**2. Fundamentals: physical, chemical, and biological aspects involved in AMD generation**

### **2.1 Some physical aspects**

From a mineral processing standpoint, the bigger the particles or rocks the less the chances of sulphide minerals from getting exposed to the environment. Such exposition is commonly associated with the concept of liberation. The liberation of a specific mineral was originally defined as the particle size threshold allowing the generation of particles composed of only one mineral [18]. From this definition, the liberation of each mineral present in an ore should be different as the presence of each mineral (or occurrence) changes from one to another. Such definition has been modified in time to better explain the efficiency of processes involved in metallurgy or mineral separation stages where the composition of the surfaces of particles is critical for their success [19]. For any geological occurrence of sulphide minerals (or any other mineral) the smaller the particle size the higher the liberation expected. The higher the liberation, the larger the exposure to the environment of surfaces containing such minerals and therefore the higher the chances of producing AMD. Nevertheless, there is in a way a trade-off between AMD flow rates and the potential of producing AMD.

**Figure 3** shows two sets of particles having significant differences in particle diameter. The group of bigger particles will exhibit larger pore sizes although the air hold up may be similar for both case scenarios [20]. The set of particles exhibiting coarser sizes presents higher permeability than that of smaller sizes. Permeability depends on both static and dynamic properties of the porous medium and fluid characteristics. In the case of finer particles, capillary forces are more relevant which allow retaining more volume of the aqueous phase inside the porous material leaving the fluid phase and dissolved species to be transported slowly across through diffusive mechanisms favouring the acidification of the aqueous phase [21]. Coarser particles can produce higher flow rates reducing the residence time of fluid in contact with the surface of the particles. Then, there might be situations where the porous structure is fulfilled or saturated with water and the permeability can be modelled using the Darcy equation, however, for most case scenarios unsaturation would be frequently observed. So, permeability would be better modelled by the Soil-Water Critical Curve (SWCC) curve (**Figure 4**) which can be evaluated using three ranges of pressure values [Eqs. (3)–(5)] [22].

The parameters *wu* and *waev* represent the water content for 1 kPa suction and for air-entry value, respectively. The first section [Eq. (3)] close to the *y*-axis represents

*Fundamentals and Practical Aspects of Acid Mine Drainage Treatment: An Overview from Mine… DOI: http://dx.doi.org/10.5772/intechopen.104507*

#### **Figure 3.**

*The impact of particle size distribution on mineral liberation and on permeability and its association with AMD generation.*

**Figure 4.** *SWCC Curve used to model the water content in an unsaturated porous media.*

the zone where the porous media is fulfilled with the aqueous phase which could represent the case of tailings coming from flotation operations when they have been freshly disposed of.

$$
\omega\_1(\varphi) = w\_u - \mathbb{S}\_1 \log \left( \psi \right) \quad \mathbb{1} \; \; \varphi < \varphi\_{ave} \tag{3}
$$

The second section [Eq. (4)] located immediately to the right-hand side represents the behaviour of the porous media when the air gets inside displacing the water

present in the pores which could correspond to an intermediate case observed in tailings where, by simple syneresis, the water drains across the material.

$$
\omega w\_2(\boldsymbol{\psi}) = \boldsymbol{w}\_{\rm aer} - \mathbb{S}\_2 \log \left(\frac{\boldsymbol{\psi}}{\boldsymbol{\psi}\_{\rm aer}}\right) \quad \boldsymbol{\psi}\_{\rm aer} \quad \boldsymbol{\psi} < \boldsymbol{\psi}\_r \tag{4}
$$

And the last section [Eq. (5)] represents the behaviour of the porous material when it gets dry leaving a certain residual water content, which describes a porous media where humidity is present mainly by wetting the surface of the particles.

$$w\_3(\varphi) = \mathcal{S}\_3 \log \left(\frac{10^6}{\varphi}\right) \quad \psi\_r \text{ } \psi < 10^6 kPa \tag{5}$$

