increases in the AMP/ATP ratio (a putative marker of apoptosis) and oxidized products (e.g., GSSH, and methionine sulfoxide), and marginal changes in polyamine levels (putative marker of necrosis), were observed [148]. It suggested that the inhibition of enolase reaction and TCA cycle progression at early stage is specific to NaF, whereas the increase of ATP utilization at later stage may be common to apoptotis-inducing agents, but not to necrosis-inducing agents [148].

## **8. Treatment and prevention of chronic fluorosis**

So far, there is no specific treatment for fluorosis. Efforts are being made to reduce the severity of the disease and improve quality of life of affected patients [149]. Medical treatment being used is mainly supplementation of vitamin (Vit) C, D, and E, calcium, antioxidants and treatment of malnutrition [150]. In recent years, some traditional Chinese medicines (TCM) have been developed to treat fluoride-induced bone lesions in China [49]. Treatment options for dental and skeletal fluorosis vary according to the severity of the disease [149]. Methods for treating dental fluorosis include micro/macro abrasion, bleaching, composite restorations, veneers, and full crowns [151]. Treatment of skeletal fluorosis may include surgical processes while treatment of deformity includes use of physiotherapy, corrective plasters, and orthoses (appropriate appliances) [149].

Clearly, chronic fluorosis is mainly caused by excess intake of fluoride through drinking water, food products, air, and industrial pollutants over a long period. Therefore, avoiding excessive intake of the fluoride is essential for the prevention of this disease. Notably, to keep fluoride intake within safe limits, one needs to consider the total daily intake, including fluoride intake from water, food, air, fluoride-rich dental products and drugs. The recommended level for daily fluoride intake is 0.05–0.07 mg F/Kg/day [152, 153]. In China, for people aged 8–16 (including 16-year-olds), the recommended values of the total daily fluoride intake per person is ≤2.4 mg; for those 16 years old, the total daily fluoride intake per person is ≤3.5 mg [154]. In water-borne fluorosis endemic areas, alternative water resources with low fluoride levels or defluorinated water can be used. Coal-burning endemic fluorosis areas need to change the way coal is burned and food is dried. Likewise, it is beneficial for a daily intake of foods, vegetables, and fruits rich in vitamin C, D, and E, calcium, and antioxidants for the prevention of chronic fluorosis in endemic regions [49, 82, 150]. Moreover, health education is a very important aspect for disease management. Knowledge regarding the harmful effects of fluoride and the causes of fluorosis can help people, especially the affected population, pay more attention to their living habits [49, 149]. Furthermore, the identification of candidate genes that affect risk factors is necessary to develop more effective measures to prevent and treat fluorosis [27].

## **9. Conclusions**

The contents of this chapter are reviewed in **Figure 1**. Excess intake of fluoride can cause chronic fluorosis, leading to dental fluorosis and skeletal fluorosis and damage

**53**

**Acknowledgements**

**Figure 1.**

**Conflict of interest**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

to nervous system, reproductive system, cardiovascular system, liver, and kidney. The possible mechanisms involved different key proteins and signal transduction pathways associated with the pathogenesis of fluorosis have been proposed. Some high-throughput methods such as proteomics, metabolomics, and transcriptomics have been used in the study of the mechanism underlying development of fluorosis. Genetic factors play a critical role in the pathogenesis of chronic fluorosis. Combined exposure to fluoride with other element such as arsenic or aluminum may result in more complicated adverse health effects than exposure to fluoride or these elements alone. Further research is needed to reveal the interaction between fluorides with these elements with regard to their toxic effects. Clearly, the mechanisms of chronic fluorosis still need further research. Prevention of chronic fluorosis is important and it can be prevented by keeping fluoride intake within safe limits. It is important to consider total exposure (i.e., exposure through air, food, and water) when evaluating adverse health effects of fluoride.

*An overview of the occurrence, influencing factors, pathogenesis, treatment, and prevention of fluorosis.*

The authors would like to acknowledge the National Natural Science Foundation of China (Grant Nos. 31870825, 81560515) and Shenzhen Bureau of Science, Technology and Information (Nos. JCYJ20170412110026229,

JCYJ20150529164656093) for funds to support this work.

The authors declare that they have no competing interests.

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

#### **Figure 1.**

*Environmental Chemistry and Recent Pollution Control Approaches*

**8. Treatment and prevention of chronic fluorosis**

plasters, and orthoses (appropriate appliances) [149].

Metabolomics can capture low-molecular weight metabolites that are the closest to the phenotype, which is believed to be one of the most powerful techniques to study the metabolic alteration associated with the treatment of environmental toxicants. However, the study on metabolic profile response to fluoride exposure is limited. A recent study carried out a metabonomics study on NaF treated human oral squamous cell carcinoma cells. The results showed that inhibition of the enolase reaction in glycolysis pathway was observed in the early stages of fluoride treatment. In the later stages, gradual increases in the AMP/ATP ratio (a putative marker of apoptosis) and oxidized products (e.g., GSSH, and methionine sulfoxide), and marginal changes in polyamine levels (putative marker of necrosis), were observed [148]. It suggested that the inhibition of enolase reaction and TCA cycle progression at early stage is specific to NaF, whereas the increase of ATP utilization at later stage may be common to apoptotis-inducing agents, but not to necrosis-inducing agents [148].

So far, there is no specific treatment for fluorosis. Efforts are being made to reduce the severity of the disease and improve quality of life of affected patients [149]. Medical treatment being used is mainly supplementation of vitamin (Vit) C, D, and E, calcium, antioxidants and treatment of malnutrition [150]. In recent years, some traditional Chinese medicines (TCM) have been developed to treat fluoride-induced bone lesions in China [49]. Treatment options for dental and skeletal fluorosis vary according to the severity of the disease [149]. Methods for treating dental fluorosis include micro/macro abrasion, bleaching, composite restorations, veneers, and full crowns [151]. Treatment of skeletal fluorosis may include surgical processes while treatment of deformity includes use of physiotherapy, corrective

Clearly, chronic fluorosis is mainly caused by excess intake of fluoride through drinking water, food products, air, and industrial pollutants over a long period. Therefore, avoiding excessive intake of the fluoride is essential for the prevention of this disease. Notably, to keep fluoride intake within safe limits, one needs to consider the total daily intake, including fluoride intake from water, food, air, fluoride-rich dental products and drugs. The recommended level for daily fluoride intake is 0.05–0.07 mg F/Kg/day [152, 153]. In China, for people aged 8–16 (including 16-year-olds), the recommended values of the total daily fluoride intake per person is ≤2.4 mg; for those 16 years old, the total daily fluoride intake per person is ≤3.5 mg [154]. In water-borne fluorosis endemic areas, alternative water resources with low fluoride levels or defluorinated water can be used. Coal-burning endemic fluorosis areas need to change the way coal is burned and food is dried. Likewise, it is beneficial for a daily intake of foods, vegetables, and fruits rich in vitamin C, D, and E, calcium, and antioxidants for the prevention of chronic fluorosis in endemic regions [49, 82, 150]. Moreover, health education is a very important aspect for disease management. Knowledge regarding the harmful effects of fluoride and the causes of fluorosis can help people, especially the affected population, pay more attention to their living habits [49, 149]. Furthermore, the identification of candidate genes that affect risk factors is necessary to develop more effective measures to prevent and treat fluorosis [27].

The contents of this chapter are reviewed in **Figure 1**. Excess intake of fluoride can cause chronic fluorosis, leading to dental fluorosis and skeletal fluorosis and damage

**52**

**9. Conclusions**

*An overview of the occurrence, influencing factors, pathogenesis, treatment, and prevention of fluorosis.*

to nervous system, reproductive system, cardiovascular system, liver, and kidney. The possible mechanisms involved different key proteins and signal transduction pathways associated with the pathogenesis of fluorosis have been proposed. Some high-throughput methods such as proteomics, metabolomics, and transcriptomics have been used in the study of the mechanism underlying development of fluorosis. Genetic factors play a critical role in the pathogenesis of chronic fluorosis. Combined exposure to fluoride with other element such as arsenic or aluminum may result in more complicated adverse health effects than exposure to fluoride or these elements alone. Further research is needed to reveal the interaction between fluorides with these elements with regard to their toxic effects. Clearly, the mechanisms of chronic fluorosis still need further research. Prevention of chronic fluorosis is important and it can be prevented by keeping fluoride intake within safe limits. It is important to consider total exposure (i.e., exposure through air, food, and water) when evaluating adverse health effects of fluoride.

## **Acknowledgements**

The authors would like to acknowledge the National Natural Science Foundation of China (Grant Nos. 31870825, 81560515) and Shenzhen Bureau of Science, Technology and Information (Nos. JCYJ20170412110026229, JCYJ20150529164656093) for funds to support this work.

## **Conflict of interest**

The authors declare that they have no competing interests.

## **Author details**

Liming Shen1 \*, Chengyun Feng2 , Sijian Xia1 , Yan Wei3 , Hua Zhang3 , Danqing Zhao4 , Fang Yao1 , Xukun Liu1 , Yuxi Zhao1 and Huajie Zhang1

1 College of Life Science and Oceanography, Shenzhen University, Shenzhen, PR China

2 Maternal and Child Health Hospital of Baoan, Shenzhen, PR China

3 School of Public Health, Guizhou Medical University, Guiyang, PR China

4 Department of Obstetrics and Gynecology, Affiliated Hospital of Guizhou Medical University, Guiyang, PR China

\*Address all correspondence to: slm@szu.edu.cn

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**55**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

Neurotoxicity Research. 2011;**19**:55-62. DOI: 10.1007/s12640-009-9139-5

[10] Wei N, Dong YT, Deng J, Wang Y,

[11] Dong YT, Wang Y, Wei N, Zhang QF, Guan ZZ. Deficit in learning and memory of rats with chronic fluorosis correlates with the decreased expressions of M1 and M3 muscarinic acetylcholine receptors. Archives of Toxicology. 2015;**89**:1981-1991. DOI:

10.1007/s00204-014-1408-2

guide\_to\_the\_literature

scitotenv.2010.10.046

[14] Chattopadhyay A, Podder S, Agarwal S, Bhattacharya S.

[12] Pain G. Mechanisms of fluoride neurotoxicity-a quick guide to the literature. 2017. Available from: https://www.researchgate.net/ publication/312057754\_Mechanisms\_ of\_Fluoride\_Neurotoxicity\_A\_quick\_

[13] Chandrajith R, Dissanayake CB, Ariyarathna T, Herath HMJMK, Padmasiri JP. Dose-dependent Na and Ca in fluoride-rich drinking water--another major cause of chronic renal failure in tropical arid regions. Science of the Total Environment. 2011;**409**:671-675. DOI: 10.1016/j.

[9] Niu R, Xue X, Zhao Y, Sun Z, Yan X, Li X, et al. Effects of fluoride on microtubule ultrastructure and expression of Tubα1a and Tubβ2a in mouse hippocampus. Chemosphere. 2015;**139**:422-427. DOI: 10.1016/j.

chemosphere.2015.07.011

jtemb.2017.09.020

Qi XL, Yu WF, et al. Changed expressions of N-methyl-d-aspartate receptors in the brains of rats and primary neurons exposed to high level of fluoride. Journal of Trace Elements in Medicine and Biology: Organ of the Society for Minerals and Trace Elements (GMS). 2018;**45**:31-40. DOI: 10.1016/j.

