Contents

#### **Preface XI**

#### **Section 3 Climate Change and Reconstruction of Natural Hazard Events 85**

Chapter 6 **Towards the Reduction of Vulnerabilities and Risks of Climate Change in the Community-Based Tourism, Namibia 87** Selma Lendelvo, Margaret N. Angula, Immaculate Mogotsi and Karl Aribeb

Preface

ronmental damage that they cause.

ous rainfall conditions, for example.

EEA member countries.

their resilience.

and reduce vulnerability; and for that there are no borders.

world should prepare for greater losses in the future.

At a time when human and material losses caused by natural disasters, such as earthquakes, tsunamis, volcanic eruptions, landslides, floods, and wildfires, are intensifying throughout the world, it is imperative to evaluate these events to mitigate the social, economic and envi‐

It is also of paramount importance to raise awareness of hazards, develop possible actions, procedures, and measures to curb the growing number of increasingly devastating events,

It is something to bear in mind that natural hazards occur in all parts of the world across different time and area scales, although some regions are more vulnerable to certain hazards than others. Earthquakes, tsunamis, flash floods, landslides, and avalanches are short-lived, violent events, affecting in general relatively small regions. Others, such as prolonged rain‐ fall, lead to floods lasting for weeks or months. Still others, such as droughts, develop slow‐ ly, but can affect entire countries for months or even years. In addition, a single-type disaster may develop into a compound disaster, as may be the case under extreme continu‐

According to the European Environment Agency (EEA) Technical Report No. 13/2010, be‐ tween 1998 and 2009 a total of 928 natural hazards and technological accidents recorded in Europe caused nearly 100,000 fatalities and, overall, a loss of about EUR 150 billion in the 32

Based on data from the Swisse Re Institute (http://www.sigma-explorer.com/) on global dis‐ asters from 1970 to 2017, collected in May 2018, the following graphs show the increase in the number of events and growth in economic loss. It seems clear from these trends that the

In addition to the increase in the number of disasters and economic loss, the growing trend in greenhouse gas emissions (to say nothing of its current value) and the consequent in‐ crease in the global temperature of our planet make it difficult to reverse the trends shown in those graphs, as many studies have shown. We must also keep in mind that human liveli‐ hoods will be affected by the chain effects of climate change, which will inevitably lead to irreversible conditions of unsustainability, especially in the second half of this century.

Therefore, on the one hand, it is urgent and essential to predict, plan, and reduce disaster risks to more effectively protect people, communities, and countries, as well as people's live‐ lihoods, health, cultural heritage, socioeconomic assets, and ecosystems, and thus strengthen


**Author details**

Ana Marisa Chudzinski-Tavassi1,2\*, Kátia Luciano Pereira Morais1,2, Jean Gabriel de Souza1 and Roger Chammas3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Laboratory of Molecular Biology, Butantan Institute, SP, Brazil

2 Department of Biochemistry, Federal University of São Paulo, SP, Brazil

3 Experimental Oncology Medical Investigation Laboratory—LIM/24, University of São Paulo School of Medicine, SP, Brazil


**Abstract**

Significant advancements have been made in Cardiac surgery during the last decades, thanks to technological evolution. The enormous progress achieved has led to a relevant improvement in terms of surgical results, and at the same time, new ethical dilemmas have been addressed. Until the 90's ethics in cardiac surgery mainly concerned significant moral problems caused by the introduction of extremely innovative techniques. However, today's ethical issue focuses essentially on the doctor-patient relationship, other aspects of doctor's practice concern relevant ethical perspectives. Ethics affects today the activity of the surgeon and the doctor in general. It is possible to distinguish clinical ethics, an ethics of health policies, and scientific research ethics. In the following chapter, we try to analyze the main ethical aspects concerning the application of cardiac surgical procedures.

**Keywords:** informed consent, LVAD (left ventricle assist device), destination therapy (DT), pandemic

#### **1. Introduction (Informed consent)**

In surgery, up to the 1960s, "paternalism" was an accepted and well-regarded tradition. The surgeon chose "the best" for his patient, who had a passive role in the choices regarding the treatment of his disease. On the other hand, in modern medicine, the patient collaborates with the surgeon to pursue the ultimate good, that is, health and recovery from illness [1].

For this reason, the act of informed consent does not represent a simple proof of the patient's consent to treatment but describes the patient's awareness of the pathology and the proposed therapies. Informed consent is not obtaining a signature on a piece of paper but a complex information process made up of different parts. Three main sets of elements characterize informed consent and are the initial conditions such as capacity and voluntariness; information, characterized by explanation and related recommendations; consent including decision and authorization [2]. For preceding conditions, we mean the ability to understand information and to make decisions voluntarily. The lack of this capacity is not uncommon and often not recognized, although many tools are available to evaluate and measure it. Voluntariness is present when the patient's choices are intentional and not conditioned by coercion or external influences. Surgeons should avoid putting pressure on patients by emphasizing benefits and minimizing risks. The information elements include a detailed explanation of the nature of the disease and the treatment options. Risks, benefits, potential consequences of the recommended therapy, and possible alternatives must be elucidated. The likelihood of success, the prognosis without treatment are other aspects to explain. The elements relating to consent conclude the informed consent process. The decision made by the patient reflects which therapeutic option among those proposed to him he considers the best based on the information obtained. The consent is signed by both parties, like a real contract [3].

As proposed by the Italian Society of Cardiac Surgery, there are five informed consents that most commonly concern the cardiac surgeon's activity:

Before signing the consent, the patient must make autonomous decisions, without external pressure (from the doctor, the family), understand the severity of the disease, and the risk/benefit ratio of the proposed interventions.

However, the surgeon remains a point of reference for the patient, who, however, informed and aware, entrusts his or her life in the hands of another person; for this reason, the surgeon must direct and advice based on their knowledge and scientific updating. This situation, for example, emerges in cardiac surgery when the patient, who is going to undergo a valve replacement, must choose between a biological and mechanical prosthesis. It is also possible that the surgeon finds himself in a condition of "paternalism, "which means he must make the decision "for the patient" who intends to leave him the choice of the valve prosthesis to be implanted. In this case, this must be reported in the written consent. Furthermore, the surgeon has to stratify the risk for each patient based on his experience and use the most recent risk calculators (EUROScore I and II - European System for Cardiac Operative Risk Evaluation, and STS score - to help him). Society of Thoracic Surgeons score) useful for risk stratification and calculating the risk/benefit ratio [4].

#### **2. Appropriateness of care**

The theory of evaluative dynamism considers each therapeutic choice as proportionate/disproportionate; ordinary/extraordinary. The use of a means of life support is considered "proportionate" to the extent that it proves adequate to achieve a specific health goal. The proportionality of the therapeutic option is given by availability, technical possibility, and efficacy. Side effects and risks, possible therapeutic alternatives must be evaluated, and required technical and economic resources should be quantified. The extraordinary judgment correlates with a reasonably high probability of severe risks to the patient's life and health concerning his clinical condition and a low overall efficacy rate. When the effort to find the therapeutic option for life support is very high, or if the choice is linked to severe physical pain and the economic costs are too burdensome for the patient or his

**57**

ensuring patient comfort [10].

*Innovation and Research in Cardiac Surgery: Bioethical Aspects*

relatives, a judgment of extraordinary nature and, therefore, sometimes of disproportionality is configured [5]. In clinical practice, it is mandatory to use this ethical axiom: it is the duty of the doctor to use any proportionate and ordinary means for the treatment of the disease, the use of disproportionate and ordinary methods or proportionate but extraordinary measures may be optional; while it is not ethical to

Professional self-regulation is a personal or collective process of supervision that reflects the ethical concept of responsibility for patient care and includes accountability for that care's scientific and clinical quality. The self-regulation process is best exemplified by introducing the clinical practice of critical reviews through

Left ventricular assist systems (LVAD) are widely used to treat patients with heart failure refractory to medical therapy as a bridge to heart transplantation [7]. In subjects who have severe comorbidities such as compromising their inclusion in the transplant list, LVADs can be used as definitive therapy (Destination Therapy - DT). This use implies a series of ethical aspects that include the correct selection of the patient, the patient's precise information, and the definition of a possible protocol for deactivating the LVAD [8]. There are three ethical considerations regarding the selection of patients: the inclusion and exclusion criteria must be clearly explained, justified by rational diagnostic-therapeutic paths, and frequently discussed with the patient during the evaluation phase: The medical criteria must be supported by the most current medical, scientific evidence; The assessment must be conducted based on an explicit weighting of the costs, benefits, and risks associated with the procedure to be applied. The physician is responsible for providing clear information to the patient and family members, clarifying the risks and benefits of different treatment options, including medical treatment, palliative care, and VAD as destination therapy. The patient must be informed about the treatment's goal as an innovative surgical procedure aimed at improving the quality of life. An advanced treatment plan must be presented to the patient, clarifying the patient's preferences in case of complications. It is advisable to propose different scenarios that may occur

during daily life through an interview with other subjects with LVAD.