Eq. (5) corresponds to later stages of tailings, the case of larger particles still retaining some water content (i.e., material coming from dump leaching or ore sorting operations) or abandoned tailings. From a thermodynamic standpoint, the suction is a function of the partial pressure of the pore-water vapour and the density of the vapour which depends on the temperature and can be computed as in Eq. (6).

$$\Psi = (pore-air\ pressure) - (pore-water\ pressure) + \text{osmotic}\tag{6}$$

And the permeability at residual water conditions can be computed from the matric (soil) suction as Eq. (7) [22].

$$k\_w = \frac{k\_s}{\left[\frac{u\_a - u\_b}{(u\_a - u\_b)\_b}\right]^{n'} + \mathbf{1}} \tag{7}$$

One of the strong capabilities of Eq. (7) is that it correctly describes porous media with small particle sizes exhibiting lower permeability and higher water retention capacity [22, 23]. The latter would reduce the chances of producing large flow rates of AMD under these conditions [20]. Large flowrates would only then be possible in these conditions when water flows over the external surface of the piled-up porous material which reduces radically the exposed surface of the particles to the aqueous phase flowing around. Certainly, the magnitude of the drying conditions will depend on the water table present in each system. As it can be observed such description is based on semi-empirical mathematical models which is an indication that this is still a quite fruitful field of research.

Finally, the transport of liquid in porous materials built up of smaller particle sizes will expose the sulphide minerals in greater extension but they usually exhibit a higher hydrophobicity due to the presence of sulphur produced by oxidation reactions. This is known as natural hydrophobicity which occurs with much lower significance in the case of mineral oxides with stronger wettability properties. The liquid phase transferred through the porous medium needs to fill the voids displacing the air. Such subprocess is commonly referred to as imbibition and will be inhibited by the presence of sulphide hydrophobic surfaces which provide a first glance of how physics and chemistry are linked in these systems, but it is usually not considered. Indeed, physics and chemistry are frequently addressed by researchers separately. The relevancy of the chemistry and biology behind this process will be examined in the next subchapter.

*Fundamentals and Practical Aspects of Acid Mine Drainage Treatment: An Overview from Mine… DOI: http://dx.doi.org/10.5772/intechopen.104507*

### **2.2 Some chemical and biological aspects**

There are several documents describing how sulphide minerals produce the so-called AMD, and the reader could refer to them for more information [4, 24]. Gas-solid and liquid-solid interactions are the major ones responsible for the significant differences between the chemical composition and structure of the bulk of the solid phase and the outmost surface layer arising from such interaction [25]. There are specific minerals that due to their instability under aerated conditions, notably metal sulphide minerals, are likely to produce enough acidity to stabilise several metals in dissolved state. Firstly, metal sulphide minerals would directly produce hydronium ions from their oxidation produced by the oxygen present in the atmosphere [Eq. (8)].

$$\text{MS}\_{m(s)} + \text{O}\_{2(g)} + (m - n)\text{H}\_2\text{O}\_{(l)} \leftrightarrow \text{M}^{n+}\_{(aq)} + m\text{SO}\_4^{2-} + (m - n)\text{H}\_3\text{O}^+\_{(aq)}\tag{8}$$

Such acidity is then enhanced by metal hydrolysis reactions occurring at the bulk of the aqueous phase. Hydrolysis can be represented by Eq. (9).