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

[1] Kanduti D, Sterbenk P, Artnik B. Fluoride: A review of use and effects on health. Mater Socio-Medica. 2016;**28**:133-137. DOI: 10.5455/

[2] Clark MB, Slayton RL. Section on oral health. Fluoride use in caries prevention in the primary care setting. Pediatrics. 2014;**134**:626-633. DOI:

**References**

msm.2016.28.133-137

10.1542/peds.2014-1699

[3] Ozsvath DL. Fluoride and environmental health: A review. Reviews in Environmental Science and Biotechnology. 2009;**8**:59-79. DOI:

10.1007/s11157-008-9136-9

2017.9104

[4] Ullah R, Zafar MS, Shahani N. Potential fluoride toxicity from oral medicaments: A review. Iranian Journal of Basic Medical Sciences. 2017;**20**: 841-848. DOI: 10.22038/IJBMS.

[5] Everett ET. Fluoride's effects on the formation of teeth and bones, and the influence of genetics. Journal of Dental Research. 2011;**90**:552-560. DOI:

[6] Wu C, Gu X, Ge Y, Zhang J, Wang J. Effects of high fluoride and arsenic on brain biochemical indexes and learning-memory in rats. Fluoride.

[7] Liu J, Gao Q, Wu CX, Guan ZZ. Alterations of nAChRs and ERK1/2 in

the brains of rats with chronic fluorosis and their connections with the decreased capacity of learning and memory. Toxicology Letters. 2010;**192**:324-329. DOI: 10.1016/j.

[8] Pereira M, Dombrowski PA, Losso EM, Chioca LR, Da Cunha C, Andreatini R. Memory impairment induced by sodium fluoride is associated with changes in brain monoamine levels.

10.1177/0022034510384626

2006;**39**:274-279

toxlet.2009.11.002

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

## **References**

*Environmental Chemistry and Recent Pollution Control Approaches*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Sijian Xia1

1 College of Life Science and Oceanography, Shenzhen University, Shenzhen,

3 School of Public Health, Guizhou Medical University, Guiyang, PR China

4 Department of Obstetrics and Gynecology, Affiliated Hospital of Guizhou

2 Maternal and Child Health Hospital of Baoan, Shenzhen, PR China

, Yan Wei3

and Huajie Zhang1

, Hua Zhang3

, Danqing Zhao4

,

**54**

**Author details**

Liming Shen1

Fang Yao1

PR China

provided the original work is properly cited.

Medical University, Guiyang, PR China

\*Address all correspondence to: slm@szu.edu.cn

\*, Chengyun Feng2

, Yuxi Zhao1

, Xukun Liu1

[1] Kanduti D, Sterbenk P, Artnik B. Fluoride: A review of use and effects on health. Mater Socio-Medica. 2016;**28**:133-137. DOI: 10.5455/ msm.2016.28.133-137

[2] Clark MB, Slayton RL. Section on oral health. Fluoride use in caries prevention in the primary care setting. Pediatrics. 2014;**134**:626-633. DOI: 10.1542/peds.2014-1699

[3] Ozsvath DL. Fluoride and environmental health: A review. Reviews in Environmental Science and Biotechnology. 2009;**8**:59-79. DOI: 10.1007/s11157-008-9136-9

[4] Ullah R, Zafar MS, Shahani N. Potential fluoride toxicity from oral medicaments: A review. Iranian Journal of Basic Medical Sciences. 2017;**20**: 841-848. DOI: 10.22038/IJBMS. 2017.9104

[5] Everett ET. Fluoride's effects on the formation of teeth and bones, and the influence of genetics. Journal of Dental Research. 2011;**90**:552-560. DOI: 10.1177/0022034510384626

[6] Wu C, Gu X, Ge Y, Zhang J, Wang J. Effects of high fluoride and arsenic on brain biochemical indexes and learning-memory in rats. Fluoride. 2006;**39**:274-279

[7] Liu J, Gao Q, Wu CX, Guan ZZ. Alterations of nAChRs and ERK1/2 in the brains of rats with chronic fluorosis and their connections with the decreased capacity of learning and memory. Toxicology Letters. 2010;**192**:324-329. DOI: 10.1016/j. toxlet.2009.11.002

[8] Pereira M, Dombrowski PA, Losso EM, Chioca LR, Da Cunha C, Andreatini R. Memory impairment induced by sodium fluoride is associated with changes in brain monoamine levels. Neurotoxicity Research. 2011;**19**:55-62. DOI: 10.1007/s12640-009-9139-5

[9] Niu R, Xue X, Zhao Y, Sun Z, Yan X, Li X, et al. Effects of fluoride on microtubule ultrastructure and expression of Tubα1a and Tubβ2a in mouse hippocampus. Chemosphere. 2015;**139**:422-427. DOI: 10.1016/j. chemosphere.2015.07.011

[10] Wei N, Dong YT, Deng J, Wang Y, Qi XL, Yu WF, et al. Changed expressions of N-methyl-d-aspartate receptors in the brains of rats and primary neurons exposed to high level of fluoride. Journal of Trace Elements in Medicine and Biology: Organ of the Society for Minerals and Trace Elements (GMS). 2018;**45**:31-40. DOI: 10.1016/j. jtemb.2017.09.020

[11] Dong YT, Wang Y, Wei N, Zhang QF, Guan ZZ. Deficit in learning and memory of rats with chronic fluorosis correlates with the decreased expressions of M1 and M3 muscarinic acetylcholine receptors. Archives of Toxicology. 2015;**89**:1981-1991. DOI: 10.1007/s00204-014-1408-2

[12] Pain G. Mechanisms of fluoride neurotoxicity-a quick guide to the literature. 2017. Available from: https://www.researchgate.net/ publication/312057754\_Mechanisms\_ of\_Fluoride\_Neurotoxicity\_A\_quick\_ guide\_to\_the\_literature

[13] Chandrajith R, Dissanayake CB, Ariyarathna T, Herath HMJMK, Padmasiri JP. Dose-dependent Na and Ca in fluoride-rich drinking water--another major cause of chronic renal failure in tropical arid regions. Science of the Total Environment. 2011;**409**:671-675. DOI: 10.1016/j. scitotenv.2010.10.046

[14] Chattopadhyay A, Podder S, Agarwal S, Bhattacharya S.

Fluoride-induced histopathology and synthesis of stress protein in liver and kidney of mice. Archives of Toxicology. 2011;**85**:327-335. DOI: 10.1007/ s00204-010-0588-7

[15] Pain G. Fluoride is a developmental nephrotoxin—Coming to a Kidney near you. 2017. Available from: https://www.researchgate.net/ publication/313025968\_Fluoride\_ is\_a\_developmental\_Nephrotoxin\_-\_ coming\_to\_a\_Kidney\_near\_you. DOI: 10.13140/RG.2.2.10999.62884

[16] Iano FG, Ferreira MC, Quaggio GB, Fernandes MS, Oliveira RC, Ximenes VF, et al. Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats. Journal of Fluorine Chemistry. 2014;**168**:212-217. DOI: 10.1016/j. jfluchem.2014.09.029

[17] Zhao Y, Li Y, Wang J, Manthari RK, Wang J. Fluoride induces apoptosis and autophagy through the IL-17 signaling pathway in mice hepatocytes. Archives of Toxicology. 2018;**92**:3277-3289. DOI: 10.1007/s00204-018-2305-x

[18] Khan ZN, Sabino IT, de Souza Melo CG, Martini T, da Silva Pereira HAB, Buzalaf MAR. Liver proteome of mice with distinct genetic susceptibilities to fluorosis treated with different concentrations of F in the drinking water. Biological Trace Element Research. 2019;**187**:107-119. DOI: 10.1007/s12011-018-1344-8

[19] Fan B, Yu Y, Zhang Y. PI3K-Akt1 expression and its significance in liver tissues with chronic fluorosis. International Journal of Clinical and Experimental Pathology. 2015;**8**:1226-1236

[20] Amini H, Taghavi Shahri SM, Amini M, Ramezani Mehrian M, Mokhayeri Y, Yunesian M. Drinking water fluoride and blood pressure? An environmental study. Biological Trace Element

Research. 2011;**144**:157-163. DOI: 10.1007/s12011-011-9054-5

[21] Sun L, Gao Y, Liu H, Zhang W, Ding Y, Li B, et al. An assessment of the relationship between excess fluoride intake from drinking water and essential hypertension in adults residing in fluoride endemic areas. Science of the Total Environment. 2013;**443**:864-869. DOI: 10.1016/j.scitotenv.20-12.11.021

[22] Liu H, Gao Y, Sun L, Li M, Li B, Sun D. Assessment of relationship on excess fluoride intake from drinking water and carotid atherosclerosis development in adults in fluoride endemic areas, China. International Journal of Hygiene and Environmental Health. 2014;**217**:413-420. DOI: 10.1016/j.ijheh.2013.08.001

[23] Sun L, Gao Y, Zhang W, Liu H, Sun D. Effect of high fluoride and high fat on serum lipid levels and oxidative stress in rabbits. Environmental Toxicology and Pharmacology. 2014;**38**:1000-1006. DOI: 10.1016/j.et-ap.2014.10.010

[24] Huo M, Han H, Sun Z, Lu Z, Yao X, Wang S, et al. Role of IL-17 pathways in immune privilege: A RNA deep sequencing analysis of the mice testis exposure to fluoride. Scientific Reports. 2016;**6**:32173. DOI: 10.1038/srep32173

[25] He X, Sun Z, Manthari RK, Wu P, Wang J. Fluoride altered rat's blood testis barrier by affecting the F-actin via IL-1α. Chemosphere. 2018;**211**:826-833. DOI: 10.1016/j. chemosphere.20-18.08.009

[26] Han H, Sun Z, Luo G, Wang C, Wei R, Wang J. Fluoride exposure changed the structure and the expressions of reproductive related genes in the hypothalamus-pituitary-testicular axis of male mice. Chemosphere. 2015;**135**:297-303. DOI: 10.1016/j. chemosphere.2015.04.012

[27] Pramanik S, Saha D. The genetic influence in fluorosis. Environmental

**57**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

pollution and testicular damage in wild mice. Chemosphere. 2015;**132**:135-141. DOI: 10.1016/j. chemosphere.20-15.03.0-17

[35] Agalakova N, Gusev G. Molecular mechanisms of cytotoxicity and

[36] Dhar V, Bhatnagar M. Physiology and toxicity of fluoride. Indian Journal of Dental Research. 2009;**20**:350-355. DOI: 10.4103/0970-9290.57379

[37] Perumal E, Paul V, Govindarajan V, Panneerselvam L. A brief review on experimental fluorosis. Toxicology Letters. 2013;**223**:236-251. DOI: 10.1016/j.toxlet.2013.09.005

[38] Barbier O, Arreola-Mendoza L, Del Razo LM. Molecular mechanisms of fluoride toxicity. Chemico-Biological Interactions. 2010;**188**:319-333. DOI:

10.1016/j.cbi.2010.07.011

lfs.2018.02.001

[39] Zuo H, Chen L, Kong M, Qiu L, Lü P, Wu P, et al. Toxic effects of fluoride on organisms. Life Sciences. 2018;**198**:18-24. DOI: 10.1016/j.

[40] Krishnamachari KA. Skeletal fluorosis in humans: A review of recent progress in the understanding of the disease. Progress in Food & Nutrition

[41] Topuz O, Akkaya N, Ardic F, Sarsan A, Cubukcu D, Gokgoz A. Bone resorption marker and ultrasound measurements in adults residing in an endemic fluorosis area of Turkey.