Another ethical aspect concerns the possible interruption of therapy with mechanical support [9]. According to the principle of self-determination, the patient is fully entitled to stop medical treatment so that natural death can occur. A natural death can occur as a consequence of the deactivation of an LVAD due to a dysfunction of the device, due to coexisting comorbidities, or as a consequence of the progression of the disease. There are two ways of deactivating the LVAD. A team can help the patient disconnect the device from the power supply to cause a stop of the pump. The device's interruption does not result in euthanasia since the ventricular function was already severely depressed before implanting the LVAD; the patient's death occurs from the underlying heart disease. The second option involves surgical removal, but this alternative is somewhat complicated because it could cause the patient's death and then. After all, it would contravene the goal of

use means that are disproportionate and extraordinary at the same time.

*DOI: http://dx.doi.org/10.5772/intechopen.94160*

**3. Professional self regulation**

**4. Ethics in LVAD as DT**

routine meetings on mortality and morbidity [6].

relatives, a judgment of extraordinary nature and, therefore, sometimes of disproportionality is configured [5]. In clinical practice, it is mandatory to use this ethical axiom: it is the duty of the doctor to use any proportionate and ordinary means for the treatment of the disease, the use of disproportionate and ordinary methods or proportionate but extraordinary measures may be optional; while it is not ethical to use means that are disproportionate and extraordinary at the same time.


**Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches**

Sara Pires, João Lopes, Inês Nunes and Elvira Gaspar *CQFB-REQUIMTE/Departamento de Química/Faculdade de Ciências e Tecnologia/Universidade Nova de Lisboa Portugal*

### **1. Introduction**

74 Ecotoxicology

Vezie, C.; Sivonen, K.; Brient, L.; Bertru, G. & Lefeuvre, J.C. (1996). Development of Toxic

Young, I.S.; Torney, J.J. & Trimble, E.R. (1992). The Effect of Ascorbate Supplementation on

Zillioux, E.J; Foulk, H. R.; Prager, J.C. & Cardin, J.A. (1973). Using *Artemia* to Assay Oil

*Medicine*, Vol.13, No.1, (July 1992), pp. 41-46, ISSN 0891-5849

ISSN 0003-4088

2389-2396, ISSN 0043-1303

Cyanobacteria in Western France-Detection of Toxicity with *Artemia salina* Tests. *Annales de Limnologie-International Journal of Limnology*, Vol.32, No.2, pp. 123-128,

Oxidative Stress in the Streptozotocin Diabetic Rat. *Free Radical Biology and*

Dispersant Toxicities. *Journal Water Pollution Control Federation*, Vol. 45, No.11, pp.

Patulin is a mycotoxin with carcinogenic, mutagenic and teratogenic potential mainly produced by several species of *Pennicillium, Aspergillus* and *Byssoclamys*. This toxin has been found in fresh food such as fruits and vegetables, but may also be present in commercial apple juices and other food products. Food contamination with this toxin had led, over the years, to intense investigations related with its occurrence, toxicity, and also to develop prevention and detoxification methods from human and animal food chains. Patulin occurrence compelled several regulating entities to establish maximum limits in some food products, and thereupon, the identification and quantification of this mycotoxin demands the development of sensitive, selective and effective analytical methods.

#### **2. Fungi and mycotoxins**

Fungus is an organism which used to be traditionally included in the plant family, but nowadays belongs to the Fungi kingdom due to their considerable differences to plants, such as their inability to produce their own food. They are cosmopolitan in distribution, generally unnoticed due to small size. Nevertheless, these organisms have been applied in the food industry, used as sources of pharmaceutical agents for the treatment of infectious and metabolic diseases, and have shown high importance in agriculture due to their ability to establish symbiotic relationships with plants roots.

Fungi obtain their food through organic matter decomposition, creating at that stage an excellent opportunity for their growth in food. Fungal growth may result in production of toxins harmful to human health causing mycotoxicoses (Bennett, 1987).

In a brief chemical description, mycotoxins can be introduced as low-molecular-weight natural products, with great structural diversity, produced as secondary metabolites by fungi (Bennett & Klich, 2003). These fungi possess the ability to infect food, causing intoxication in animals and humans through contamination of crops or foods prepared from them, reason why the term mycotoxin comes from the fusion of the Greek word *mykes* and the Latin *toxicum*, meaning fungus and toxin, respectively. At this point, it is clear that mycotoxins are toxic compounds; but the definition of mycotoxin refers specifically to

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 77

the current trade patterns, mycotoxicoses resulting from contaminated food, locally grown or imported, could occur worldwide. And as consequence, it would be wrong to state that mycotoxin exposure only occurs in underdeveloped countries in which the population is malnourished and there is no regards in food handling and storage (Peraica et al., 1999).

The use of preventive techniques for the control of fungal growth in agricultural commodities and to improve methods for food preparation in industrial standards and food spoilage are never successful. For instance, taste, size, shape, texture and appearance are the basic features of evaluation of a fruit, these will dictate the fruit final destination, consumption or processing. In other hand, food products made from fruits are classified according to their quality using indicators of contamination. Specifically in food products, quality can be characterized by the growth of observable fungi, the existence of unpleasant odor and decomposition of the fruit. In the presence of a fungal contamination, a fruit can exhibit a profound change due to production of disintegrative pectinolytic enzymes

As it was mentioned before, food contamination with mycotoxins is dependent of the influence of environmental and biological factors that determine the fungi production. These factors include the composition of the substrate, temperature, humidity, pH, microbial competition and insect damage (Songsermsakul & Razzazi-Fazeli, 2008; Sant'Ana et al., 2008). Contamination occurs during growing, harvesting, transportation, storage and/or processing of food products (Sant'Ana et al., 2008). As an example, *Aspergillus* and *Penicillium* species are usually associated with contamination during storage, whereas *Fusarium* species can produce mycotoxins before or right after harvesting (Abramson et al.,

Contaminated feed for animals may also have deleterious effects on humans through food chain. In fact, the animal derived products like milk, eggs and meat may appear

Beyond the devastating effect of agricultural growth of fungi, toxins produced by them have harmful effects on human health, including carcinogenicity, teratogenicity and growth retardation (Shephard, 2008). Aflatoxins, citrinin, fumonisin (Figure 1), ochratoxin, patulin, trichothecenes and zearalenone are among the mycotoxins that have received more attention in recent years due to their frequent occurrence and their effects on human health

Patulin (Figure 2) was initially classified as broad-spectrum antifungal antibiotic. Later, it was found to inhibit more than seventy five different species of bacteria. Further investigations also revealed patulin's violent toxicity to animals and plants (Ciegler et al.,

Patulin (PAT), IUPAC name 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, is characterized by being a hemiacetal lactone, with empirical formula C7H6O4, molecular weight 154.12 g/mol, with an appearance of white powder and a melting point of 110ºC. Patulin presents a very intense maximum UV absorption at 276 nm (Ciegler et al., 1971; Nielsen & Smedsgaard,

contaminated with chemicals produced by fungi (Shephard, 2008; Krska et al., 2008).

responsible for the fruit tissues degradation.

1997).

(Bennett & Klich, 2003).

**2.3 Patulin**

1971).

2003).

metabolites toxic to vertebrates in low concentration (Bennett, 1987). Nevertheless, not all toxic compounds produced by fungi can be defined as mycotoxins, and a good example of this is the antibiotic penicillin, produced by *Penicillium* fungi, which is especially toxic to bacteria.

Common observation one can find in fungi kingdom is that mould species produces more than one type of mycotoxin, and the same mycotoxin can be produced by different types of moulds (Robbins et al., 2000, as cited in Ciegler & Bennett, 1980).

#### **2.1 Toxicology and human health**

Mycoses are infections caused by fungal growth on a host, and mycotoxicoses are intoxications, which occur as a consequence from different exposures sources such as food intake, respiratory and dermal contacts. Mycotoxicoses are similar to several other types of intoxications and their symptoms are quite dependent on the specific mycotoxin which causes the illness, the extent of exposure, age, gender and health of the individual, and other aspects like genetics and drug interactions.

Mycotoxicoses syndromes can be categorized as acute or chronic, and can range from rapid death to tumor development. Other less revealing diseases may occur when the mycotoxin interferes with immune system, leaving it susceptible to infectious diseases. The recognition of a mycotoxicoses is a long way to run. To do so, a dose-response relationship is required to recognize correctly the illness and associate it with the mycotoxin which caused it. Therefore, it is imperative to perform epidemiologic studies in the human population. The common way to execute these studies is by analogy, where a controlled animal population is subjected to a mycotoxin and human syndromes are reproduced. Human exposure to mycotoxins can also be determined by environmental or biological monitoring. In environmental monitoring, mycotoxins are measured in food, air, or other samples that may be in contact with the subjects, while in biological monitoring quantitative and qualitative evaluations of residues are made, and adducts and metabolites in tissue fragments, bodily fluids and excreta (Bennet & Klich, 2003, as cited in Hsieh, 1988) are evaluated.

Fungal diseases are considered to be a severe health problem worldwide.

#### **2.2 Food contaminants**

Nowadays, the use of botanical products is increasing due to the importance of diets and life style for human beings and, consequently, the quality of food products is fundamental. In developed countries, botanical products are recommended for medicinal and general health-promoting purposes. However, numerous occurrences of natural mycotoxins in botanicals and fruits have been reported, bringing along food spoilage and considerable economic losses.