$$\mathrm{M}\_{(aq)}^{n+} + m\mathrm{H}\_{2}\mathrm{O}\_{(l)} \leftrightarrow \mathrm{M}(\mathrm{OH})\_{\mathrm{T}^{(l)}}^{\left(n-\frac{m}{2}\right)} + \frac{m}{2}\mathrm{H}\_{3}\mathrm{O}^{+}\_{(aq)}\tag{9}$$

where the variable *m* ¼ 0, 2, 4, 6, 8,*etc:* Depending on the concentration, *i* may refer to a solid phase for *n* ¼ *m* leading to the precipitation of the metal hydroxide. If the dissolved metal is polyvalent, it may undergo subsequent oxidation stages due to the presence of dissolved oxygen in the system. The latter may raise other stronger mechanisms of oxidation of the sulphide minerals. That is the case of dissolved iron which can go from Fe(II) to Fe(III) in acidic aqueous solutions due to dissolved oxygen reduction. The latter increases the rate at which the metal sulphide dissolves producing more acidity simultaneously rising the concentration of sulphate ions in solution.

### **2.3 Application to pyrite and marcasite (***FeS***2)**

Probably the most reported case study that exemplifies AMD generation is that of pyrite and marcasite (*FeS*2) which encompasses a series of processes that up to now are not fully understood, especially with regards to the state of surface or surface mediator being formed [26].

In any case, the oxidation reaction is usually described as Eq. (10).

$$\text{FeS}\_{2(s)} + \text{Ox}\_{(aq)} + 2\text{H}\_2\text{O}\_{(l)} \leftrightarrow \text{Fe}^{2+}\_{(aq)} + \text{H}\_3\text{O}^+\_{(aq)} + \text{SO}^{2-}\_{4(aq)}\tag{10}$$

In Eq. (10) the oxidant, represented by the symbol *Ox*, can be oxygen or ferric ions if the thermodynamic potentials are suitable. The *FeS*2ð Þ*s* in the reactants is only an over-simplification of what is really happening. Indeed, Holmes and Crundwell (2000) succeed in describing the oxidation of pyrite using the classic mixed potential theory but claimed that the state of surface also plays a relevant role in the process and is still not well understood [27]. **Figure 5** attempts to summarise the kinetics of the major electrochemical reactions taking place in the system. The oxidation reactions of pyrite in presence of sulphate ions start at potentials about 0.54 V vs SHE, which as mentioned later in this text becomes wider in presence of chloride ions. The oxygen reduction reaction in acidic conditions exhibits a Nernst potential of about 1.23 V vs SHE. Such reduction reaction is known to be from an electrochemical point of view

### **Figure 5.**

*Electrochemical scheme of the I vs E curve presenting the most relevant electrochemical reactions as well as the mixed potentials.*

rather irreversible, so currents are only observed below 0.61 V vs SHE which is explained by the authors in terms of the low conductivity n-type pyrite semiconductor properties [28]. The latter is not observed in **Figure 5**. Instead, due to the significant reduction in overvoltage of the oxygen reduction, the current density would reach a maximum value of about 38*:*5 *mAcm*2 considering a layer thickness of 0.05 cm, a concentration of 8 *mgL*1 , and a diffusion coefficient of the unstirred aqueous phase of 2 105 *cm*2*s* 1. The current density observed by the authors is about 2% or that maximum value which is an indication that the diffusion layer thickness is much higher than that assumed. This only allows an exiguous net current that can be barely detected (in fact, in **Figure 5** it was enhanced for the reader to be able to see it!) at a mixed potential labelled as (1), which is controlled by the cathodic reaction [27]. The low current is also obtained in the situation presented by the authors where both cathodic and anodic reactions exhibit a thermodynamic potential close to each other. In fact, under these conditions, the reversibility of both reactions needs to be high to attain any relevant reaction rates. Other authors have indicated that the oxidation potential in presence of chloride ions, especially relevant in mineral processes implemented using seawater, ranges between 70 V and 530 V vs SHE [29]. Strikingly, under such a range of potentials, a series of reversible electrochemical adsorption/desorption steps take place whenever sweep rates of30 *mVs*1 or higher is used. At sweep rates below 10 *mVs*1 the authors found that oxidation steps are triggered indicating that precursors need time to be formed to undertake oxidation reactions. Under these experimental conditions, the pyrite oxidation takes place as a sequence of electron losses promoting the formation of several sulphite-like precursors occurring at both solid surface and bulk of aqueous phase.