[42] Mousny M, Omelon S, Wise L, Everett ET, Dumitriu M, Holmyard DP, et al. Fluoride effects on bone formation and mineralization are influenced by genetics. Bone. 2008;**43**:1067-1074. DOI:

Science. 1986;**10**:279-314

Fluoride. 2006;**39**:138-144

10.1016/j.bone.2008.07.248

apoptosis induced by inorganic fluoride. ISRN Cell Biology. 2012;**2012**. Article ID 403835. DOI: 10.5402/2012/403835

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

[28] Xu Y, Huang H, Zeng Q , Yu C, Yao M, Hong F, et al. The effect of elemental content on the risk of dental fluorosis and the exposure of the environment and population to fluoride produced by coal-burning. Environmental Toxicology and Pharmacology. 2017;**56**:329-339. DOI: 10.1016/j.etap.2017.10.011

[29] Qin X, Wang S, Yu M, Zhang L, Li X, Zuo Z, et al. Child skeletal fluorosis from indoor burning of coal in southwestern China. Journal of Environmental and Public Health. 2009;**2009**:969764. DOI:

[30] Chen J, Liu G, Kang Y, Wu B, Sun R, Zhou C, et al. Coal utilization in China: Environmental impacts and human health. Environmental Geochemistry and Health. 2014;**36**:735-753. DOI:

10.1155/2009/9-69764

10.1007/s10653-013-9592-1

bmjopen-2012-001564

10.2188/jea.JE20150037

[31] Wang C, Gao Y, Wang W, Zhao L, Zhang W, Han H, et al. A national cross-sectional study on effects of fluoride-safe water supply on the prevalence of fluorosis in China. BMJ Open. 2012;**2**:e001564. DOI: 10.1136/

[32] Fan Z, Gao Y, Wang W, Gong H, Guo M, Zhao S, et al. Prevalence of brick tea-type fluorosis in the Tibet autonomous region. Journal of Epidemiology. 2016;**26**:57-63. DOI:

[33] Linhares D, Camarinho R, Garcia PV, Rodrigues ADS. Mus musculus bone fluoride concentration as a useful biomarker for risk assessment of skeletal fluorosis in volcanic areas. Chemosphere. 2018;**205**:540-544. DOI: 10.1016/j.chemosphere.2018.04.144

[34] Ferreira AF, Garcia PV, Camarinho R, Rodrigues A dos S. Volcanogenic

Toxicology and Pharmacology. 2017;**56**:157-162. DOI: 10.1016/j.

etap.2017.09.008

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

Toxicology and Pharmacology. 2017;**56**:157-162. DOI: 10.1016/j. etap.2017.09.008

*Environmental Chemistry and Recent Pollution Control Approaches*

Research. 2011;**144**:157-163. DOI: 10.1007/s12011-011-9054-5

[21] Sun L, Gao Y, Liu H, Zhang W, Ding Y, Li B, et al. An assessment of the relationship between excess fluoride intake from drinking water and essential hypertension in adults residing in fluoride endemic areas. Science of the Total Environment. 2013;**443**:864-869. DOI: 10.1016/j.scitotenv.20-12.11.021

[22] Liu H, Gao Y, Sun L, Li M, Li B, Sun D. Assessment of relationship on excess fluoride intake from drinking water and carotid atherosclerosis development in adults in fluoride endemic areas, China. International Journal of Hygiene and Environmental Health. 2014;**217**:413-420.

DOI: 10.1016/j.ijheh.2013.08.001

DOI: 10.1016/j.et-ap.2014.10.010

[25] He X, Sun Z, Manthari RK, Wu P, Wang J. Fluoride altered rat's blood testis barrier by affecting the F-actin via IL-1α. Chemosphere. 2018;**211**:826-833. DOI: 10.1016/j. chemosphere.20-18.08.009

[23] Sun L, Gao Y, Zhang W, Liu H, Sun D. Effect of high fluoride and high fat on serum lipid levels and oxidative stress in rabbits. Environmental Toxicology and Pharmacology. 2014;**38**:1000-1006.

[24] Huo M, Han H, Sun Z, Lu Z, Yao X, Wang S, et al. Role of IL-17 pathways in immune privilege: A RNA deep sequencing analysis of the mice testis exposure to fluoride. Scientific Reports. 2016;**6**:32173. DOI: 10.1038/srep32173

[26] Han H, Sun Z, Luo G, Wang C, Wei R, Wang J. Fluoride exposure changed the structure and the expressions of reproductive related genes in the hypothalamus-pituitary-testicular axis of male mice. Chemosphere. 2015;**135**:297-303. DOI: 10.1016/j. chemosphere.2015.04.012

[27] Pramanik S, Saha D. The genetic influence in fluorosis. Environmental

Fluoride-induced histopathology and synthesis of stress protein in liver and kidney of mice. Archives of Toxicology.

[15] Pain G. Fluoride is a developmental nephrotoxin—Coming to a Kidney near you. 2017. Available from: https://www.researchgate.net/ publication/313025968\_Fluoride\_ is\_a\_developmental\_Nephrotoxin\_-\_ coming\_to\_a\_Kidney\_near\_you. DOI:

2011;**85**:327-335. DOI: 10.1007/

10.13140/RG.2.2.10999.62884

jfluchem.2014.09.029

10.1007/s00204-018-2305-x

[16] Iano FG, Ferreira MC, Quaggio GB, Fernandes MS, Oliveira RC, Ximenes VF, et al. Effects of chronic fluoride intake on the antioxidant systems of the liver and kidney in rats. Journal of Fluorine Chemistry. 2014;**168**:212-217. DOI: 10.1016/j.

[17] Zhao Y, Li Y, Wang J, Manthari RK, Wang J. Fluoride induces apoptosis and autophagy through the IL-17 signaling pathway in mice hepatocytes. Archives of Toxicology. 2018;**92**:3277-3289. DOI:

[18] Khan ZN, Sabino IT, de Souza Melo CG, Martini T, da Silva Pereira HAB, Buzalaf MAR. Liver proteome of mice with distinct genetic susceptibilities to fluorosis treated with different concentrations of F in the drinking water. Biological Trace Element Research. 2019;**187**:107-119. DOI: 10.1007/s12011-018-1344-8

[19] Fan B, Yu Y, Zhang Y. PI3K-Akt1 expression and its significance in liver tissues with chronic fluorosis. International Journal of Clinical and Experimental Pathology.

[20] Amini H, Taghavi Shahri SM, Amini M, Ramezani Mehrian M, Mokhayeri Y, Yunesian M. Drinking water fluoride and blood pressure? An environmental

study. Biological Trace Element

s00204-010-0588-7

**56**

2015;**8**:1226-1236

[28] Xu Y, Huang H, Zeng Q , Yu C, Yao M, Hong F, et al. The effect of elemental content on the risk of dental fluorosis and the exposure of the environment and population to fluoride produced by coal-burning. Environmental Toxicology and Pharmacology. 2017;**56**:329-339. DOI: 10.1016/j.etap.2017.10.011

[29] Qin X, Wang S, Yu M, Zhang L, Li X, Zuo Z, et al. Child skeletal fluorosis from indoor burning of coal in southwestern China. Journal of Environmental and Public Health. 2009;**2009**:969764. DOI: 10.1155/2009/9-69764

[30] Chen J, Liu G, Kang Y, Wu B, Sun R, Zhou C, et al. Coal utilization in China: Environmental impacts and human health. Environmental Geochemistry and Health. 2014;**36**:735-753. DOI: 10.1007/s10653-013-9592-1

[31] Wang C, Gao Y, Wang W, Zhao L, Zhang W, Han H, et al. A national cross-sectional study on effects of fluoride-safe water supply on the prevalence of fluorosis in China. BMJ Open. 2012;**2**:e001564. DOI: 10.1136/ bmjopen-2012-001564

[32] Fan Z, Gao Y, Wang W, Gong H, Guo M, Zhao S, et al. Prevalence of brick tea-type fluorosis in the Tibet autonomous region. Journal of Epidemiology. 2016;**26**:57-63. DOI: 10.2188/jea.JE20150037

[33] Linhares D, Camarinho R, Garcia PV, Rodrigues ADS. Mus musculus bone fluoride concentration as a useful biomarker for risk assessment of skeletal fluorosis in volcanic areas. Chemosphere. 2018;**205**:540-544. DOI: 10.1016/j.chemosphere.2018.04.144

[34] Ferreira AF, Garcia PV, Camarinho R, Rodrigues A dos S. Volcanogenic

pollution and testicular damage in wild mice. Chemosphere. 2015;**132**:135-141. DOI: 10.1016/j. chemosphere.20-15.03.0-17

[35] Agalakova N, Gusev G. Molecular mechanisms of cytotoxicity and apoptosis induced by inorganic fluoride. ISRN Cell Biology. 2012;**2012**. Article ID 403835. DOI: 10.5402/2012/403835

[36] Dhar V, Bhatnagar M. Physiology and toxicity of fluoride. Indian Journal of Dental Research. 2009;**20**:350-355. DOI: 10.4103/0970-9290.57379

[37] Perumal E, Paul V, Govindarajan V, Panneerselvam L. A brief review on experimental fluorosis. Toxicology Letters. 2013;**223**:236-251. DOI: 10.1016/j.toxlet.2013.09.005

[38] Barbier O, Arreola-Mendoza L, Del Razo LM. Molecular mechanisms of fluoride toxicity. Chemico-Biological Interactions. 2010;**188**:319-333. DOI: 10.1016/j.cbi.2010.07.011

[39] Zuo H, Chen L, Kong M, Qiu L, Lü P, Wu P, et al. Toxic effects of fluoride on organisms. Life Sciences. 2018;**198**:18-24. DOI: 10.1016/j. lfs.2018.02.001

[40] Krishnamachari KA. Skeletal fluorosis in humans: A review of recent progress in the understanding of the disease. Progress in Food & Nutrition Science. 1986;**10**:279-314

[41] Topuz O, Akkaya N, Ardic F, Sarsan A, Cubukcu D, Gokgoz A. Bone resorption marker and ultrasound measurements in adults residing in an endemic fluorosis area of Turkey. Fluoride. 2006;**39**:138-144

[42] Mousny M, Omelon S, Wise L, Everett ET, Dumitriu M, Holmyard DP, et al. Fluoride effects on bone formation and mineralization are influenced by genetics. Bone. 2008;**43**:1067-1074. DOI: 10.1016/j.bone.2008.07.248

[43] Song Y, Tan H, Liu K, Zhang Y, Liu Y, Lu C, et al. Effect of fluoride exposure on bone metabolism indicators ALP, BALP, and BGP. Environmental Health and Preventive Medicine. 2011;**16**:158-163. DOI: 10.1007/ s12199-010-0181-y

[44] Jianping Z, Benli Y. The application of bone metabolism biochemical markers in the study of skeletal fluorosis. Chinese Journal of Endemiology. 2003;**22**:186-188

[45] Debiński A, Nowicka G. Effect of sodium fluoride on ectopic induction of bone tissue. Annales Academiae Medicae Stetinensis. 2004;**50**(Suppl 1): 23-27

[46] Ma L, Lin ZQ , Liu KT. The effects of sodium fluoride on the resorptive activity of isolated osteoclasts. Journal of Xinjiang Medical University. 2004;**27**:334-336 (In Chinese)

[47] Okuda A, Kanehisa J, Heersche JN. The effects of sodium fluoride on the resorptive activity of isolated osteoclasts. Journal of Bone and Mineral Research. 1990;**5**(Suppl 1):S115-S120. DOI: 10.1002/jbmr.5650051381

[48] Junrui P, Bingyun L, Yanhui G, Xu J, Darko GM, Dianjun S. Relationship between fluoride exposure and osteoclast markers during RANKLinduced osteoclast differentiation. Environmental Toxicology and Pharmacology. 2016;**46**:241-245. DOI: 10.1016/j.etap.2016.08.001

[49] Chen Y, Yan W, Hui X. Treatment and prevention of skeletal fluorosis. Biomedical and Environmental Sciences. 2017;**30**:147-149. DOI: 10.3967/ bes2017.020

[50] Lai LP, Lotinun S, Bouxsein ML, Baron R, McMahon AP. Stk11 (Lkb1) deletion in the osteoblast lineage leads to high bone turnover, increased trabecular bone density and cortical

porosity. Bone. 2014;**69**:98-108. DOI: 10.1016/j.bone.2014.09.010

[51] Ito M, Nakagawa H, Okada T, Miyazaki S, Matsuo S. ER-stress caused by accumulated intracistanal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride. Archives of Toxicology. 2009;**83**:151-159. DOI: 10.1007/s00204-008-0341-7