Despite the development and industrialization of a country, mycotoxins can occur when environmental, social and economic conditions combined with meteorological conditions (humidity, temperature) promote growth of moulds. Therefore, from an ecological point of view, the role of mycotoxins in disease inducement should not be disregarded. For example, the current trade patterns, mycotoxicoses resulting from contaminated food, locally grown or imported, could occur worldwide. And as consequence, it would be wrong to state that mycotoxin exposure only occurs in underdeveloped countries in which the population is malnourished and there is no regards in food handling and storage (Peraica et al., 1999).

The use of preventive techniques for the control of fungal growth in agricultural commodities and to improve methods for food preparation in industrial standards and food spoilage are never successful. For instance, taste, size, shape, texture and appearance are the basic features of evaluation of a fruit, these will dictate the fruit final destination, consumption or processing. In other hand, food products made from fruits are classified according to their quality using indicators of contamination. Specifically in food products, quality can be characterized by the growth of observable fungi, the existence of unpleasant odor and decomposition of the fruit. In the presence of a fungal contamination, a fruit can exhibit a profound change due to production of disintegrative pectinolytic enzymes responsible for the fruit tissues degradation.

As it was mentioned before, food contamination with mycotoxins is dependent of the influence of environmental and biological factors that determine the fungi production. These factors include the composition of the substrate, temperature, humidity, pH, microbial competition and insect damage (Songsermsakul & Razzazi-Fazeli, 2008; Sant'Ana et al., 2008). Contamination occurs during growing, harvesting, transportation, storage and/or processing of food products (Sant'Ana et al., 2008). As an example, *Aspergillus* and *Penicillium* species are usually associated with contamination during storage, whereas *Fusarium* species can produce mycotoxins before or right after harvesting (Abramson et al., 1997).

Contaminated feed for animals may also have deleterious effects on humans through food chain. In fact, the animal derived products like milk, eggs and meat may appear contaminated with chemicals produced by fungi (Shephard, 2008; Krska et al., 2008).

Beyond the devastating effect of agricultural growth of fungi, toxins produced by them have harmful effects on human health, including carcinogenicity, teratogenicity and growth retardation (Shephard, 2008). Aflatoxins, citrinin, fumonisin (Figure 1), ochratoxin, patulin, trichothecenes and zearalenone are among the mycotoxins that have received more attention in recent years due to their frequent occurrence and their effects on human health (Bennett & Klich, 2003).

#### **2.3 Patulin**

76 Ecotoxicology

metabolites toxic to vertebrates in low concentration (Bennett, 1987). Nevertheless, not all toxic compounds produced by fungi can be defined as mycotoxins, and a good example of this is the antibiotic penicillin, produced by *Penicillium* fungi, which is especially toxic to

Common observation one can find in fungi kingdom is that mould species produces more than one type of mycotoxin, and the same mycotoxin can be produced by different types of

Mycoses are infections caused by fungal growth on a host, and mycotoxicoses are intoxications, which occur as a consequence from different exposures sources such as food intake, respiratory and dermal contacts. Mycotoxicoses are similar to several other types of intoxications and their symptoms are quite dependent on the specific mycotoxin which causes the illness, the extent of exposure, age, gender and health of the individual, and other aspects like genetics and drug interactions. In the worst cases, mycotoxin poisoning can be amplified due to factors like sub nutrition, alcohol abuse and the presence of other diseases

Mycotoxicoses syndromes can be categorized as acute or chronic, and can range from rapid death to tumor development. Other less revealing diseases may occur when the mycotoxin interferes with immune system, leaving it susceptible to infectious diseases. The recognition of a mycotoxicoses is a long way to run. To do so, a dose-response relationship is required to recognize correctly the illness and associate it with the mycotoxin which caused it. Therefore, it is imperative to perform epidemiologic studies in the human population. The common way to execute these studies is by analogy, where a controlled animal population is subjected to a mycotoxin and human syndromes are reproduced. Human exposure to mycotoxins can also be determined by environmental or biological monitoring. In environmental monitoring, mycotoxins are measured in food, air, or other samples that may be in contact with the subjects, while in biological monitoring quantitative and qualitative evaluations of residues are made, and adducts and metabolites in tissue fragments, bodily

Nowadays, the use of botanical products is increasing due to the importance of diets and life style for human beings and, consequently, the quality of food products is fundamental. In developed countries, botanical products are recommended for medicinal and general health-promoting purposes. However, numerous occurrences of natural mycotoxins in botanicals and fruits have been reported, bringing along food spoilage and considerable

Despite the development and industrialization of a country, mycotoxins can occur when environmental, social and economic conditions combined with meteorological conditions (humidity, temperature) promote growth of moulds. Therefore, from an ecological point of view, the role of mycotoxins in disease inducement should not be disregarded. For example,

fluids and excreta (Bennet & Klich, 2003, as cited in Hsieh, 1988) are evaluated.

Fungal diseases are considered to be a severe health problem worldwide.

moulds (Robbins et al., 2000, as cited in Ciegler & Bennett, 1980).

**2.1 Toxicology and human health**

(Bennett & Klich, 2003).

**2.2 Food contaminants**

economic losses.

bacteria.

Patulin (Figure 2) was initially classified as broad-spectrum antifungal antibiotic. Later, it was found to inhibit more than seventy five different species of bacteria. Further investigations also revealed patulin's violent toxicity to animals and plants (Ciegler et al., 1971).

Patulin (PAT), IUPAC name 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, is characterized by being a hemiacetal lactone, with empirical formula C7H6O4, molecular weight 154.12 g/mol, with an appearance of white powder and a melting point of 110ºC. Patulin presents a very intense maximum UV absorption at 276 nm (Ciegler et al., 1971; Nielsen & Smedsgaard, 2003).

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 79

to the fungi ability to survive the heat treatments which are applied to food products

Fungal strains which produce patulin have been isolated over the years from various fruits and vegetables. Although patulin can occur in several fruits, grains and other foods infected by fungi, the main concern is with apples (Sommer et al., 1974, as cited in Brian et al., 1956; Harwig et al., 1973), apple cider (Sands et al., 1976, as cited in Stott &.Bullernan, 1975) and apple juice (Scott et al., 1972) due to their higher consumption. It is also found and isolated from other fruits and vegetables; they are: grapes, pears, apricots (Sommer et al., 1974), cherries, strawberries, nectarines, raspberries, peaches, plums, tomatoes, bananas, almonds, hazelnuts and peanuts (Leggott & Shephard, 2001; Moake et al., 2005, as cited in Harvey et al., 1972; Buchanan et al., 1974; Lovett et al., 1974; Akerstrand et al., 1976; Andersson et al., 1977; Frank et al., 1977; Harwig et al., 1978; Brackett & Marth, 1979a; Jelinek et al., 1989;

Studies have been developed in several countries related with the occurrence of patulin; it has been identified in apples from Canada, England, United States, Australia (Sommer et al., 1974), South Africa (Leggott & Shephard, 2001), New Zealand (Moake et al., 2005, as cited in Walker, 1969) and Portugal (Gaspar & Lucena, 2009), and had also been found in apple juices from Canada (Moake et al., 2005, as cited in Scott et al., 1972), United States (Moake et al., 2005, as cited in Ware et al., 1974), Sweden (Moake et al., 2005, as cited in Josefsson & Andersson, 1976), South Africa (Leggott & Shephard, 2001), Turkey (Gokmen & Acar, 1998), Brazil (Moake et al., 2005, as cited in de Sylos & Rodriquez-Amaya, 1999), Austria (Moake et al., 2005, as cited in Steiner et al., 1999a), Italy (Moake et al., 2005, as cited in Ritieni, 2003), Belgium (Moake et al., 2005, as cited in Tangi et al., 2003), and Portugal

In the last decades, attempts have been made to carry out the work on the risk caused by patulin on human health. In the early 1940's, a work reported a metabolic compound derived from a *Penicillium* species, called patulin, which had the potential of being applied in the treatment to the common cold (Medical Research Council, 2004, as cited in Raistrick, 1943), but no evidence was found that patulin would be effective in the treatment of that disease. Due to its toxic evidenced effects, such as nausea, vomiting and gastrointestinal

In the same decade, under the common name clavacin, a study of its antibiotic properties was performed. Preliminary results from this study showed that it was active against Gramnegative and Gram-positive bacteria, but it was also highly toxic to animal organisms

Since then, several *in vitro* and *in vivo* studies have been performed to evaluate the toxicological risk associated with patulin consumption through fresh and processed food products, and other exposure types. In one of these chemical researches, patulin showed a strong affinity for sulfhydryl groups. The compound detains a great ability to form adducts with cysteine, although these adducts are less toxic than the unmodified toxin, respecting acute toxicity, teratogenicity, and mutagenicity studies. Its affinity for sulfhydryl groups explains the inhibition of many enzymes (Ciegler et al., 1976). Although the data on

Jiminez et al., 1991; Prieta et al., 1994; Demirci et al., 2003; Ritieni, 2003).

(Gaspar & Lucena, 2009; Barreira et al, 2010).

**2.3.2 Ecotoxicology and legal relevance**

disturbances, patulin did not prove to be an efficient drug.