Once ferric ions are formed in the aqueous phase, the oxidation rate of pyrite increases significantly not only because of the increase of the anodic overvoltage but also because of the high reversibility of the reduction reaction of ferric ions exhibits. Indeed, the mixed potential moves to higher voltages represented as (2) in **Figure 5**. Then, an accumulation of ferrous ions in solution may arise. The chances of

*Fundamentals and Practical Aspects of Acid Mine Drainage Treatment: An Overview from Mine… DOI: http://dx.doi.org/10.5772/intechopen.104507*

regenerating the oxidant only by introducing oxygen would not be enough since the latter reaction still is mass transfer controlled. This is one of the most crucial issues the leaching of copper sulphide minerals presents which has been partially solved by microorganisms. In effect, it has been proved that bacteria, specifically, *thiobacillus ferroxidans*, would catalyse this reaction [30]. For many years it was not clear whether the catalysis is achieved by direct bacteria adsorption and oxidation of the mineral sulphide surface or indirect reaction through oxidation of ferrous ions happening at the bulk of the aqueous phase. Nowadays, such matter has been sorted out and it is known that it is the indirect oxidation mechanism that governs the oxidation of ferrous ions to ferric ions, and it constitutes the foundations of bioleaching of sulphide minerals [31]. During such studies on bioleaching, it has been learnt that the *thiobacillus ferroxidans* have a relatively narrow pH range in which they may adapt at their best and the presence of chloride ions at 2M or higher inhibits its growth [32].

**Figure 6** summarises the main role of the two major types of bacteria, *thiobacillus ferrooxidans* and *thiobacillus thiooxidans*. Eqs. (10) and (11) are commonly coupled and a global reaction is obtained indicating how oxygen can oxidise elemental sulphur. These two reactions may also be studied separately. Dissolved oxygen oxidises a number of reduced chemical species such as Fe(II) or any other sulphide more susceptible to be oxidised than pyrite while sulphur can be at least oxidised by Fe(III). A simple stoichiometric analysis of both routes of reaction, without considering the cycle Fe(II)/Fe(III), indicates the simultaneous oxidation of 1 mol of elemental sulphur and the reduction of 2 mols of molecular oxygen would neutralise the local pH. In acidic aqueous solutions (as it may happen with AMD) it would be desirable to have more than 2 mols of oxygen reacting per mol of sulphur being oxidise. This rather simplistic analysis attempts to prove that the generation of AMD is not the inevitable outcome coming from these systems. Understanding and tuning the relevancy of the reactions at a fundamental level may also lead to inhibiting its formation.

$$\text{O}\_{2(g)} + 4\text{H}\_3\text{O}^+\_{(aq)} + 4e^- \to \text{6H}\_2\text{O}\_{(l)}\tag{11}$$

$$\text{SO}^{0}\_{(s)} + \text{12}H\_{2}\text{O}\_{(l)} \rightarrow \text{SO}^{2-}\_{4(aq)} + \text{8H}\_{3}\text{O}^{+}\_{(aq)} + \text{6e}^{-}\tag{12}$$

**Figure 6.** *Conceptual simplified model for generation of AMD.*

Another more realistic analysis would involve coupling these two reactions. In this case, 2 mols of elemental sulphur would react with 3 mols of molecular oxygen producing 4 mols of hydronium ions and 2 mols of sulphate which is also troublesome for AMD (to be discussed in Section 5). Simultaneous oxidation of 12 mols of ferrous ions using 3 mols of molecular oxygen would then neutralise the acidity provided by the overall sulphur oxidation reaction by oxygen.