[52] Daiwile AP, Sivanesan S, Tarale P, Naoghare PK, Bafana A, Parmar D, et al. Role of fluoride induced histone trimethylation in development of skeletal fluorosis. Environmental Toxicology and Pharmacology. 2018;**57**:159-165. DOI: 10.1016/j.etap.2017.12.015

[53] Iwatsuki M, Matsuoka M. Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells. Toxicology Mechanisms and Methods. 2016;**26**:132-138. DOI: 10.3109/15376516.2015.1129570

[54] Wei Y, Zeng B, Zhang H, Chen C, Wu Y, Wang N, et al. Comparative proteomic analysis of fluoride treated rat bone provides new insights into the molecular mechanisms of fluoride toxicity. Toxicology Letters. 2018;**291**:39-50. DOI: 10.1016/j. toxlet.2018.04.006

[55] Miao Q , Xu M, Liu B, You B. In vivo and in vitro study on the effect of excessive fluoride on type I collagen of rats. Wei Sheng Yan Jiu. 2002;**31**:145-147

[56] Yan X, Feng C, Chen Q , Li W, Wang H, Lv L, et al. Effects of sodium fluoride treatment in vitro on cell proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression by neonatal rat osteoblasts. Archives of Toxicology. 2009;**83**:451-458. DOI: 10.1007/ s00204-008-0365-z

[57] Yan X, Yan X, Morrison A, Han T, Chen Q , Li J, et al. Fluoride

**59**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

[65] Waddington RJ, Langley MS. Altered expression of matrix

2003;**44**:88-95

metalloproteinases within mineralizing bone cells in vitro in the presence of fluoride. Connective Tissue Research.

[66] Dec K, Łukomska A, Maciejewska D, Jakubczyk K, Baranowska-Bosiacka I,

Chlubek D, et al. The influence of fluorine on the disturbances of homeostasis in the central nervous system. Biological Trace Element Research. 2017;**177**:224-234. DOI: 10.1007/s12011-016-0871-4

[67] Choi AL, Sun G, Zhang Y, Grandjean P. Developmental fluoride neurotoxicity: A systematic review and meta-analysis. Environmental Health Perspectives. 2012;**120**:1362-1368. DOI:

[68] Zhang S, Zhang X, Liu H, Qu W, Guan Z, Zeng Q , et al. Modifying effect of COMT gene polymorphism and a predictive role for proteomics analysis in children's intelligence in endemic fluorosis area in Tianjin, China.

Toxicological Sciences. 2015;**144**: 238-245. DOI: 10.1093/toxsci/kfu311

[69] Choi AL, Zhang Y, Sun G, Bellinger DC, Wang K, Yang XJ, et al. Association of lifetime exposure to fluoride and cognitive functions in Chinese children: A pilot study. Neurotoxicology and Teratology. 2015;**47**:96-101. DOI: 10.1016/j.

[70] Whitford GM, Whitford JL, Hobbs SH. Appetitive-based learning in rats: Lack of effect of chronic exposure to fluoride. Neurotoxicology and Teratology. 2009;**31**:210-215. DOI:

[71] Gao Q , Liu YJ, Guan ZZ. Decreased learning and memory ability in rats with fluorosis: Increased oxidative stress and reduced cholinesterase activity in the brain. Fluoride. 2009;**42**:277-285

10.1016/j.ntt.20-09.02.003

ntt.2014.11.001

10.1289/ehp.1104912

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

induces apoptosis and alters collagen I expression in rat osteoblasts. Toxicology

Letters. 2011;**200**:133-138. DOI: 10.1016/j.toxlet.20-10.11.005

[58] Yan X, Hao X, Nie Q , Feng C, Wang H, Sun Z, et al. Effects of fluoride on the ultrastructure and expression of type I collagen in rat hard tissue. Chemosphere. 2015;**128**:36-41. DOI: 10.1016/j.chemosphere.2014.12.090

[59] Zhong DB, Sun LT, Wu PF, Su JL,

[60] Li W, Yang L, Ren Y, Yan X, Wang J. Quantification of rib COL1A2 gene expression in healthy and fluorosed Inner Mongolia cashmere goats.

[61] Nair M, Belak ZR, Ovsenek N. Effects of fluoride on expression of bone-specific genes in developing *Xenopus laevis* larvae. Biochemistry and Cell Biology. 2011;**89**:377-386. DOI:

[62] Kobayashi CAN, Leite AL, Peres-Buzalaf C, Carvalho JG, Whitford GM, Everett ET, et al. Bone response to fluoride exposure is influenced by genetics. PLoS One. 2014;**9**:e114343. DOI: 10.1371/journal.pone.0114343

[63] Waddington RJ, Langley MS. Structural analysis of proteoglycans synthesized by mineralizing bone cells in vitro in the presence of

fluoride. Matrix Biology: Journal of the International Society for Matrix Biology.

[64] Slompo C, Buzalaf CP, Damante CA, Martins GM, Hannas AR, Buzalaf MAR, et al. Fluoride

modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities. Brazilian Dental Journal.

Han B. Effect of fluoride on proliferation, differentiation, and apoptosis of cultured caprine (goat) osteoblasts. Fluoride. 2005;**38**:230-231

Fluoride. 2007;**40**:13-18

10.1139/o11-034

1998;**17**:255-268

2012;**23**:629-634

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

induces apoptosis and alters collagen I expression in rat osteoblasts. Toxicology Letters. 2011;**200**:133-138. DOI: 10.1016/j.toxlet.20-10.11.005

*Environmental Chemistry and Recent Pollution Control Approaches*

porosity. Bone. 2014;**69**:98-108. DOI:

[51] Ito M, Nakagawa H, Okada T, Miyazaki S, Matsuo S. ER-stress caused by accumulated intracistanal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride. Archives of Toxicology. 2009;**83**:151-159. DOI:

10.1016/j.bone.2014.09.010

10.1007/s00204-008-0341-7

10.1016/j.etap.2017.12.015

toxlet.2018.04.006

s00204-008-0365-z

[57] Yan X, Yan X, Morrison A, Han T, Chen Q , Li J, et al. Fluoride

[53] Iwatsuki M, Matsuoka M. Fluoride-induced c-Fos expression in MC3T3-E1 osteoblastic cells. Toxicology Mechanisms and Methods. 2016;**26**:132-138. DOI: 10.3109/15376516.2015.1129570

[54] Wei Y, Zeng B, Zhang H, Chen C, Wu Y, Wang N, et al. Comparative proteomic analysis of fluoride treated rat bone provides new insights into the molecular mechanisms of fluoride toxicity. Toxicology Letters. 2018;**291**:39-50. DOI: 10.1016/j.

[55] Miao Q , Xu M, Liu B, You B. In vivo and in vitro study on the effect of excessive fluoride on type I collagen of rats. Wei Sheng Yan Jiu. 2002;**31**:145-147

[56] Yan X, Feng C, Chen Q , Li W, Wang H, Lv L, et al. Effects of sodium fluoride treatment in vitro on cell proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression by neonatal rat osteoblasts. Archives of Toxicology. 2009;**83**:451-458. DOI: 10.1007/

[52] Daiwile AP, Sivanesan S, Tarale P, Naoghare PK, Bafana A, Parmar D, et al. Role of fluoride induced histone trimethylation in development of skeletal fluorosis. Environmental Toxicology and Pharmacology. 2018;**57**:159-165. DOI:

[43] Song Y, Tan H, Liu K, Zhang Y, Liu Y, Lu C, et al. Effect of fluoride exposure on bone metabolism indicators ALP, BALP, and BGP. Environmental Health and Preventive Medicine. 2011;**16**:158-163. DOI: 10.1007/

s12199-010-0181-y

23-27

[44] Jianping Z, Benli Y. The application of bone metabolism biochemical markers in the study of skeletal fluorosis. Chinese Journal of Endemiology. 2003;**22**:186-188

[45] Debiński A, Nowicka G. Effect of sodium fluoride on ectopic induction of bone tissue. Annales Academiae Medicae Stetinensis. 2004;**50**(Suppl 1):

[46] Ma L, Lin ZQ , Liu KT. The effects of sodium fluoride on the resorptive activity of isolated osteoclasts. Journal of Xinjiang Medical University. 2004;**27**:334-336 (In Chinese)

[47] Okuda A, Kanehisa J, Heersche JN. The effects of sodium fluoride on the resorptive activity of isolated osteoclasts. Journal of Bone and Mineral Research. 1990;**5**(Suppl 1):S115-S120. DOI: 10.1002/jbmr.5650051381

[48] Junrui P, Bingyun L, Yanhui G, Xu J, Darko GM, Dianjun S. Relationship between fluoride exposure and osteoclast markers during RANKLinduced osteoclast differentiation. Environmental Toxicology and Pharmacology. 2016;**46**:241-245. DOI:

[49] Chen Y, Yan W, Hui X. Treatment and prevention of skeletal fluorosis. Biomedical and Environmental

Sciences. 2017;**30**:147-149. DOI: 10.3967/

[50] Lai LP, Lotinun S, Bouxsein ML, Baron R, McMahon AP. Stk11 (Lkb1) deletion in the osteoblast lineage leads to high bone turnover, increased trabecular bone density and cortical

10.1016/j.etap.2016.08.001

**58**

bes2017.020

[58] Yan X, Hao X, Nie Q , Feng C, Wang H, Sun Z, et al. Effects of fluoride on the ultrastructure and expression of type I collagen in rat hard tissue. Chemosphere. 2015;**128**:36-41. DOI: 10.1016/j.chemosphere.2014.12.090

[59] Zhong DB, Sun LT, Wu PF, Su JL, Han B. Effect of fluoride on proliferation, differentiation, and apoptosis of cultured caprine (goat) osteoblasts. Fluoride. 2005;**38**:230-231

[60] Li W, Yang L, Ren Y, Yan X, Wang J. Quantification of rib COL1A2 gene expression in healthy and fluorosed Inner Mongolia cashmere goats. Fluoride. 2007;**40**:13-18

[61] Nair M, Belak ZR, Ovsenek N. Effects of fluoride on expression of bone-specific genes in developing *Xenopus laevis* larvae. Biochemistry and Cell Biology. 2011;**89**:377-386. DOI: 10.1139/o11-034

[62] Kobayashi CAN, Leite AL, Peres-Buzalaf C, Carvalho JG, Whitford GM, Everett ET, et al. Bone response to fluoride exposure is influenced by genetics. PLoS One. 2014;**9**:e114343. DOI: 10.1371/journal.pone.0114343

[63] Waddington RJ, Langley MS. Structural analysis of proteoglycans synthesized by mineralizing bone cells in vitro in the presence of fluoride. Matrix Biology: Journal of the International Society for Matrix Biology. 1998;**17**:255-268

[64] Slompo C, Buzalaf CP, Damante CA, Martins GM, Hannas AR, Buzalaf MAR, et al. Fluoride modulates preosteoblasts viability and matrix metalloproteinases-2 and -9 activities. Brazilian Dental Journal. 2012;**23**:629-634

[65] Waddington RJ, Langley MS. Altered expression of matrix metalloproteinases within mineralizing bone cells in vitro in the presence of fluoride. Connective Tissue Research. 2003;**44**:88-95

[66] Dec K, Łukomska A, Maciejewska D, Jakubczyk K, Baranowska-Bosiacka I, Chlubek D, et al. The influence of fluorine on the disturbances of homeostasis in the central nervous system. Biological Trace Element Research. 2017;**177**:224-234. DOI: 10.1007/s12011-016-0871-4