(Katzman et al., 1944, as cited in Waksman et al., 1942).

(Sant'Ana et al., 2008).

Fig. 1. Chemical structures of some common mycotoxins: aflotoxin B1 (A), citrinin (B) and fumonisin B1 (C).

Fig. 2. Patulin chemical structure.

Patulin is an organic compound very soluble in water and in most organic solvents. It is stable in diluted acids and resistant to temperatures at 125ºC in the 3.5 - 5.5 pH range (Collin et al., 2008, as cited in Lovett & Peeler, 1973).

#### **2.3.1 Sources and natural occurrence**

The main species of fungi which produce patulin in food are *Aspergillus*, *Penicillium, Byssochlamys*. *Penicillium expansum* has been identified as the major producer of patulin from the *Penicillium* species (Ciegler et al., 1971, as cited in Shibata et al., 1964) which is responsible for the vast majority of spoiled fruit. Both *Penicillium* and *Aspergillus* have the ability to grow at low temperatures. On the other hand, the fungi of the genus *Byssochlamys* are responsible for patulin production in post-pasteurization stages. This production is due to the fungi ability to survive the heat treatments which are applied to food products (Sant'Ana et al., 2008).

Fungal strains which produce patulin have been isolated over the years from various fruits and vegetables. Although patulin can occur in several fruits, grains and other foods infected by fungi, the main concern is with apples (Sommer et al., 1974, as cited in Brian et al., 1956; Harwig et al., 1973), apple cider (Sands et al., 1976, as cited in Stott &.Bullernan, 1975) and apple juice (Scott et al., 1972) due to their higher consumption.

Studies have been developed in several countries related with the occurrence of patulin; it has been identified in apples from Canada, England, United States, Australia (Sommer et al., 1974), South Africa (Leggott & Shephard, 2001), New Zealand (Moake et al., 2005, as cited in Walker, 1969) and Portugal (Gaspar & Lucena, 2009), and had also been found in apple juices from Canada (Moake et al., 2005, as cited in Scott et al., 1972), United States (Moake et al., 2005, as cited in Ware et al., 1974), Sweden (Moake et al., 2005, as cited in Josefsson & Andersson, 1976), South Africa (Leggott & Shephard, 2001), Turkey (Gokmen & Acar, 1998), Brazil (Moake et al., 2005, as cited in de Sylos & Rodriquez-Amaya, 1999), Austria (Moake et al., 2005, as cited in Steiner et al., 1999a), Italy (Moake et al., 2005, as cited in Ritieni, 2003), Belgium (Moake et al., 2005, as cited in Tangi et al., 2003), and Portugal (Gaspar & Lucena, 2009; Barreira et al, 2010).

#### **2.3.2 Ecotoxicology and legal relevance**

78 Ecotoxicology

Fig. 1. Chemical structures of some common mycotoxins: aflotoxin B1 (A), citrinin (B) and

Patulin is an organic compound very soluble in water and in most organic solvents. It is stable in diluted acids and resistant to temperatures at 125ºC in the 3.5 - 5.5 pH range

The main species of fungi which produce patulin in food are *Aspergillus*, *Penicillium, Byssochlamys*. *Penicillium expansum* has been identified as the major producer of patulin from the *Penicillium* species (Ciegler et al., 1971, as cited in Shibata et al., 1964) which is responsible for the vast majority of spoiled fruit. Both *Penicillium* and *Aspergillus* have the ability to grow at low temperatures. On the other hand, the fungi of the genus *Byssochlamys* are responsible for patulin production in post-pasteurization stages. This production is due

fumonisin B1 (C).

Fig. 2. Patulin chemical structure.

**2.3.1 Sources and natural occurrence**

(Collin et al., 2008, as cited in Lovett & Peeler, 1973).

In the last decades, attempts have been made to carry out the work on the risk caused by patulin on human health. In the early 1940's, a work reported a metabolic compound derived from a *Penicillium* species, called patulin, which had the potential of being applied in the treatment to the common cold (Medical Research Council, 2004, as cited in Raistrick, 1943), but no evidence was found that patulin would be effective in the treatment of that disease. Due to its toxic evidenced effects, such as nausea, vomiting and gastrointestinal disturbances, patulin did not prove to be an efficient drug.

In the same decade, under the common name clavacin, a study of its antibiotic properties was performed. Preliminary results from this study showed that it was active against Gramnegative and Gram-positive bacteria, but it was also highly toxic to animal organisms (Katzman et al., 1944, as cited in Waksman et al., 1942).

Since then, several *in vitro* and *in vivo* studies have been performed to evaluate the toxicological risk associated with patulin consumption through fresh and processed food products, and other exposure types. In one of these chemical researches, patulin showed a strong affinity for sulfhydryl groups. The compound detains a great ability to form adducts with cysteine, although these adducts are less toxic than the unmodified toxin, respecting acute toxicity, teratogenicity, and mutagenicity studies. Its affinity for sulfhydryl groups explains the inhibition of many enzymes (Ciegler et al., 1976). Although the data on

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 81

includes an initial multi-extraction step with ethyl acetate and a posterior extraction with an aqueous sodium carbonate solution. The combined extracts were dried and evaporated, then being dissolved in acetic acid solution and chromatographed for patulin analysis. Several improved methodologies of the official method have been reported over the years (Gökmen & Acar, 1998; Iha & Sabino, 2006). One of them was described for cloudy apple juice and apple purees, and includes a pretreatment procedure with pectinase to improve

Solid phase extraction (SPE) emerged as an extremely attractive versatile technique for sample preparation of patulin containing matrices. The principles of the technique are based on chromatography: solutes are dissolved or suspended in a liquid and eluted through a solid phase; depending on their affinity, a separation occurs (Figure 3). SPE has been used to clean-up matrices and concentrate trace substances, such as patulin. Like any other technique it has also limitations: matrix effect and the undesired competition between analyte(s) and the other matrix components. As can be seen in Figure 3, SPE uses prepacked cartridges containing up to 500 mg of stationary phase - silica or silica bonded to specific functional groups such as hydrocarbons of variable chain length, reversed phase SPE, or quaternary ammonium or amino groups, anion-exchange SPE, or sulfonic acid or

For patulin analysis in apple juices (Gökmen et al., 2005) two SPE systems using a tandem polyvinylpolypyrrolidone-octadecyl (PVPP-C18) cartridge and a hydrophilic lipophilic balanced (HLB) macroporous copolymer sorbent cartridge, were described. The technique required few mL of juice and the elution was done with diethyl ether. After solvent

the clarity of juices and purees before patulin analysis (MacDonald et al., 2000).

**<IP_ADDRESS> Solid Phase Extraction (SPE)**

carboxylic groups, cation-exchange SPE.

Fig. 3. SPE procedure steps (Supelco, 1998).

genotoxicity were variable, most assays carried out with mammalian cells were positive, while assays with bacteria were mainly negative. After acute administration and short-term studies, the main signals of toxicity caused by patulin were gastrointestinal hyperemia, distension, hemorrhage and ulceration. *In vitro* and *in vivo* experiments had showed immunosuppressive properties at high dosages of patulin (Wouter & Speijers, 1996).

The International Agency for Research on Cancer (IARC) concluded that no evaluation was made respecting the carcinogenicity of patulin to humans and that there was inadequate evidence in experimental animals (IARC, 1986).

Even though no epidemic outbreak in humans and animals has been attributed to patulin contamination, increasing concern with public health forced the regulating authorities to establish maximum limits. As a consequence, international recommendations and regulations were established for patulin maximum levels allowed in food products; patulin content warnings in food products have to be displayed, acting like quality markers. Within European countries the regulation (EC) No 1425/2003 was adopted and setting as maximum levels of 50 µg/L for fruit juices and derived products, 25 µg/L for solid apple products and 10 µg/L for juices and foods aimed for babies and young infants (Commission regulation (EC) No 1881/2006). Joint FAO/WHO Expert Committee on Food Additives (JECFA) also established the provisional maximum tolerable daily intake 0.4 µg/kg body mass/day (JECFA, 1995). Nowadays, the US Food and Drug Administration (FDA) limit patulin to 50 µg/L.

#### **2.4 Analysis**

Patulin analysis encompass several important steps: sampling, sample preparation, isolation and/or identification, quantitation and, sometimes, statistical evaluation. Each step is equally important to obtain good results to fulfill the analytical purpose.

Sampling includes deciding on sampling points and choosing a method to get appropriate amounts of samples, which can be solid or liquid with very different structural and chemical complexity.

#### **2.4.1 Sample preparation techniques**

Most of the time, sample preparation is necessary to isolate and/or concentrate patulin from sample matrices, having into account its chemical properties and the complexity of matrices. As mentioned before, patulin can be found in human food and beverages, animal feed, biological and environmental samples.

Matrix solid-phase dispersion (MSPD) was reported (Wu et al., 2008, 2009) as a suitable method to extract patulin from apples, A small portion of apple was blended homogeneously in a C18-bonded silica solid support. After the washing step with hexane, patulin was extracted by elution with dichloromethane. After solvent evaporation, the extract was dissolved in acetic acid buffer solution and the patulin was analyzed by High Performance Liquid Chromatography (HPLC).