Supposedly, in real systems oxygen is slowly transferred to surface sites inside the porous media where the interaction with sulphide minerals exposed would produce elemental sulphur, one of the most relevant products generated at the surface of the particles, which then is oxidized due to the presence of microorganisms. Additionally, bacteria require oxygen and eventually carbon dioxide for growth, which is also responsible for producing both sulphur and acid [33]. It looks like the key would lie in inhibiting the formation of elemental sulphur which is quite insoluble (about 0*:*6 *ngL*1 ) [34]. Nevertheless, the role of sulphur though is much more complicated than just the formation of sulphate ions [29]. Research studies have proved that thiosulphate would be an intermediate species that in acid media would be disproportionate to sulphite or sulphur dioxide and elemental sulphur, going back to the initial state. Then, handling better the presence of sulphite or sulphur dioxide then might be crucial. Thermodynamically, elemental sulphur could be avoided but to do that an increase in temperature or the reduction of total amount of sulphur in the system is needed which is something not easy to accomplish considering the scale at these systems are commonly implemented [35].

From all the above, it may infer that the chances of producing AMD cannot only be observed from a fluid dynamic or physical perspective. The gathering of key reactants needs to occur to produce it. Delays in the interaction between reactants given by the transport of oxygen, or carbon dioxide in less importance, will slow down the generation of acidity and therefore that of AMD.

It is precise because of this that many of the strategies to prevent AMD obey to block the reagents from coming into the porous material (**Figure 7**). For instance, with regards to the sulphide minerals present in the porous material authors have suggested removing it before piling up the solid wastes using froth flotation or any other selective separation

**Figure 7.**

*Scheme of various strategies to prevent or treat AMD [36–55].*

### *Fundamentals and Practical Aspects of Acid Mine Drainage Treatment: An Overview from Mine… DOI: http://dx.doi.org/10.5772/intechopen.104507*

method [36]. Bear in mind that this requires preparing the material for the separation process such as milling to certain particle diameter and the use of appropriate reagents at certain dosages to run froth flotation operations adequately. Another option would be to cover up only the surface of the particles exposing the sulphide mineral to the gaseous phase to avoid any contact with oxygen inhibiting the AMD formation [40]. A similar but more extensive blockage would entail forming a cap enclosing the whole porous material preventing oxygen from entering the system [41]. Even more, some researchers have suggested using materials known as not acid producers (or NAP) and directly carrying out some neutralisation of the AMD. It also has been suggested to introduce some positive pressure on inert gases to keep the oxygen from entering the system [42]. From the use of water, perhaps the most studied strategy for suspensions of mineral particles involves reducing the water content of slurries to dose lower amounts of neutralisation reagents [43]. And finally, with respect to the microorganisms, it has been recommended the use of some bactericides to prevent the acid-forming bacteria to appear in the system [44] while other authors have focused their attention in using some bacteria growth inhibitors [45].

However, having implemented any of these paths to prevent AMD from appearing does not secure that it will not take place and if it occurs, several actions have been studied to deal with it. Furthermore, in many cases, these solid wastes are not appropriately disposed of over geomembranes or other impervious materials which forces continuously monitor surface- and ground-waters at the mining location outskirts. Whenever these waters acquire any properties resembling AMD, the wastewater treatment must act as a barrier to bringing the parameters of water quality back to their usual values. Even using such geomembranes does not ensure that the eventual AMD produced will be appropriately contained as the properties of these covers may also detrimentally evolve in time [56].

### **3. AMD in mine closure conditions**

Mine closure is one of the subjects in the field of mine management that has gained notoriety over the last few decades [57]. The people's perspective of mining activities is negative when abandoned mine sites impact adversely the environment or the nearby communities [58]. **Figure 8A** presents the iconic case of the Grand Canyon, in the United States, where mining activities were developed between 1957 to early 1960s. It can be observed that a section of the original plant is still in place and the exploitation has increased the surface area exposed to the atmosphere. In the same picture the before and after of a gold exploitation mine site located in the northern part of Chile shows a similar situation. In this case, the open-pit mine, unfortunately, made the entire town of *Churrumata* move without procuring better conditions for the villagers (**Figure 8B**).