[67] Choi AL, Sun G, Zhang Y, Grandjean P. Developmental fluoride neurotoxicity: A systematic review and meta-analysis. Environmental Health Perspectives. 2012;**120**:1362-1368. DOI: 10.1289/ehp.1104912

[68] Zhang S, Zhang X, Liu H, Qu W, Guan Z, Zeng Q , et al. Modifying effect of COMT gene polymorphism and a predictive role for proteomics analysis in children's intelligence in endemic fluorosis area in Tianjin, China. Toxicological Sciences. 2015;**144**: 238-245. DOI: 10.1093/toxsci/kfu311

[69] Choi AL, Zhang Y, Sun G, Bellinger DC, Wang K, Yang XJ, et al. Association of lifetime exposure to fluoride and cognitive functions in Chinese children: A pilot study. Neurotoxicology and Teratology. 2015;**47**:96-101. DOI: 10.1016/j. ntt.2014.11.001

[70] Whitford GM, Whitford JL, Hobbs SH. Appetitive-based learning in rats: Lack of effect of chronic exposure to fluoride. Neurotoxicology and Teratology. 2009;**31**:210-215. DOI: 10.1016/j.ntt.20-09.02.003

[71] Gao Q , Liu YJ, Guan ZZ. Decreased learning and memory ability in rats with fluorosis: Increased oxidative stress and reduced cholinesterase activity in the brain. Fluoride. 2009;**42**:277-285

[72] Liu F, Ma J, Zhang H, Liu P, Liu YP, Xing B, et al. Fluoride exposure during development affects both cognition and emotion in mice. Physiology & Behavior. 2014;**124**:1-7. DOI: 10.1016/j. physbeh.20-13.10.027

[73] Zhang Z, Xu X, Shen X, Xu X. Effect of fluoride exposure on synaptic structure of brain areas related to learning-memory in mice. Wei Sheng Yan Jiu. 1999;**28**:210-212 (In Chinese)

[74] Zhang KL, Lou DD, Guan ZZ. Activation of the AGE/RAGE system in the brains of rats and in SH-SY5Y cells exposed to high level of fluoride might connect to oxidative stress. Neurotoxicology and Teratology. 2015;**48**:49-55. DOI: 10.1016/j. ntt.2015.01.007

[75] Yan N, Liu Y, Liu S, Cao S, Wang F, Wang Z, et al. Fluoride-induced neuron apoptosis and expressions of inflammatory factors by activating microglia in rat brain. Molecular Neurobiology. 2016;**53**:4449-4460. DOI: 10.1007/s12035-015-9380-2

[76] Scheff SW, Price DA, Hicks RR, Baldwin SA, Robinson S, Brackney C. Synaptogenesis in the hippocampal CA1 field following traumatic brain injury. Journal of Neurotrauma. 2005;**22**:719- 732. DOI: 10.1089/neu.2005.22.719

[77] Saxena R, Meena B, Chouhan VS, Bhatnagar M, et al. Biochemical changes in brain and other tissues of young adult female mice from fluoride in their drinking water. In: Fluoride (Res Rep November 1, 2). Vol. 39. 2006. pp. 280-284

[78] Chirumari K, Reddy PK. Dosedependent effects of fluoride on neurochemical milieu in the hippocampus and neocortex of rat brain. Fluoride. 2007;**40**:101-110

[79] Gui CZ, Ran LY, Li JP, Guan ZZ. Changes of learning and memory ability and brain nicotinic receptors of rat offspring with coal burning fluorosis. Neurotoxicology and Teratology. 2010;**32**:536-541. DOI: 10.1016/j. ntt.2010.03.010

[80] Bartos M, Gumilar F, Bras C, Gallegos CE, Giannuzzi L, Cancela LM, et al. Neurobehavioural effects of exposure to fluoride in the earliest stages of rat development. Physiology & Behavior. 2015;**147**:205-212. DOI: 10.1016/j.physbeh.2015.04.044

[81] Schulz JA, Lamb AR. The effect of fluorine as sodium fluoride on the growth and reproduction of albino rats. Science. 1925;**61**:93-94. DOI: 10.1126/ science.61.1569.93

[82] Tian Y, Xiao Y, Wang B, Sun C, Tang K, Sun F. Vitamin E and lycopene reduce coal burning fluorosis-induced spermatogenic cell apoptosis via oxidative stress-mediated JNK and ERK signaling pathways. Bioscience Reports. 2018;**38**:BSR20171003. DOI: 10.1042/ BSR20171003

[83] Pati P, Bhunya S. Genotoxic effect of an environmental-pollutant, sodiumfluoride, in mammalian in vivo test system. Caryologia. 1987;**40**:79-87. DOI: 10.1080/00087114.1987.10797811

[84] Zhang J, Li Z, Qie M, Zheng R, Shetty J, Wang J. Sodium fluoride and sulfur dioxide affected male reproduction by disturbing blood-testis barrier in mice. Food and Chemical Toxicology. 2016;**94**:103-111. DOI: 10.1016/j.fct.2016.05.017

[85] Inkielewicz I, Krechniak J. Fluoride effects on glutathione peroxidase and lipid peroxidation in rats. Fluoride. 2004;**37**:7-12

[86] Dhurvey V, Patil V, Thakare M. Effect of sodium fluoride on the structure and function of the thyroid and ovary in albino rats (rattus Norvegicus). Fluoride. 2017;**50**:235-246

**61**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

Stookey GK. Severe dental fluorosis in a Tanzanian population consuming water with negligible fluoride concentration. Community Dentistry and Oral Epidemiology. 1998;**26**:382-393

[96] Choubisa SL, Choubisa L, Choubisa DK. Endemic fluorosis in Rajasthan. Indian Journal of Environmental

Health. 2001;**43**:177-189

10.1177/0810794

[97] Everett ET, McHenry MAK, Reynolds N, Eggertsson H, Sullivan J, Kantmann C, et al. Dental fluorosis: Variability among different inbred mouse strains. Journal of Dental Research. 2002;**81**:794-798. DOI:

[98] Zeng BB, Zhang YF, Xia M, et al. Comparison of dental fluorosis susceptibility between SD rats and Wistar rats. Journal of Environment & Health. 2015;**32**:867-871 (In Chinese)

[99] Huang H, Ba Y, Cui L, Cheng X, Zhu J, Zhang Y, et al. COL1A2 gene polymorphisms (Pvu II and Rsa I), serum calciotropic hormone

[100] Jarquín-Yñezá L, Alegría-Torres JA, Castillo CG, de Jesús Mejía-Saavedra J. Dental fluorosis and a polymorphism in the COL1A2 gene in Mexican children. Archives of Oral Biology. 2018;**96**:21-25. DOI: 10.1016/j.

[101] Ba Y, Zhang H, Wang G, Wen S, Yang Y, Zhu J, et al. Association of dental fluorosis with polymorphisms of estrogen receptor gene in Chinese children. Biological Trace Element Research. 2011;**143**:87-96. DOI: 10.1007/

[102] Küchler EC, Dea Bruzamolin C, Ayumi Omori M, Costa MC, Antunes LS, Pecharki GD, et al. Polymorphisms

levels, and dental fluorosis. Community Dentistry and Oral Epidemiology. 2008;**36**:517-522. DOI: 10.1111/j.1600-0528.2007.00424

archoralbio.2018.08.010

s12011-010-8848-1

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

potentiates a K (+)-selective ion channel in G292 osteoblastic cells. The Journal of Membrane Biology. 1996;**149**:211-219

[89] Sun Z, Wei R, Luo G, Niu R, Wang J. Proteomic identification of sperm from mice exposed to sodium fluoride. Chemosphere. 2018;**207**:676-681. DOI: 10.1016/j.chemosphere.2018.05.153

[90] Sun Z, Zhang W, Li S, Xue X, Niu R, Shi L, et al. Altered miRNAs expression profiling in sperm of mice induced by fluoride. Chemosphere. 2016;**155**:109-114. DOI: 10.1016/j. chemosphere.2016.04.053

[91] Buzalaf CP, Leite ADL, Buzalaf MAR. Fluoride Metabolism. In: Preedy VR, editor. Fluorine: Chemistry, Analysis, Function and Effects. Royal Society of Chemistry; 2015. p. 54-74. DOI: 10.10-39/9781782628507-00054

[92] Niu R, Han H, Sun Z, Zhang Y, Yin W, Wang J, et al. Effects of fluoride exposure on the antioxidative status in the kidneys. Fluoride. 2016;**49**:5-12

[93] Ma Y, Ma Z, Yin S, Yan X, Wang J. Arsenic and fluoride induce apoptosis, inflammation and oxidative stress in cultured human umbilical vein endothelial cells. Chemosphere. 2017;**167**:454-461. DOI: 10.1016/j.

chemosphere.2016.10.025

[94] Dequeker J, Declerck K. Fluor in the treatment of osteoporosis. An overview of thirty years clinical research. Schweizerische Medizinische Wochenschrift. 1993;**123**:2228-2234

[95] Yoder KM, Mabelya L, Robison VA, Dunipace AJ, Brizendine EJ,

[87] Gofa A, Davidson RM. NaF

[88] Wei R, Luo G, Sun Z, Wang S, Wang J. Chronic fluoride exposureinduced testicular toxicity is associated with inflammatory response in mice. Chemosphere. 2016;**153**:419-425. DOI: 10.1016/j.chemosphere.2016.03.045

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

[87] Gofa A, Davidson RM. NaF potentiates a K (+)-selective ion channel in G292 osteoblastic cells. The Journal of Membrane Biology. 1996;**149**:211-219

*Environmental Chemistry and Recent Pollution Control Approaches*

and brain nicotinic receptors of rat offspring with coal burning fluorosis. Neurotoxicology and Teratology. 2010;**32**:536-541. DOI: 10.1016/j.

[80] Bartos M, Gumilar F, Bras C, Gallegos CE, Giannuzzi L, Cancela LM, et al. Neurobehavioural effects of exposure to fluoride in the earliest stages of rat development. Physiology & Behavior. 2015;**147**:205-212. DOI: 10.1016/j.physbeh.2015.04.044

[81] Schulz JA, Lamb AR. The effect of fluorine as sodium fluoride on the growth and reproduction of albino rats. Science. 1925;**61**:93-94. DOI: 10.1126/

[82] Tian Y, Xiao Y, Wang B, Sun C, Tang K, Sun F. Vitamin E and lycopene reduce coal burning fluorosis-induced spermatogenic cell apoptosis via

oxidative stress-mediated JNK and ERK signaling pathways. Bioscience Reports. 2018;**38**:BSR20171003. DOI: 10.1042/

[83] Pati P, Bhunya S. Genotoxic effect of an environmental-pollutant, sodiumfluoride, in mammalian in vivo test system. Caryologia. 1987;**40**:79-87. DOI:

10.1080/00087114.1987.10797811

10.1016/j.fct.2016.05.017

2004;**37**:7-12

[84] Zhang J, Li Z, Qie M, Zheng R, Shetty J, Wang J. Sodium fluoride and sulfur dioxide affected male reproduction by disturbing blood-testis barrier in mice. Food and Chemical Toxicology. 2016;**94**:103-111. DOI:

[85] Inkielewicz I, Krechniak J. Fluoride effects on glutathione peroxidase and lipid peroxidation in rats. Fluoride.

[86] Dhurvey V, Patil V, Thakare M. Effect of sodium fluoride on the structure and function of the thyroid and ovary in albino rats (rattus

Norvegicus). Fluoride. 2017;**50**:235-246

ntt.2010.03.010

science.61.1569.93

BSR20171003

[72] Liu F, Ma J, Zhang H, Liu P, Liu YP, Xing B, et al. Fluoride exposure during development affects both cognition and emotion in mice. Physiology & Behavior. 2014;**124**:1-7. DOI: 10.1016/j.