The Association of Official Analytical Chemists (AOAC) adopted a liquid-liquid extraction as sample preparation methodology for patulin analysis of apple juices and concentrates in its Official Method 995.10 (Sewram et al., 2000, as cited in Brause et al., 1996). The method includes an initial multi-extraction step with ethyl acetate and a posterior extraction with an aqueous sodium carbonate solution. The combined extracts were dried and evaporated, then being dissolved in acetic acid solution and chromatographed for patulin analysis. Several improved methodologies of the official method have been reported over the years (Gökmen & Acar, 1998; Iha & Sabino, 2006). One of them was described for cloudy apple juice and apple purees, and includes a pretreatment procedure with pectinase to improve the clarity of juices and purees before patulin analysis (MacDonald et al., 2000).

#### **<IP_ADDRESS> Solid Phase Extraction (SPE)**

80 Ecotoxicology

genotoxicity were variable, most assays carried out with mammalian cells were positive, while assays with bacteria were mainly negative. After acute administration and short-term studies, the main signals of toxicity caused by patulin were gastrointestinal hyperemia, distension, hemorrhage and ulceration. *In vitro* and *in vivo* experiments had showed

The International Agency for Research on Cancer (IARC) concluded that no evaluation was made respecting the carcinogenicity of patulin to humans and that there was inadequate

Even though no epidemic outbreak in humans and animals has been attributed to patulin contamination, increasing concern with public health forced the regulating authorities to establish maximum limits. As a consequence, international recommendations and regulations were established for patulin maximum levels allowed in food products; patulin content warnings in food products have to be displayed, acting like quality markers. Within European countries the regulation (EC) No 1425/2003 was adopted and setting as maximum levels of 50 µg/L for fruit juices and derived products, 25 µg/L for solid apple products and 10 µg/L for juices and foods aimed for babies and young infants (Commission regulation (EC) No 1881/2006). Joint FAO/WHO Expert Committee on Food Additives (JECFA) also established the provisional maximum tolerable daily intake 0.4 µg/kg body mass/day (JECFA, 1995). Nowadays, the US Food and Drug Administration (FDA) limit patulin to 50

Patulin analysis encompass several important steps: sampling, sample preparation, isolation and/or identification, quantitation and, sometimes, statistical evaluation. Each step is

Sampling includes deciding on sampling points and choosing a method to get appropriate amounts of samples, which can be solid or liquid with very different structural and chemical

Most of the time, sample preparation is necessary to isolate and/or concentrate patulin from sample matrices, having into account its chemical properties and the complexity of matrices. As mentioned before, patulin can be found in human food and beverages, animal feed,

Matrix solid-phase dispersion (MSPD) was reported (Wu et al., 2008, 2009) as a suitable method to extract patulin from apples, A small portion of apple was blended homogeneously in a C18-bonded silica solid support. After the washing step with hexane, patulin was extracted by elution with dichloromethane. After solvent evaporation, the extract was dissolved in acetic acid buffer solution and the patulin was analyzed by High

The Association of Official Analytical Chemists (AOAC) adopted a liquid-liquid extraction as sample preparation methodology for patulin analysis of apple juices and concentrates in its Official Method 995.10 (Sewram et al., 2000, as cited in Brause et al., 1996). The method

equally important to obtain good results to fulfill the analytical purpose.

immunosuppressive properties at high dosages of patulin (Wouter & Speijers, 1996).

evidence in experimental animals (IARC, 1986).

µg/L.

**2.4 Analysis**

complexity.

**2.4.1 Sample preparation techniques**

biological and environmental samples.

Performance Liquid Chromatography (HPLC).

Solid phase extraction (SPE) emerged as an extremely attractive versatile technique for sample preparation of patulin containing matrices. The principles of the technique are based on chromatography: solutes are dissolved or suspended in a liquid and eluted through a solid phase; depending on their affinity, a separation occurs (Figure 3). SPE has been used to clean-up matrices and concentrate trace substances, such as patulin. Like any other technique it has also limitations: matrix effect and the undesired competition between analyte(s) and the other matrix components. As can be seen in Figure 3, SPE uses prepacked cartridges containing up to 500 mg of stationary phase - silica or silica bonded to specific functional groups such as hydrocarbons of variable chain length, reversed phase SPE, or quaternary ammonium or amino groups, anion-exchange SPE, or sulfonic acid or carboxylic groups, cation-exchange SPE.

Fig. 3. SPE procedure steps (Supelco, 1998).

For patulin analysis in apple juices (Gökmen et al., 2005) two SPE systems using a tandem polyvinylpolypyrrolidone-octadecyl (PVPP-C18) cartridge and a hydrophilic lipophilic balanced (HLB) macroporous copolymer sorbent cartridge, were described. The technique required few mL of juice and the elution was done with diethyl ether. After solvent

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 83

Using MEPS as sample preparation technique, a new methodology for patulin analysis is described, for the first time, in this book chapter (Section <IP_ADDRESS>). It aims the identification

The most suitable analytical methods respecting accuracy, low detection limits and simple procedures for patulin analysis in food products, especially apple juice and its derivatives, involve chromatographic techniques such as thin-layer chromatography (TLC), gas

The pioneering methodology used in the identification and quantification of patulin, in apple juice, was thin-layer chromatography (TLC) with the advantages of being simple and low cost. One official method of the Association of Official Analytical Chemists (AOAC) for the analysis of mycotoxins (1974) uses TLC followed by silica gel column chromatography as sample preparation procedures. The analysis was carried out in silica gel plates, using, for detection, the reaction of patulin with 3-methyl-2-hydrazone benzothiazolinone and HCl (MBTH). The limit of detection (LOD) was described as 20 μg/L (Shephard & Leggott, 2000, as cited in Scott, 1974). Alternative detection and quantification methods were described using fluorodensiometry (Shephard & Leggott, 2000, as cited in Duraković et al., 1993), or absorbance–transmittance followed by densitometry at 275nm (Shephard & Leggott, 2000, as cited in Leming et al., 1993). Reversed phase TLC was also investigated, but despite the patulin elution with a wide variety of solvents, the technique was not applied to

HPLC coupled with ultraviolet (UV) or photodiodes (DAD) detection is the most used chromatographic technique for patulin analysis; it allows an easy identification and quantification of patulin through its characteristic absorption spectrum. The AOAC also adopted HPLC-UV method (official method 995.10), for the detection and quantification of patulin in apple juices. As mentioned before, liquid-liquid extraction was used as sample preparation technique. The HPLC was done using a C18 reversed phase column, with particle size of 5 μm and pore size ranging from 12 to 25 nm, and isocratic elution with a flow of 1mL/min, and the eluent was composed by acetonitrile in acidified water. UV detection at 276 nm (Moake et al., 2005, as cited in AOAC official method 995.10) was used.

Improved AOAC methods were described in the literature for the determination of patulin in apple juices (Gökmen & Acar, 2005) and in apple solid derivatives (Katerere et al., 2008). Over the years, HPLC methodologies associated with mass spectrometry (MS) were also developed. A work published in 2000 described patulin detection by collision-induced dissociation (CID) using atmospheric pressure chemical ionization (APCI); the LOD was

Gas chromatography (GC) has also been applied in the analysis of patulin. The literature reported GC-MS analysis by electron impact ionization, using a silylated patulin derivative (Moukas et al., 2008). Raw patulin (not derivatized) was also determined in apple juice by GC-MS using negative chemical ionization (Roach et al., 2000). Other GC methods were reported using on-column injection and selected ion monitoring (SIM) detection (Moake et

chromatography (GC) and high-performance liquid chromatography (HPLC).

and quantification of this mycotoxin in real matrices.

contaminated samples (Shephard & Leggott, 2000).

good, with values near 4 μg/L (Sewram et al., 2000).

The LOD achieved is low (5 μg/L).

al., 2005, as cited in Llovera et al., 1999).

**2.4.2 Chromatographic methodologies**

evaporation the residue was dissolved in a mixture of acetonitrile/water with a small portion of acetic acid and then chromatographed.

Another work reported the patulin analysis of infant's apple-based products involving a SPE cleanup step using a unconditioned silica gel cartridge (Arranz et al., 2005).

For the analysis of an apple juice syrup it was described a pretreatment procedure using C18- SPE cartridges (Li et al., 2007). The syrup sample was diluted with acetic acid buffer solution and loaded onto the column. After the washing procedure using hexane, the cartridge was dried with a strong stream of air for 15 min, and eluted with the solvent mixture (hexane/ethyl acetate/acetone) in a gradient mode. After acidification, patulin was analyzed by HPLC.

#### **<IP_ADDRESS> Microextraction by packed sorbent or packaged syringe (MEPS)**

The development of miniaturized analytical techniques has been done to fulfill many requirements and also enable rapid analysis at lower operation costs, with lower environmental pollution problems. Microextraction by packed sorbent or packaged syringe (MEPS) methodology seems to be a very promising solution in patulin analysis. MEPS technique uses a small amount of stationary phase, packed in the drum called BIN (Barrel Insert and Needle Assembly, Figure 4) of a gastight syringe (100μL – 250μL). Like SPE, MEPS aims the elimination of interferences and the selective isolation and concentration of the target compounds, but in a micro scale. MEPS procedure steps are similar to SPE laboratorial approaches and are illustrated in Figure 5.