All mine sites have a definite lifetime. At the end of the mining exploitation, the site needs to be rehabilitated, ideally eluding any threat to the environment, or living organisms, vegetation, and nearby communities.

Behind this topic there are many concepts to address and it is difficult to summarise them in just a few lines. For instance, researchers have differentiated the terms mine closure and mine completion [59]. On one hand, mine closure is a procedure over a timespan where plant operation stops, and decommissioning is undertaken. On the other hand, mine completion refers to an aim of mine closure where the ownership is renounced by the mining lease and accepted by the next user of the land for a different purpose. These and other perspectives are still an ongoing theme for the whole society.

### **Figure 8.**

Despite the latter, for this quest to be successful, every government has stated a plan which considers not only technical requirements but also regulatory and legislation guidelines to prevent the sites to become hazardous reducing eventual further contamination in many years to come.

It was not necessary to go by a great deal of time before governments, mining companies and the whole society realised that the major threat behind the cease of a mining operation is the lack of planning. There are many reasons why mine sites close such as economics, geological, technical, regulatory, policy changes, social pressure, end of markets, etc. Therefore, it is not surprising that this stage of mine development requires a multidisciplinary set of actions. For instance, the actions relevant to the present subject might include:

i. Focused brainstorming aims at identifying environmental values, gains or losses inherent to AMD.

As the Mine Closure conditions refer to the situation where the mining activities cease to take place, it is expected that energy, material, or personnel are not going to be available to deal with AMD. Therefore, unassisted wastewater treatment needs to be implemented.


**Acknowledgments:** Conversations with Arnie Bendich about organelle DNA replication and repair in meristem and vegetative cells were interesting and illuminating. We are grateful to Emily Jezewski for finding time in her busy golf schedule to do some of the qPCR experiments. Beth Rowan provided advice on Illumina library preparations from Arabidopsis leaves and many insights and helpful conversations about plant mitochondrial genomes. Daniel Sloan also provided insights and useful discussions. We thank Christian Elowski and the Nebraska Center for Biotechnology Core Research Facility for Microscopy for confocal fluorescent microscopy. This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant. Daniel Schachtman helped with disposal of leaf tissues from generations 2–9. The use of product and company names is necessary to accurately report the methods and results; however, the United States Department of Agriculture (USDA) neither guarantees nor warrants the standard of the products, and the use of names by the USDA implies no approval of the product to the exclusion of others that may also be suitable. The USDA is an equal opportunity provider and employer.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**7.6 With or without 'reactive oxygen species (ROS) and Antioxidant dynamics'**

A 'two-hit' theory has been postulated to help explain the mechanisms underlying the development of advanced NAFLD. The 'second hit' has yet to be completely described; extensive research has identified several possible mechanisms, including oxidative stress (OS)-induced inflammation with lipid peroxidation, cytokine activation and excess production of reactive oxygen and nitrogen species (ROS/RNS) [124].

The main source of radicals in biological systems is molecular oxygen, which readily accepts electrons, the most important of which being the hydroxyl radical (•OH), the superoxide anion (O2 •−) and nitric oxide radical (NO•). These unstable and reactive radicals are natural by-products of the intracellular metabolism and from exogenous substances, which have the ability to react with biological compounds including proteins, FFA and DNA [136, 137]. On the other hand, the main endogenous intracellular sources of ROS are mitochondria, the endoplasmic reticulum (ER) and peroxisomes, superoxide anion radicals (O2 •−) are produced because of enzymatic activity, such as with xanthine oxidase (XO) and cytochrome P450 metabolism [107, 138].