[73] Zhang Z, Xu X, Shen X, Xu X. Effect

of fluoride exposure on synaptic structure of brain areas related to learning-memory in mice. Wei Sheng Yan Jiu. 1999;**28**:210-212 (In Chinese)

[74] Zhang KL, Lou DD, Guan ZZ. Activation of the AGE/RAGE system in the brains of rats and in SH-SY5Y cells exposed to high level of fluoride might connect to oxidative stress. Neurotoxicology and Teratology. 2015;**48**:49-55. DOI: 10.1016/j.

[75] Yan N, Liu Y, Liu S, Cao S, Wang F, Wang Z, et al. Fluoride-induced neuron apoptosis and expressions of inflammatory factors by activating microglia in rat brain. Molecular

Neurobiology. 2016;**53**:4449-4460. DOI:

[76] Scheff SW, Price DA, Hicks RR, Baldwin SA, Robinson S, Brackney C. Synaptogenesis in the hippocampal CA1 field following traumatic brain injury. Journal of Neurotrauma. 2005;**22**:719- 732. DOI: 10.1089/neu.2005.22.719

[77] Saxena R, Meena B, Chouhan VS, Bhatnagar M, et al. Biochemical changes in brain and other tissues of young adult female mice from fluoride in their drinking water. In: Fluoride (Res Rep November 1, 2). Vol. 39. 2006.

[78] Chirumari K, Reddy PK. Dosedependent effects of fluoride on neurochemical milieu in the hippocampus and neocortex of rat brain. Fluoride. 2007;**40**:101-110

[79] Gui CZ, Ran LY, Li JP, Guan ZZ. Changes of learning and memory ability

10.1007/s12035-015-9380-2

physbeh.20-13.10.027

ntt.2015.01.007

**60**

pp. 280-284

[88] Wei R, Luo G, Sun Z, Wang S, Wang J. Chronic fluoride exposureinduced testicular toxicity is associated with inflammatory response in mice. Chemosphere. 2016;**153**:419-425. DOI: 10.1016/j.chemosphere.2016.03.045

[89] Sun Z, Wei R, Luo G, Niu R, Wang J. Proteomic identification of sperm from mice exposed to sodium fluoride. Chemosphere. 2018;**207**:676-681. DOI: 10.1016/j.chemosphere.2018.05.153

[90] Sun Z, Zhang W, Li S, Xue X, Niu R, Shi L, et al. Altered miRNAs expression profiling in sperm of mice induced by fluoride. Chemosphere. 2016;**155**:109-114. DOI: 10.1016/j. chemosphere.2016.04.053

[91] Buzalaf CP, Leite ADL, Buzalaf MAR. Fluoride Metabolism. In: Preedy VR, editor. Fluorine: Chemistry, Analysis, Function and Effects. Royal Society of Chemistry; 2015. p. 54-74. DOI: 10.10-39/9781782628507-00054

[92] Niu R, Han H, Sun Z, Zhang Y, Yin W, Wang J, et al. Effects of fluoride exposure on the antioxidative status in the kidneys. Fluoride. 2016;**49**:5-12

[93] Ma Y, Ma Z, Yin S, Yan X, Wang J. Arsenic and fluoride induce apoptosis, inflammation and oxidative stress in cultured human umbilical vein endothelial cells. Chemosphere. 2017;**167**:454-461. DOI: 10.1016/j. chemosphere.2016.10.025

[94] Dequeker J, Declerck K. Fluor in the treatment of osteoporosis. An overview of thirty years clinical research. Schweizerische Medizinische Wochenschrift. 1993;**123**:2228-2234

[95] Yoder KM, Mabelya L, Robison VA, Dunipace AJ, Brizendine EJ,

Stookey GK. Severe dental fluorosis in a Tanzanian population consuming water with negligible fluoride concentration. Community Dentistry and Oral Epidemiology. 1998;**26**:382-393

[96] Choubisa SL, Choubisa L, Choubisa DK. Endemic fluorosis in Rajasthan. Indian Journal of Environmental Health. 2001;**43**:177-189

[97] Everett ET, McHenry MAK, Reynolds N, Eggertsson H, Sullivan J, Kantmann C, et al. Dental fluorosis: Variability among different inbred mouse strains. Journal of Dental Research. 2002;**81**:794-798. DOI: 10.1177/0810794

[98] Zeng BB, Zhang YF, Xia M, et al. Comparison of dental fluorosis susceptibility between SD rats and Wistar rats. Journal of Environment & Health. 2015;**32**:867-871 (In Chinese)

[99] Huang H, Ba Y, Cui L, Cheng X, Zhu J, Zhang Y, et al. COL1A2 gene polymorphisms (Pvu II and Rsa I), serum calciotropic hormone levels, and dental fluorosis. Community Dentistry and Oral Epidemiology. 2008;**36**:517-522. DOI: 10.1111/j.1600-0528.2007.00424

[100] Jarquín-Yñezá L, Alegría-Torres JA, Castillo CG, de Jesús Mejía-Saavedra J. Dental fluorosis and a polymorphism in the COL1A2 gene in Mexican children. Archives of Oral Biology. 2018;**96**:21-25. DOI: 10.1016/j. archoralbio.2018.08.010

[101] Ba Y, Zhang H, Wang G, Wen S, Yang Y, Zhu J, et al. Association of dental fluorosis with polymorphisms of estrogen receptor gene in Chinese children. Biological Trace Element Research. 2011;**143**:87-96. DOI: 10.1007/ s12011-010-8848-1

[102] Küchler EC, Dea Bruzamolin C, Ayumi Omori M, Costa MC, Antunes LS, Pecharki GD, et al. Polymorphisms in nonamelogenin enamel matrix genes are associated with dental fluorosis. Caries Research. 2018;**52**:1-6. DOI: 10.1159/000479826

[103] Küchler EC, Tannure PN, de ODSB, Charone S, Nelson-Filho P, da Silva RAB, et al. Polymorphisms in genes involved in enamel development are associated with dental fluorosis. Archives of Oral Biology. 2017;**76**:66-69. DOI: 10.1016/j. archoralbio.2017.01.009

[104] Yang Y, Zhao Q , Liu Y, Liu X, Chu Y, Yan H, et al. FRZB1 rs2242070 polymorphisms is associated with brick tea type skeletal fluorosis in Kazakhs, but not in Tibetans, China. Archives of Toxicology. 2018;**92**:2217-2225. DOI: 10.1007/s00204-018-2217-9

[105] Escobar-García D, Mejía-Saavedra J, Jarquín-Yáñez L, Molina-Frechero N, Pozos-Guillén A. Collagenase 1A2 (COL1A2) gene A/C polymorphism in relation to severity of dental fluorosis. Community Dentistry and Oral Epidemiology. 2016;**44**:162-168. DOI: 10.1111/cdoe.12201

[106] Wu J, Wang W, Liu Y, Sun J, Ye Y, Li B, et al. Modifying role of GSTP1 polymorphism on the association between tea fluoride exposure and the brick-tea type fluorosis. PLoS One. 2015;**10**:e0128280. DOI: 10.1371/journal. pone.0128280

[107] Li BY, Yang YM, Liu Y, Sun J, Ye Y, Liu XN, et al. Prolactin rs1341239 T allele may have protective role against the brick tea type skeletal fluorosis. PLoS One. 2017;**12**:e0171011. DOI: 10.1371/journal.pone.0171011

[108] Pei J, Li B, Liu Y, Liu X, Li M, Chu Y, et al. Matrix metallopeptidase-2 gene rs2287074 polymorphism is associated with brick tea skeletal fluorosis in Tibetans and Kazaks, China. Scientific Reports. 2017;**7**:40086. DOI: 10.1038/ srep40086

[109] Chouhan S, Flora SJS. Arsenic and fluoride: Two major ground water pollutants. Indian Journal of Experimental Biology. 2010;**48**:666-678

[110] Wang GQ , Huang YZ, Xiao BY, Qian XC, Yao H, Hu Y, et al. Toxicity from water containing arsenic and fluoride in Xinjiang. Fluoride. 1997;**30**:81-84

[111] González-Horta C, Ballinas-Casarrubias L, Sánchez-Ramírez B, Ishida MC, Barrera-Hernández A, Gutiérrez-Torres D, et al. A concurrent exposure to arsenic and fluoride from drinking water in Chihuahua, Mexico. International Journal of Environmental Research and Public Health. 2015;**12**:4587-4601. DOI: 10.3390/ ijerph120504587

[112] Lin G, Gong S, Wei C, Chen J, Golka K, Shen J. Co-occurrence of arseniasis and fluorosis due to indoor combustion of high fluorine and arsenic content coal in a rural township in Northwest China: Epidemiological and toxicological aspects. Archives of Toxicology. 2012;**86**:839-847. DOI: 10.1007/s00204-011-0792-0

[113] An D, Ho GY, Hu XQ. Chronic arsenic-fluorine intoxication from burning coals with high arsenic and fluorine content. Chinese Journal of Preventive Medicine. 1994;**28**:312-313 (In Chinese)

[114] Zeng Q , Xu Y, Yu X, Yang J, Hong F, Zhang A. The combined effects of fluorine and arsenic on renal function in a Chinese population. Toxicology Research. 2014;**3**:359-366. DOI: 10.1039/ C4TX00038B

[115] Zeng Q , Xu Y, Yu X, Yang J, Hong F, Zhang A. Arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway. Environmental Toxicology and Pharmacology. 2014;**37**:228-233. DOI: 10.1016/j.etap.2013.11.027

**63**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

[124] Yang X, Huo H, Xiu C, Song M, Han Y, Li Y, et al. Inhibition of osteoblast differentiation by aluminum trichloride exposure is associated with inhibition of BMP-2/Smad pathway component expression. Food and Chemical Toxicology. 2016;**97**:120-126.

DOI: 10.1016/j.fct.2016.09.004

Communications in Molecular Pathology and Pharmacology.

[126] Zhang H, Wei Y, Xie C, et al. Experimental study on the relationship between fluorosis and different dosage proportion of fluorine to aluminum. Chinese Jouranl of Endemiology.

[127] Lubkowska A, Chlubek D, Machoy-Mokrzyniska A. The effect of alternating administration of

Medicae Stetinensis. 2006;**52**

[128] Guo X, Cai R, Wu S, He Y, Sun G. Combined effect of fluoride and aluminum on the expression of Runx2 and Osterix mRNA in MC3T3-E1 cells. Wei Sheng Yan Jiu.

[129] Lubkowska A, Zyluk B, Chlubeka D. Interactions between fluorine and aluminum. Fluoride. 2002;**35**:73-77

[130] Ghorbel I, Amara IB, Ktari N, Elwej A, Boudawara O, Boudawara T, et al. Aluminium and acrylamide disrupt cerebellum redox states, cholinergic function and membranebound ATPase in adult rats and their offspring. Biological Trace Element Research. 2016;**174**:335-346. DOI: 10.1007/s12011-016-0716-1

aluminum chloride and sodium fluoride in drinking water on the concentration of fluoride in serum and its content in bones of rats. Annales Academiae

1996;**91**:225-231

2001;**20**:426-428

(Suppl 1):67-71

2011;**40**:164-166

[125] Allain P, Gauchard F, Krari N. Enhancement of aluminum digestive absorption by fluoride in rats. Research

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

[117] Ma Y, Niu R, Sun Z, Wang J, Luo G, Zhang J, et al. Inflammatory responses induced by fluoride and arsenic at toxic concentration in rabbit aorta. Archives

[118] Jiang S, Su J, Yao S, Zhang Y, Cao F, Wang F, et al. Fluoride and arsenic exposure impairs learning and memory and decreases mGluR5 expression in the hippocampus and cortex in rats. PLoS One. 2014;**9**:e96041. DOI: 10.1371/

[119] Li FC. Report on disease caused by kaolin mixed with coal for roasting corn. Chinese Journal of Preventive Medicine. 1988;**22**:225-229 (In Chinese)

[120] Wong MH, Fung KF, Carr HP. Aluminium and fluoride contents of tea, with emphasis on brick tea and their health implications. Toxicology Letters.