Fig. 5. MEPS procedure steps (SGE, 2009).

Using MEPS as sample preparation technique, a new methodology for patulin analysis is described, for the first time, in this book chapter (Section <IP_ADDRESS>). It aims the identification and quantification of this mycotoxin in real matrices.

#### **2.4.2 Chromatographic methodologies**

82 Ecotoxicology

evaporation the residue was dissolved in a mixture of acetonitrile/water with a small

Another work reported the patulin analysis of infant's apple-based products involving a

For the analysis of an apple juice syrup it was described a pretreatment procedure using C18- SPE cartridges (Li et al., 2007). The syrup sample was diluted with acetic acid buffer solution and loaded onto the column. After the washing procedure using hexane, the cartridge was dried with a strong stream of air for 15 min, and eluted with the solvent mixture (hexane/ethyl acetate/acetone) in a gradient mode. After acidification, patulin was

The development of miniaturized analytical techniques has been done to fulfill many requirements and also enable rapid analysis at lower operation costs, with lower environmental pollution problems. Microextraction by packed sorbent or packaged syringe (MEPS) methodology seems to be a very promising solution in patulin analysis. MEPS technique uses a small amount of stationary phase, packed in the drum called BIN (Barrel Insert and Needle Assembly, Figure 4) of a gastight syringe (100μL – 250μL). Like SPE, MEPS aims the elimination of interferences and the selective isolation and concentration of the target compounds, but in a micro scale. MEPS procedure steps are similar to SPE

SPE cleanup step using a unconditioned silica gel cartridge (Arranz et al., 2005).

**<IP_ADDRESS> Microextraction by packed sorbent or packaged syringe (MEPS)**

portion of acetic acid and then chromatographed.

laboratorial approaches and are illustrated in Figure 5.

Fig. 4. MEPS Syringe and BIN (SGE, 2009).

Fig. 5. MEPS procedure steps (SGE, 2009).

analyzed by HPLC.

The most suitable analytical methods respecting accuracy, low detection limits and simple procedures for patulin analysis in food products, especially apple juice and its derivatives, involve chromatographic techniques such as thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC).

The pioneering methodology used in the identification and quantification of patulin, in apple juice, was thin-layer chromatography (TLC) with the advantages of being simple and low cost. One official method of the Association of Official Analytical Chemists (AOAC) for the analysis of mycotoxins (1974) uses TLC followed by silica gel column chromatography as sample preparation procedures. The analysis was carried out in silica gel plates, using, for detection, the reaction of patulin with 3-methyl-2-hydrazone benzothiazolinone and HCl (MBTH). The limit of detection (LOD) was described as 20 μg/L (Shephard & Leggott, 2000, as cited in Scott, 1974). Alternative detection and quantification methods were described using fluorodensiometry (Shephard & Leggott, 2000, as cited in Duraković et al., 1993), or absorbance–transmittance followed by densitometry at 275nm (Shephard & Leggott, 2000, as cited in Leming et al., 1993). Reversed phase TLC was also investigated, but despite the patulin elution with a wide variety of solvents, the technique was not applied to contaminated samples (Shephard & Leggott, 2000).

HPLC coupled with ultraviolet (UV) or photodiodes (DAD) detection is the most used chromatographic technique for patulin analysis; it allows an easy identification and quantification of patulin through its characteristic absorption spectrum. The AOAC also adopted HPLC-UV method (official method 995.10), for the detection and quantification of patulin in apple juices. As mentioned before, liquid-liquid extraction was used as sample preparation technique. The HPLC was done using a C18 reversed phase column, with particle size of 5 μm and pore size ranging from 12 to 25 nm, and isocratic elution with a flow of 1mL/min, and the eluent was composed by acetonitrile in acidified water. UV detection at 276 nm (Moake et al., 2005, as cited in AOAC official method 995.10) was used. The LOD achieved is low (5 μg/L).

Improved AOAC methods were described in the literature for the determination of patulin in apple juices (Gökmen & Acar, 2005) and in apple solid derivatives (Katerere et al., 2008). Over the years, HPLC methodologies associated with mass spectrometry (MS) were also developed. A work published in 2000 described patulin detection by collision-induced dissociation (CID) using atmospheric pressure chemical ionization (APCI); the LOD was good, with values near 4 μg/L (Sewram et al., 2000).

Gas chromatography (GC) has also been applied in the analysis of patulin. The literature reported GC-MS analysis by electron impact ionization, using a silylated patulin derivative (Moukas et al., 2008). Raw patulin (not derivatized) was also determined in apple juice by GC-MS using negative chemical ionization (Roach et al., 2000). Other GC methods were reported using on-column injection and selected ion monitoring (SIM) detection (Moake et al., 2005, as cited in Llovera et al., 1999).

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 85

3. The sample (0.5 mL) was introduced and eluted at a flow rate of 2–3 mL/min (Figure 3,

7. After dissolution in (0.5 mL) acidified water the sample was analyzed using the previously mentioned improved HPLC methodology (Gaspar & Lucena, 2009);

This improved sample preparation changed the previously described (Li et al., 2007) washing solvent - acidified water instead of hexane - and also the eluent composition,

This optimized SPE-HPLC/DAD methodology showed an average recovery of 82% with a RSD value of 6% in the linear dynamic range 200 to 600 ppb (Table 2). The precision of the method showed a repeatability of 1.2% and a reproducibility value of 2.2%. According to the literature (Miller & Miller, 1988; IPQ, 2000) being these values below 10%, they indicate a good method performance. The overall SPE-HPLC methodology represents an economical,

200 - 600 82 (6) 1.2 2.2 Table 2. Analytical parameters of improved SPE-HPLC methodology for patulin analysis.

Using the recent MEPS analytical tool (Abdel-Rehim, 2004), a new, simple, sample preparation method was developed for patulin analysis. MEPS methodology can be rationalized as a miniaturization of SPE analytical system; it employs smaller quantities of sample and eluent (few microliters), being also adequate to remove interferences,

For patulin analysis, this new sample preparation methodology was performed using the

1. MEPS was carried out by means of a SGE Analytical Science (SGE Analytical Science, Germany) apparatus, consisting of a 250 μL HPLC syringe with a removable needle; the syringe was fitted with a BIN (Barrel Insert and Needle) containing the C18 sorbent and was used to draw and discharge samples and solutions through the BIN (Figure 4); 2. The sorbent was activated/conditioned three times with (3 × 100 μL) methanol and (5 ×

3. Sampling was done using a volume of 2x25 μL of sample solution, being introduced through the stationary phase for three times, in order to remove the interferences and

4. The analyte was eluted using methanol (2 × (3 × 25 μL)) and after analyzed by HPLC using the previously described conditions (Gaspar & Lucena, 2009) (illustrated in

This method development evidenced the relevance of several factors in this sample preparation methodology, MEPS: sampling speed was around 10 μL/s, sample introduction

100 μL) MilliQ water-perchloric acid ((100): 0.01 (v/v)) (Figure 5);

retain the patulin (illustrated in Figure 5, step 1);

Repeatability (RSD,%), n = 3 Reproducibility (RSD,%), n = 3

methanol, a greener solvent, instead of the mixture hexane/ethyl acetate/acetone.

4. The following washing step was done with (0.5 mL) acidified water (Figure 3, step 4);

5. Sample elution was done with (3 × 1 mL) methanol (Figure 3, step 5); 6. The combined eluates were evaporated to dryness under vacuum;

> Recovery (RSD,%), n = 3

step 3);

faster and routine usable methodology.

**2.5.2 MEPS-HPLC/DAD methodology**

Range (μg/L)

simplifying the analysis.

following sequential steps:

Figure 5, steps 3 and 4);

A protocol including biphasic dialysis extraction, *in situ* acylation, as sample preparation and GC-MS (SIM) analysis was developed for detection and quantification of patulin (acetylated) in fruit juices (Sheu & Shyu, 1999). Another work presented the detection and quantification of C13 labeled patulin (Rychlik & Schieberle, 1999).

Capillary electrophoresis (CE) was also described as an alternative method for patulin analysis. In 2000, a capillary electrophoresis method was published as an analytical tool for rapid and highly sensitive analysis of patulin in cider; a fused silica capillary column was used, and the elution was done with an aqueous. The separation was achieved by migration of charged particles in the buffer. The cations migrated to the cathode and anions migrated to the anode under the influence of an electro-osmotic flow (EOF). In fact, compounds or mixtures of neutral and charged compounds can be analyzed by micellar electrokinetic capillary electrophoresis or micellar electrokinetic capillary chromatography (MECC). The method was described as having the advantage of requiring small amounts of sample and smaller amount of organic solvents when compared with HPLC (Tsao & Zhou, 2000).

#### **2.5 Development of improved analytical methodologies**

For patulin analysis, two sample preparation procedures using SPE and MEPS were developed and associated with an improved reversed-phase HPLC methodology (Gaspar & Lucena, 2009). The purpose was to simplify the sample, liberating the patulin from its original matrix, and allowing therefore a better performance of the overall analytical method. Table 1 shows the analytical parameters of the HPLC methodology.