In a normal situation, a fine balance exists between prooxidant and antioxidant mechanisms, and OS, which has been long recognised as a key mechanism responsible for liver damage and disease progression in NAFLD, is believed to occur due to an imbalance in favour of prooxidation [139]. Numerous pieces of evidence accumulated over the past decade suggest that mitochondrial dysfunction plays a significant role in steatosis and steatohepatitis. ROS overproduction is induced by mitochondrial dysfunction, and the ensuing increase in the lipid peroxidation and protein oxidation has a detrimental effect on fat homeostasis in the liver. Mitochondria remain the main source of ROS in hepatocytes, although other subcellular organelles have also been shown to participate in the process [140, 141].

As a matter of fact, peroxisomes can oxidise long-chain FFA more rapidly than mitochondria, thereby increasing the cell's capacity to metabolise FFA. However, H2O2, which is an end-product of peroxisomal β-oxidation, is converted into the highly reactive OH radical with ease. By promoting toxic accumulation of ROS, which *Regulation of Iron Metabolism in NAFLD/NASH DOI: http://dx.doi.org/10.5772/intechopen.107221*

triggers other signalling pathways within the cell, chronic ER stress may also contribute to OS. The relationship between ER stress and OS works both ways because ROS generated through inflammation or damage to organelles (e.g., mitochondria) may also accelerate ER dysfunction [140, 141].

Because of either excessive production of ROS within the hepatocyte or reduced antioxidant defences, oxidative stress occurs and accumulates within the hepatocytes. Most antioxidant enzymes, copper/zinc superoxide dismutase (Cu/Zn SOD) and manganese-superoxide dismutase (MnSOD), which are mainly present in the cytoplasm and mitochondria, promote the reduction of O2 •− to H2O2. Another antioxidant enzyme, glutathione peroxidase (GPx), facilitates the subsequent conversion of H2O2 to H2O [137]. In correlation with disease severity, a breakdown in the antioxidant defences plays a significant role in OS associated with NASH, as evidenced by decreased hepatic glutathione (GSH) and diminished SOD, GPx, catalase and glutathione transferase activities [107].

Lipid peroxidation to release more reactive aldehydes is augmented by the resultant increase in mitochondrial ROS, which further damages the mitochondrial DNA (mtDNA) and respiratory chain polypeptides [142].

In summary, mitochondrial dysfunction not only impairs fat homeostasis in the liver but also leads to an overproduction of ROS, which is deliberated to be an important factor in producing lethal hepatocyte injury associated with NAFLD [107].


**2. Factors that affect nutritional diagnosis**

It is important to know the main factors that interfere in the diagnosis of plant nutritional status, so that confident analytic results are obtained and compared to pre-established litera‐ ture standards. The composition of the vegetal tissue reflects the interaction of factors acting up to the moment the samples are collected for analysis.

Initially, it is necessary to exclude biotic and abiotic factors that affect nutrient concentra‐ tions in plants. Among these should be considered, lack or excess of water, high or low tem‐ peratures winds, pests and illnesses, compacted or poorly plowed soils, mechanical damage and herbicide toxicity [1]. These and other factors may produce deficiency symptoms in the plant by preventing absorption and/or translocation of nutrients. In such cases symptoms of deficiency are only eliminated by removing the stress factors.

Table 1 shows some of the factors directly involved in the appearance of real or apparent symptoms, which are similar and confound typical deficiency and toxic patterns.

**Table 1.** Effects of biotic and abiotic factors, that may directly or indirectly induce typical deficiency or toxicity patterns. (Adapted from [1]).

To exclude the factors in Table 1, the professional must know the interactions "plant-envi‐ ronment-soil – farming activity" before proceeding to sampling.


**Placental Related Disorders of Pregnancy**

Editors **Hiten D. Mistry Eun Lee**

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*Editors* Hiten D. Mistry Department of Women and Children's Health, School of Life Course and Population Health Sciences King's College London London United Kingdom

Eun Lee Department of Obstetrics and Gynecology Virginia Commonwealth University Health System Richmond, VA United States

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This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: www.mdpi.com/journal/ ijms/special issues/Placental Pregnancy).

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