[121] Zhu Y, Xu F, Yan X, Miao L, Li H, Hu C, et al. The suppressive effects of aluminum chloride on the osteoblasts function. Environmental Toxicology and Pharmacology. 2016;**48**:125-129. DOI: 10.1016/j.

[122] Sun X, Liu J, Zhuang C, Yang X, Han Y, Shao B, et al. Aluminum trichloride induces bone impairment through TGF-β1/Smad signaling pathway. Toxicology. 2016;**371**:49-57. DOI: 10.1016/j.tox.2016.10.002

[123] Sun X, Wang H, Huang W, Yu H, Shen T, Song M, et al. Inhibition of bone formation in rats by aluminum exposure via Wnt/β-catenin pathway. Chemosphere. 2017;**176**:1-7. DOI: 10.1016/j.chemosphere.2017.02.086

[116] Jia L, Jin TY. Combined effect of fluoride and arsenate on gene expression of osteoclast differentiation factor and osteoprotegerin. Biomedical

and Environmental Sciences.

of Toxicology. 2012;**86**:849-856

journal.pone.0096041

2003;**137**:111-120

etap.20-16.10.009

2006;**19**:375-379

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

[116] Jia L, Jin TY. Combined effect of fluoride and arsenate on gene expression of osteoclast differentiation factor and osteoprotegerin. Biomedical and Environmental Sciences. 2006;**19**:375-379

*Environmental Chemistry and Recent Pollution Control Approaches*

[109] Chouhan S, Flora SJS. Arsenic and fluoride: Two major ground water pollutants. Indian Journal of Experimental Biology. 2010;**48**:666-678

[110] Wang GQ , Huang YZ, Xiao BY, Qian XC, Yao H, Hu Y, et al. Toxicity from water containing arsenic and fluoride in Xinjiang. Fluoride.

[111] González-Horta C, Ballinas-Casarrubias L, Sánchez-Ramírez B, Ishida MC, Barrera-Hernández A, Gutiérrez-Torres D, et al. A concurrent exposure to arsenic and fluoride from drinking water in Chihuahua, Mexico. International Journal of Environmental

Research and Public Health. 2015;**12**:4587-4601. DOI: 10.3390/

10.1007/s00204-011-0792-0

[113] An D, Ho GY, Hu XQ. Chronic arsenic-fluorine intoxication from burning coals with high arsenic and fluorine content. Chinese Journal of Preventive Medicine. 1994;**28**:312-313

[114] Zeng Q , Xu Y, Yu X, Yang J, Hong F, Zhang A. The combined effects of fluorine and arsenic on renal function in a Chinese population. Toxicology Research. 2014;**3**:359-366. DOI: 10.1039/

[115] Zeng Q , Xu Y, Yu X, Yang J, Hong F, Zhang A. Arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway. Environmental Toxicology and Pharmacology. 2014;**37**:228-233. DOI:

10.1016/j.etap.2013.11.027

[112] Lin G, Gong S, Wei C, Chen J, Golka K, Shen J. Co-occurrence of arseniasis and fluorosis due to indoor combustion of high fluorine and arsenic content coal in a rural township in Northwest China: Epidemiological and toxicological aspects. Archives of Toxicology. 2012;**86**:839-847. DOI:

ijerph120504587

(In Chinese)

C4TX00038B

1997;**30**:81-84

in nonamelogenin enamel matrix genes are associated with dental fluorosis. Caries Research. 2018;**52**:1-6. DOI:

[103] Küchler EC, Tannure PN, de ODSB, Charone S, Nelson-Filho P, da Silva RAB, et al. Polymorphisms

[104] Yang Y, Zhao Q , Liu Y, Liu X, Chu Y, Yan H, et al. FRZB1 rs2242070 polymorphisms is associated with brick tea type skeletal fluorosis in Kazakhs, but not in Tibetans, China. Archives of Toxicology. 2018;**92**:2217-2225. DOI:

[105] Escobar-García D, Mejía-Saavedra J, Jarquín-Yáñez L, Molina-Frechero N, Pozos-Guillén A. Collagenase 1A2 (COL1A2) gene A/C polymorphism in relation to severity of dental fluorosis. Community Dentistry and Oral Epidemiology. 2016;**44**:162-168. DOI:

[106] Wu J, Wang W, Liu Y, Sun J, Ye Y, Li B, et al. Modifying role of GSTP1 polymorphism on the association between tea fluoride exposure and the brick-tea type fluorosis. PLoS One. 2015;**10**:e0128280. DOI: 10.1371/journal.

[107] Li BY, Yang YM, Liu Y, Sun J, Ye Y, Liu XN, et al. Prolactin rs1341239 T allele may have protective role against the brick tea type skeletal fluorosis. PLoS One. 2017;**12**:e0171011. DOI: 10.1371/journal.pone.0171011

[108] Pei J, Li B, Liu Y, Liu X, Li M, Chu Y, et al. Matrix metallopeptidase-2 gene rs2287074 polymorphism is associated with brick tea skeletal fluorosis in Tibetans and Kazaks, China. Scientific Reports. 2017;**7**:40086. DOI: 10.1038/

in genes involved in enamel development are associated with dental fluorosis. Archives of Oral Biology. 2017;**76**:66-69. DOI: 10.1016/j.

archoralbio.2017.01.009

10.1007/s00204-018-2217-9

10.1111/cdoe.12201

pone.0128280

10.1159/000479826

**62**

srep40086

[117] Ma Y, Niu R, Sun Z, Wang J, Luo G, Zhang J, et al. Inflammatory responses induced by fluoride and arsenic at toxic concentration in rabbit aorta. Archives of Toxicology. 2012;**86**:849-856

[118] Jiang S, Su J, Yao S, Zhang Y, Cao F, Wang F, et al. Fluoride and arsenic exposure impairs learning and memory and decreases mGluR5 expression in the hippocampus and cortex in rats. PLoS One. 2014;**9**:e96041. DOI: 10.1371/ journal.pone.0096041

[119] Li FC. Report on disease caused by kaolin mixed with coal for roasting corn. Chinese Journal of Preventive Medicine. 1988;**22**:225-229 (In Chinese)

[120] Wong MH, Fung KF, Carr HP. Aluminium and fluoride contents of tea, with emphasis on brick tea and their health implications. Toxicology Letters. 2003;**137**:111-120

[121] Zhu Y, Xu F, Yan X, Miao L, Li H, Hu C, et al. The suppressive effects of aluminum chloride on the osteoblasts function. Environmental Toxicology and Pharmacology. 2016;**48**:125-129. DOI: 10.1016/j. etap.20-16.10.009

[122] Sun X, Liu J, Zhuang C, Yang X, Han Y, Shao B, et al. Aluminum trichloride induces bone impairment through TGF-β1/Smad signaling pathway. Toxicology. 2016;**371**:49-57. DOI: 10.1016/j.tox.2016.10.002

[123] Sun X, Wang H, Huang W, Yu H, Shen T, Song M, et al. Inhibition of bone formation in rats by aluminum exposure via Wnt/β-catenin pathway. Chemosphere. 2017;**176**:1-7. DOI: 10.1016/j.chemosphere.2017.02.086

[124] Yang X, Huo H, Xiu C, Song M, Han Y, Li Y, et al. Inhibition of osteoblast differentiation by aluminum trichloride exposure is associated with inhibition of BMP-2/Smad pathway component expression. Food and Chemical Toxicology. 2016;**97**:120-126. DOI: 10.1016/j.fct.2016.09.004

[125] Allain P, Gauchard F, Krari N. Enhancement of aluminum digestive absorption by fluoride in rats. Research Communications in Molecular Pathology and Pharmacology. 1996;**91**:225-231

[126] Zhang H, Wei Y, Xie C, et al. Experimental study on the relationship between fluorosis and different dosage proportion of fluorine to aluminum. Chinese Jouranl of Endemiology. 2001;**20**:426-428

[127] Lubkowska A, Chlubek D, Machoy-Mokrzyniska A. The effect of alternating administration of aluminum chloride and sodium fluoride in drinking water on the concentration of fluoride in serum and its content in bones of rats. Annales Academiae Medicae Stetinensis. 2006;**52** (Suppl 1):67-71

[128] Guo X, Cai R, Wu S, He Y, Sun G. Combined effect of fluoride and aluminum on the expression of Runx2 and Osterix mRNA in MC3T3-E1 cells. Wei Sheng Yan Jiu. 2011;**40**:164-166

[129] Lubkowska A, Zyluk B, Chlubeka D. Interactions between fluorine and aluminum. Fluoride. 2002;**35**:73-77

[130] Ghorbel I, Amara IB, Ktari N, Elwej A, Boudawara O, Boudawara T, et al. Aluminium and acrylamide disrupt cerebellum redox states, cholinergic function and membranebound ATPase in adult rats and their offspring. Biological Trace Element Research. 2016;**174**:335-346. DOI: 10.1007/s12011-016-0716-1

[131] Lubkowska A, Chlubeka D, Machoy-Mokrzynska A, et al. Distribution of fluoride in selected structures of the central nervous system in rats exposed to NaF and AlCl3 in drinking water. Trace Elements & Electrolytes. 2012;**29**:162-171

[132] Akinrinade ID, Memudu AE, Ogundele OM, Ajetunmobi OI. Interplay of glia activation and oxidative stress formation in fluoride and aluminium exposure. Pathophysiology. 2015;**22**:39-48. DOI: 10.1016/j. pathophys.2014.12.001

[133] Akinrinade ID, Memudu AE, Ogundele OM. Fluoride and aluminium disturb neuronal morphology, transport functions, cholinesterase, lysosomal and cell cycle activities. Pathophysiology, the Official Journal of the International Society for Pathophysiology. 2015;**22**:105-115. DOI: 10.1016/j. pathophys.20-15.03.001

[134] Ge QD, Xie C, Zhang H, Tan Y, Wan CW, Wang WJ, et al. Differential expression of miRNAs in the hippocampi of offspring rats exposed to fluorine combined with aluminum during the embryonic stage and into adulthood. Biological Trace Element Research. 2018:1-15. DOI: 10.1007/ s12011-018-1445-4

[135] Ge QD, Tan Y, Luo Y, Wang WJ, Zhang H, Xie C. MiR-132, miR-204 and BDNF-TrkB signaling pathway may be involved in spatial learning and memory impairment of the offspring rats caused by fluorine and aluminum exposure during the embryonic stage and into adulthood. Environmental Toxicology and Pharmacology. 2018;**63**:60-68. DOI: 10.1016/j.etap.2018.08.011

[136] Chinoy NJ, Patel TN. Effects of sodium fluoride and aluminium chloride on ovary and uterus of mice and their reversal by some antidotes. Fluoride. 2001;**34**:9-20

[137] Dong C, Cao J, Cao C, Han Y, Wu S, Wang S, et al. Effects of fluoride and aluminum on expressions of StAR and P450scc of related steroidogenesis in Guinea pigs' testis. Chemosphere. 2016;**147**:345-351. DOI: 10.1016/j. chemosphere.2015.12.064

[138] Wasana HM, Perera GD, De Gunawardena PS, Bandara J. The impact of aluminum, fluoride, and aluminum– fluoride complexes in drinking water on chronic kidney disease. Environmental Science and Pollution Research. 2015;**22**:11001-11009

[139] Dugo G, Mondello L, Costa R, Albergamo A, Gomes T. Potential use of proteomics in shellfish aquaculture: From assessment of environmental toxicity to evaluation of seafood quality and safety. Current Organic Chemistry. 2017;**21**:402-425. DOI: 10.2174/13852728 20666161102121232

[140] Paul D, Kumar A, Gajbhiye A, Santra MK, Srikanth R. Mass spectrometry-based proteomics in molecular diagnostics: Discovery of cancer biomarkers using tissue culture. BioMed Research International. 2013;**2013**:783131. DOI: 10.1155/2013/783131

[141] Xu H, Jing L, Li G-S. Proteomic analysis of osteoblasts exposed to fluoride in vitro. Biological Trace Element Research. 2008;**123**:91-97. DOI: 10.1007/s12011-007-8086-3

[142] Kobayashi CAN, Leite A de L, da Silva TL, dos Santos LD, Nogueira FCS, Santos KS, et al. Proteomic analysis of urine in rats chronically exposed to fluoride. Journal of Biochemical and Molecular Toxicology. 2011;**25**:8-14. DOI: 10.1002/jbt.20353

[143] Carvalho JG, Leite A de L, Peres-Buzalaf C, Salvato F, Labate CA, Everett ET, et al. Renal proteome in mice with different susceptibilities to fluorosis.