Table 1. Analytical parameters of improved HPLC methodology for patulin analysis (Elvira & Lucena, 2009).

#### **2.5.1 SPE-HPLC/DAD methodology**

Aiming a small scale sample preparation procedure, a previously described SPE approach (Li et al., 2007) was improved and optimized. The purpose was the analysis of patulin in fresh fruits, namely apples, but also in apple juices commercialized in Portugal. The SPE sample preparation optimization was done by spiking fresh (healthy) apples extracts with patulin, in order to establish the best SPE analytical conditions, such as the choice of stationary phase and its conditioning, washing and extraction steps.

The SPE optimized sequential procedure is:

This improved sample preparation changed the previously described (Li et al., 2007) washing solvent - acidified water instead of hexane - and also the eluent composition, methanol, a greener solvent, instead of the mixture hexane/ethyl acetate/acetone.

This optimized SPE-HPLC/DAD methodology showed an average recovery of 82% with a RSD value of 6% in the linear dynamic range 200 to 600 ppb (Table 2). The precision of the method showed a repeatability of 1.2% and a reproducibility value of 2.2%. According to the literature (Miller & Miller, 1988; IPQ, 2000) being these values below 10%, they indicate a good method performance. The overall SPE-HPLC methodology represents an economical, faster and routine usable methodology.

Table 2. Analytical parameters of improved SPE-HPLC methodology for patulin analysis.

#### **2.5.2 MEPS-HPLC/DAD methodology**

84 Ecotoxicology

A protocol including biphasic dialysis extraction, *in situ* acylation, as sample preparation and GC-MS (SIM) analysis was developed for detection and quantification of patulin (acetylated) in fruit juices (Sheu & Shyu, 1999). Another work presented the detection and

Capillary electrophoresis (CE) was also described as an alternative method for patulin analysis. In 2000, a capillary electrophoresis method was published as an analytical tool for rapid and highly sensitive analysis of patulin in cider; a fused silica capillary column was used, and the elution was done with an aqueous. The separation was achieved by migration of charged particles in the buffer. The cations migrated to the cathode and anions migrated to the anode under the influence of an electro-osmotic flow (EOF). In fact, compounds or mixtures of neutral and charged compounds can be analyzed by micellar electrokinetic capillary electrophoresis or micellar electrokinetic capillary chromatography (MECC). The method was described as having the advantage of requiring small amounts of sample and smaller amount of organic solvents when

For patulin analysis, two sample preparation procedures using SPE and MEPS were developed and associated with an improved reversed-phase HPLC methodology (Gaspar & Lucena, 2009). The purpose was to simplify the sample, liberating the patulin from its original matrix, and allowing therefore a better performance of the overall analytical

Linearity LOD

Table 1. Analytical parameters of improved HPLC methodology for patulin analysis (Elvira

Aiming a small scale sample preparation procedure, a previously described SPE approach (Li et al., 2007) was improved and optimized. The purpose was the analysis of patulin in fresh fruits, namely apples, but also in apple juices commercialized in Portugal. The SPE sample preparation optimization was done by spiking fresh (healthy) apples extracts with patulin, in order to establish the best SPE analytical conditions, such as the choice of

1. Using C18-SPE cartridges, pre-washed with (3 mL) methanol, methanol:water (3 mL, 10% methanol) and acidified water (3 mL MilliQ water-perchloric acid ((100):

(μg/L)

2161.5 0.9996 2.0 6.0 3.5

LOQ (μg/L)

Precision (RSD,%), n = 4

method. Table 1 shows the analytical parameters of the HPLC methodology.

Calibration

y = 136592x-

stationary phase and its conditioning, washing and extraction steps.

0.01,(v/v))) sequentially (illustrated in Figure 3, steps 1 and 2);

equation R2

quantification of C13 labeled patulin (Rychlik & Schieberle, 1999).

compared with HPLC (Tsao & Zhou, 2000).

Range (mg/L)

0.50

The SPE optimized sequential procedure is:

2. The column was not allowed to run dry;

**2.5.1 SPE-HPLC/DAD methodology**

16.8 0.03 –

Retention time (min)

& Lucena, 2009).

**2.5 Development of improved analytical methodologies**

Using the recent MEPS analytical tool (Abdel-Rehim, 2004), a new, simple, sample preparation method was developed for patulin analysis. MEPS methodology can be rationalized as a miniaturization of SPE analytical system; it employs smaller quantities of sample and eluent (few microliters), being also adequate to remove interferences, simplifying the analysis.

For patulin analysis, this new sample preparation methodology was performed using the following sequential steps:

This method development evidenced the relevance of several factors in this sample preparation methodology, MEPS: sampling speed was around 10 μL/s, sample introduction

(Figure 6).

<IP_ADDRESS>).

**3. Conclusions**

extraction.

for human health.

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 87

As it was mentioned before, patulin is formed in bruised apples as a result of contamination with fungi. The analysis of an infected (mouldy) apple revealed that it contains a very high amount of patulin (894 ppm in SPE-HPLC/DAD and 919 ppm in MEPS-HPLC/DAD), much higher than its LD50 (5 mg/Kg = 5 ppm). This result indicates how dangerous is the direct consumption of damaged fruits and their use in the production of fruit juices

Fig. 7. SPE-HPLC/DAD (A) and MEPS-HPLC/DAD (B) patulin analysis from a spiked commercial apple juice; spike 7.7 ηg /50 µL (conditions described in Section <IP_ADDRESS> and

Apple juice (cloudy) was also analysed by both methods showing a level of patulin lower than 6 ppb, the limit of quantification (LOQ) of both methods for this compound. Spiked matrices (Figure 7) were also analyzed, by both methods, to test the methodologies. The results revealed that the analyzed juice was produced with good sanitation criteria, satisfying the legislation limits (Commission Regulation (EC) No 1881/2006). These results show the necessity of constant surveillance of the occurrence of this toxin in fruit juices and evidenced that national public institutions should be capable of evaluating and determining food content in toxic substances in real time, due to the risk associated with consumption.

Patulin analysis is an important subject with social relevance. This chapter describes the most suitable analytical methods respecting accuracy, low detection limits and simple procedures for application in quality control, developed for the determination of patulin in food products, especially apple juice and its derivatives. Techniques such as thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC) were described together with sample preparation methodologies like liquid–liquid extraction (LLE) and solid-phase extraction (SPE) to determine the patulin. This chapter also describes, for the first time, an improved, simple, no time consuming, trace analysis micro extraction in packed syringe (MEPS) methodology for the determination of patulin in food products, and compares and discusses the use of MEPS and SPE in patulin

Having into account the importance of detecting this mycotoxin in food chain as marker of quality, this work will contribute to a better characterization and quantification of its presence in human diet and will make possible to determine the toxicological relevance of human exposure to patulin, its biological role and long-term implications of its consumption

was done in a fractionated mode (2 × 25 μL) and the washing step was not used; these procedures revealed a better recovery. The extracting solvent was of crucial importance methanol was much better than acetonitrile for the patulin analysis.

The method performance was achieved using five replicates, spiking the target samples (apples) with different quantities of patulin standard ranging from 200 to 600 ppb (Table 3). The average recovery was 69% with a RSD of 4%. The precision of the method showed a repeatability of 3.2% and a reproducibility value of 4.0%. Also here these values are below 10%, indicating a good precision for this new sample preparation methodology (Miller & Miller, 1988; IPQ, 2000). MEPS can be an excellent alternative to the SPE technique, being faster, less solvent consuming and less expensive than SPE.

Table 3. Analytical parameters of MEPS-HPLC methodology for patulin analysis.

#### **2.5.3 Analysis of food matrices**

Two food matrices: an infected apple and a commercial apple juice were analyzed using both sample preparation methodologies: SPE-HPLC/DAD and MEPS-HPLC/DAD. These analyses are shown in Figures 6 and 7.

Fig. 6. SPE-HPLC/DAD (A) and MEPS-HPLC/DAD (B) patulin analysis from a naturally infected apple; the UV spectrum is shown (conditions described in Section <IP_ADDRESS> and <IP_ADDRESS>).

As it was mentioned before, patulin is formed in bruised apples as a result of contamination with fungi. The analysis of an infected (mouldy) apple revealed that it contains a very high amount of patulin (894 ppm in SPE-HPLC/DAD and 919 ppm in MEPS-HPLC/DAD), much higher than its LD50 (5 mg/Kg = 5 ppm). This result indicates how dangerous is the direct consumption of damaged fruits and their use in the production of fruit juices (Figure 6).

Fig. 7. SPE-HPLC/DAD (A) and MEPS-HPLC/DAD (B) patulin analysis from a spiked commercial apple juice; spike 7.7 ηg /50 µL (conditions described in Section <IP_ADDRESS> and <IP_ADDRESS>).

Apple juice (cloudy) was also analysed by both methods showing a level of patulin lower than 6 ppb, the limit of quantification (LOQ) of both methods for this compound. Spiked matrices (Figure 7) were also analyzed, by both methods, to test the methodologies. The results revealed that the analyzed juice was produced with good sanitation criteria, satisfying the legislation limits (Commission Regulation (EC) No 1881/2006). These results show the necessity of constant surveillance of the occurrence of this toxin in fruit juices and evidenced that national public institutions should be capable of evaluating and determining food content in toxic substances in real time, due to the risk associated with consumption.