**65**

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis*

[150] Darchen A, Sivasankar V, Mamba BB, Narayanasamy R. Treatment of fluorosis disease and prevention of negative effects of fluoride ingestion. In: Sivasankar V, editor. Surface Modified Carbons as Scavengers for Fluoride from Water. Cham: Springer; 2016. pp. 197-210. DOI: 10.1007/978-3-319-40686-2\_10

[151] Sherwood IA. Fluorosis varied treatment options. Journal of

DOI: 10.4103/0972-0707.62631

[153] Levy SM. Review of fluoride exposures and ingestion. Community Dentistry and Oral Epidemiology. 1994;**22**(3):173-180. DOI: 10.1111/ j.1600-0528.1994.tb01836.x

[154] Ministry of Health of the People's Republic of China. Standard of

Population Total Fluoride Intake (WS/T 87-2016) [Internet]. 2016. Available from: http://www.nhfpc.gov.cn/fzs/s785 2d/201606/94e135cb57b1495bb1a16f631

2009;**14**(2):E103-E107

d2265ae.shtml

Conservative Dentistry. 2010;**13**(1):47.

[152] Abanto AJ, Rezende KM, Marocho SM, Cellerti P, Ciamponi AL. Dental fluorosis: Exposure, prevention and management. Medicina Oral, Patología Oral y Cirugía Bucal.

*DOI: http://dx.doi.org/10.5772/intechopen.84548*

PLoS One. 2013;**8**:e53261. DOI: 10.1371/

[144] Leite AL, Lobo JGVM, da Silva Pereira HAB, Fernandes MS, Martini T, Zucki F, et al. Proteomic analysis of gastrocnemius muscle in rats with streptozotocin-induced diabetes and chronically exposed to fluoride. PloS One. 2014;**9**:e106646. DOI: 10.1371/

[145] Kobayashi CAN, Leite AL, Silva TL, Santos LD, Nogueira FCS, Oliveira RC, et al. Proteomic analysis of kidney in rats chronically exposed to fluoride. Chemico-Biological Interactions. 2009;**180**:305-311. DOI: 10.1016/j.

[146] Pan Y, Lü P, Yin L, Chen K, He Y. Effect of fluoride on the proteomic profile of the hippocampus in rats. Zeitschrift fur Naturforschung. C, Journal of Bbiosciences. 2015;**70**:151-157.

[147] Wei Y, Zeng B, Zhang H, Chen C, Wu Y, Wang N, et al. iTRAQ-based proteomics analysis of serum proteins in Wistar rats treated with sodium fluoride: Insight into the potential mechanism and candidate biomarkers of fluorosis. International Journal of Molecular Sciences. 2016;**17**:E1644.

[148] Sakagami H, Sugimoto M, Tanaka S, et al. Metabolomic profiling of sodium fluoride-induced cytotoxicity in an oral squamous cell carcinoma cell line. Metabolomics. 2014;**10**:270- 279. DOI: https://doi.org/10.1007/

DOI: 10.1515/znc-2014-4158

DOI:10.3390/ij-ms17101644

s11306-013-0576-z

[149] Ministry of Health & Family Welfare Government of India. National Programme for Prevention and Control of Fluorosis (NPPCF) [Internet]. 2014. Available from: http://cghealth. nic.in/ehealth/2017/Instructions/ NPPCFnewguidelinebyGOI.pdf

journal.pone.0053261

journal.pone.0106646

cbi.2009.03.009

*Progressive Research in the Molecular Mechanisms of Chronic Fluorosis DOI: http://dx.doi.org/10.5772/intechopen.84548*

PLoS One. 2013;**8**:e53261. DOI: 10.1371/ journal.pone.0053261

*Environmental Chemistry and Recent Pollution Control Approaches*

[137] Dong C, Cao J, Cao C, Han Y, Wu S, Wang S, et al. Effects of fluoride and aluminum on expressions of StAR and P450scc of related steroidogenesis in Guinea pigs' testis. Chemosphere. 2016;**147**:345-351. DOI: 10.1016/j. chemosphere.2015.12.064

[138] Wasana HM, Perera GD, De Gunawardena PS, Bandara J. The impact of aluminum, fluoride, and aluminum– fluoride complexes in drinking water on chronic kidney disease. Environmental Science and Pollution Research.

[139] Dugo G, Mondello L, Costa R, Albergamo A, Gomes T. Potential use of proteomics in shellfish aquaculture: From assessment of environmental toxicity to evaluation of seafood quality and safety. Current Organic Chemistry. 2017;**21**:402-425. DOI: 10.2174/13852728

[140] Paul D, Kumar A, Gajbhiye A, Santra MK, Srikanth R. Mass spectrometry-based proteomics in molecular diagnostics: Discovery of cancer biomarkers using tissue culture. BioMed Research International. 2013;**2013**:783131. DOI:

[141] Xu H, Jing L, Li G-S. Proteomic analysis of osteoblasts exposed to fluoride in vitro. Biological Trace Element Research. 2008;**123**:91-97. DOI:

2015;**22**:11001-11009

20666161102121232

10.1155/2013/783131

10.1002/jbt.20353

10.1007/s12011-007-8086-3

[142] Kobayashi CAN, Leite A de L, da Silva TL, dos Santos LD, Nogueira FCS, Santos KS, et al. Proteomic analysis of urine in rats chronically exposed to fluoride. Journal of Biochemical and Molecular Toxicology. 2011;**25**:8-14. DOI:

[143] Carvalho JG, Leite A de L, Peres-Buzalaf C, Salvato F, Labate CA, Everett ET, et al. Renal proteome in mice with different susceptibilities to fluorosis.

[131] Lubkowska A, Chlubeka D, Machoy-Mokrzynska A, et al. Distribution of fluoride in selected structures of the central nervous system in rats exposed to NaF and AlCl3 in drinking water. Trace Elements & Electrolytes. 2012;**29**:162-171

[132] Akinrinade ID, Memudu AE, Ogundele OM, Ajetunmobi OI.

stress formation in fluoride and

2015;**22**:39-48. DOI: 10.1016/j. pathophys.2014.12.001

[133] Akinrinade ID, Memudu AE, Ogundele OM. Fluoride and aluminium disturb neuronal morphology, transport functions, cholinesterase, lysosomal and cell cycle activities. Pathophysiology, the Official Journal of the International

Society for Pathophysiology. 2015;**22**:105-115. DOI: 10.1016/j.

expression of miRNAs in the

s12011-018-1445-4

10.1016/j.etap.2018.08.011

Fluoride. 2001;**34**:9-20

[136] Chinoy NJ, Patel TN. Effects of sodium fluoride and aluminium chloride on ovary and uterus of mice and their reversal by some antidotes.

[134] Ge QD, Xie C, Zhang H, Tan Y, Wan CW, Wang WJ, et al. Differential

hippocampi of offspring rats exposed to fluorine combined with aluminum during the embryonic stage and into adulthood. Biological Trace Element Research. 2018:1-15. DOI: 10.1007/

[135] Ge QD, Tan Y, Luo Y, Wang WJ, Zhang H, Xie C. MiR-132, miR-204 and BDNF-TrkB signaling pathway may be involved in spatial learning and memory impairment of the offspring rats caused by fluorine and aluminum exposure during the embryonic stage and into adulthood. Environmental Toxicology and Pharmacology. 2018;**63**:60-68. DOI:

pathophys.20-15.03.001

Interplay of glia activation and oxidative

aluminium exposure. Pathophysiology.

**64**

[144] Leite AL, Lobo JGVM, da Silva Pereira HAB, Fernandes MS, Martini T, Zucki F, et al. Proteomic analysis of gastrocnemius muscle in rats with streptozotocin-induced diabetes and chronically exposed to fluoride. PloS One. 2014;**9**:e106646. DOI: 10.1371/ journal.pone.0106646

[145] Kobayashi CAN, Leite AL, Silva TL, Santos LD, Nogueira FCS, Oliveira RC, et al. Proteomic analysis of kidney in rats chronically exposed to fluoride. Chemico-Biological Interactions. 2009;**180**:305-311. DOI: 10.1016/j. cbi.2009.03.009

[146] Pan Y, Lü P, Yin L, Chen K, He Y. Effect of fluoride on the proteomic profile of the hippocampus in rats. Zeitschrift fur Naturforschung. C, Journal of Bbiosciences. 2015;**70**:151-157. DOI: 10.1515/znc-2014-4158

[147] Wei Y, Zeng B, Zhang H, Chen C, Wu Y, Wang N, et al. iTRAQ-based proteomics analysis of serum proteins in Wistar rats treated with sodium fluoride: Insight into the potential mechanism and candidate biomarkers of fluorosis. International Journal of Molecular Sciences. 2016;**17**:E1644. DOI:10.3390/ij-ms17101644

[148] Sakagami H, Sugimoto M, Tanaka S, et al. Metabolomic profiling of sodium fluoride-induced cytotoxicity in an oral squamous cell carcinoma cell line. Metabolomics. 2014;**10**:270- 279. DOI: https://doi.org/10.1007/ s11306-013-0576-z

[149] Ministry of Health & Family Welfare Government of India. National Programme for Prevention and Control of Fluorosis (NPPCF) [Internet]. 2014. Available from: http://cghealth. nic.in/ehealth/2017/Instructions/ NPPCFnewguidelinebyGOI.pdf

[150] Darchen A, Sivasankar V, Mamba BB, Narayanasamy R. Treatment of fluorosis disease and prevention of negative effects of fluoride ingestion. In: Sivasankar V, editor. Surface Modified Carbons as Scavengers for Fluoride from Water. Cham: Springer; 2016. pp. 197-210. DOI: 10.1007/978-3-319-40686-2\_10

[151] Sherwood IA. Fluorosis varied treatment options. Journal of Conservative Dentistry. 2010;**13**(1):47. DOI: 10.4103/0972-0707.62631

[152] Abanto AJ, Rezende KM, Marocho SM, Cellerti P, Ciamponi AL. Dental fluorosis: Exposure, prevention and management. Medicina Oral, Patología Oral y Cirugía Bucal. 2009;**14**(2):E103-E107

[153] Levy SM. Review of fluoride exposures and ingestion. Community Dentistry and Oral Epidemiology. 1994;**22**(3):173-180. DOI: 10.1111/ j.1600-0528.1994.tb01836.x

[154] Ministry of Health of the People's Republic of China. Standard of Population Total Fluoride Intake (WS/T 87-2016) [Internet]. 2016. Available from: http://www.nhfpc.gov.cn/fzs/s785 2d/201606/94e135cb57b1495bb1a16f631 d2265ae.shtml

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Section 2

Pollution Control

Approaches

Section 2