#### **3. Conclusions**

86 Ecotoxicology

was done in a fractionated mode (2 × 25 μL) and the washing step was not used; these procedures revealed a better recovery. The extracting solvent was of crucial importance -

The method performance was achieved using five replicates, spiking the target samples (apples) with different quantities of patulin standard ranging from 200 to 600 ppb (Table 3). The average recovery was 69% with a RSD of 4%. The precision of the method showed a repeatability of 3.2% and a reproducibility value of 4.0%. Also here these values are below 10%, indicating a good precision for this new sample preparation methodology (Miller & Miller, 1988; IPQ, 2000). MEPS can be an excellent alternative to the SPE technique, being

200 - 600 69 (4) 3.2 4.0

Two food matrices: an infected apple and a commercial apple juice were analyzed using both sample preparation methodologies: SPE-HPLC/DAD and MEPS-HPLC/DAD. These

Fig. 6. SPE-HPLC/DAD (A) and MEPS-HPLC/DAD (B) patulin analysis from a naturally infected apple; the UV spectrum is shown (conditions described in Section <IP_ADDRESS> and <IP_ADDRESS>).

Table 3. Analytical parameters of MEPS-HPLC methodology for patulin analysis.

Repeatability (RSD,%), n = 5 Reproducibility (RSD,%), n = 3

methanol was much better than acetonitrile for the patulin analysis.

faster, less solvent consuming and less expensive than SPE.

Recovery (RSD,%), n = 5

Range (μg/L)

**2.5.3 Analysis of food matrices**

analyses are shown in Figures 6 and 7.

Patulin analysis is an important subject with social relevance. This chapter describes the most suitable analytical methods respecting accuracy, low detection limits and simple procedures for application in quality control, developed for the determination of patulin in food products, especially apple juice and its derivatives. Techniques such as thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC) were described together with sample preparation methodologies like liquid–liquid extraction (LLE) and solid-phase extraction (SPE) to determine the patulin. This chapter also describes, for the first time, an improved, simple, no time consuming, trace analysis micro extraction in packed syringe (MEPS) methodology for the determination of patulin in food products, and compares and discusses the use of MEPS and SPE in patulin extraction.

Having into account the importance of detecting this mycotoxin in food chain as marker of quality, this work will contribute to a better characterization and quantification of its presence in human diet and will make possible to determine the toxicological relevance of human exposure to patulin, its biological role and long-term implications of its consumption for human health.

Patulin Analysis: Sample Preparation Methodologies and Chromatographic Approaches 89

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#### **4. Acknowledgements**

Portuguese Foundation for Science and Technology is acknowledged for funding PhD grants: FCT SFRH/33809/2009 (Inês Nunes) and FCT SFRH/SFRH/BD/40564/2007 (João Lopes).

#### **5. References**

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**4. Acknowledgements**

**5. References**

**5**

*Italy*

**Utilization of Marine Crustaceans as Study**

During last decades the productive activities, the increasing energy demand and the massive resource exploitation have caused extensive pollution phenomena that are to date spread on a worldwide scale. Therefore, the monitoring and assessment of environmental pollution is a subject of high concern owing to the implications that pollutants can exert on the environment, organisms and ecosystems, as well as on the quality of life of humans and

Pollution problems affect greatly the aquatic environments that are mainly sensitive to several typologies of contamination, such as chemical pollution, oil dumping, microbiological contamination from sewers, etc. These inputs can exert devastating effects

To date a lot of chemicals are utilized in productive processes and many new substances are synthesized every year; the utilization and introduction of these newly synthesized chemicals into the environment and in production cycles must be approved after an accurate evaluation of their eventual toxic properties against selected organisms with the main purpose to protect the safety of plants and animals and the human health. To do this several experiments useful to test the effects consequent to contact, inhalation and ingestion and to estimate the risks connected to the acute/chronic exposition in the natural and work environments have been proposed with the aim to define some fundamental parameters,

These evaluations need to be carried out using test-species which are representative of the environmental compartment under consideration; in this connection, the availability of testspecies able to furnish reliable and cheap results and to evaluate the activity of pollutants at the individual and ecosystem level is essential. Nevertheless, it is known that the tests on animals have ethical implications and often show problems connected to the reliability and to the application of results to the natural conditions. As a matter of fact, the test-organisms

on ecosystems with long-term consequences (Mille et al., 1998).

such as the acceptable dose, the risk dose, the lethal dose, etc.

**1. Introduction**

on public health.

**Models: A New Approach in Marine**

**Ecotoxicology for European**

Pane Luigi, Agrone Chiara, Giacco Elisabetta, Somà Alessandra and Mariottini Gian Luigi

**(REACH) Regulation**

*DIP.TE.RIS, University of Genova, Genova,*


Contents

**III**

**Chapter 16 281**

**Chapter 17 299**

Drugs Therapeutics Treatments **317**

**Chapter 18 319**

**Chapter 19 353**

**Chapter 20 377**

**Chapter 21 397**

**Chapter 22 413**

**Chapter 23 443**

**Chapter 24 453**

Convalescent Plasma: An Evidence-Based Old Therapy to Treat Novel

*by Saurabh Kumar, Chandra Devi, Subhabrata Sarkar, Vivek Kumar Garg, Priyanka Choudhary, Madhu Chopra, Vinit Sharma and Ravi Prakash*

Frequency of Hyperglycemia in Patients with Covid-19 Infection

Refocusing Functional Anatomy and Immunology of the Respiratory

Cellular Therapy as Promising Choice of Treatment for COVID-19

Repurposed Therapeutic Strategies towards COVID-19 Potential Targets Based on Genomics and Protein Structure Remodeling *by Ashok K. Singh, Aakansha Singh and Ankit Kumar Dubey*

Role of Anti-Viral Drugs in Combating SARS-CoV-2

Targeting Mononuclear Phagocytes to Treat COVID-19

Potential Therapeutics Pathways in Solving the Challenges

*by Meemansha Sharma, Thakur Uttam Singh,*

*Madhu Cholenahalli Lingaraju and Subhashree Parida*

Different Therapeutic Strategies to Tackle the Infection Associated

*by Sweta Kamboj, Rohit Kamboj, Shikha Kamboj, Rohit Dutt,*

*by Valeriy Ivanovich Vechorko, Evgeny Mikhailovich Evsikov, Oksana Alekseevna Baykova, Natalya Vadimovna Teplova*

*and Dmitriy Aleksandrovich Doroshenko*

*by Duygu Koyuncu Irmak and Erdal Karaoz*

*Reeva Chabbra and Priyanka Kriplani*

*by Brandt D. Pence and Theodore J. Cory*

Coronavirus Patients

of the COVID-19 Pandemic *by Tafirenyika Mafugu*

with COVID-19

Mucosa in the Advent of Covid-19

*by Humphrey Simukoko*

**Section 6**

and Pneumonia

**V**

**Chapter 32 581**

**Chapter 33 607**

COVID-19 Lockdown and the Aerosphere in India: Lessons Learned

*by Subhasmita Panda, Priyadatta Satpathy, Trutpi Das*

A Qualitative Study of Pre-Vaccine Decrease of Mortality

on How to Reduce Air Pollution

*by Vugar Mammadov and Lala Jafarova*

*and Boopathy Ramasamy*

from COVID-19


*2.7. Diagnostic Checking*

The final step in the development of model is diagnosing the ARIMA model. It is one of the significant steps of model development it point towards the appropriateness of model that inspects the assumptions of model unquestionably. The acceptability or appropriateness of model guarantees that the time series is in the time with model assumptions and that the prophecy of values is well founded. To examine the correlation of residuals with error terms, various diagnostic statistics and plots of residuals have been inspected to make it sure that whether these residuals correspond with error terms or not.

$$\text{MAE} = \frac{1}{N} \sum\_{i=1}^{N} |(X\_m)\_i - (X\_s)\_i| \tag{7}$$

$$\text{RMSE} = \sqrt{\frac{1}{N} \sum\_{i=1}^{N} [(X\_m)\_i - (X\_s)\_i]^2} \tag{8}$$

where *N* is the number of forecasting events, *Xm* the observed SPEI and *Xs* the predicted SPEI [49].

$$\text{MAE} = \frac{1}{N} \sum\_{i=1}^{N} |\mathbf{x}\_i - \mathbf{\hat{x}}\_i| \tag{9}$$

$$\text{MPE} = \frac{100\%}{N} \sum\_{i=1}^{N} \left( \frac{\mathbf{x}\_i - \mathbf{x}\_i}{\mathbf{x}\_i} \right) \tag{10}$$

$$\text{MAPE} = \frac{100\%}{N} \sum\_{i=1}^{N} \left| \frac{\mathbf{x}\_i - \hat{\mathbf{x}}\_i}{\mathbf{x}\_i} \right| \tag{11}$$

$$\text{MSE} = \frac{1}{N} \sum\_{i=1}^{N} |\mathbf{x}\_i - \mathbf{\hat{x}}\_i|^2 \tag{12}$$

where 1 *N N* ∑ *i*=1 is test set, *xi* predicted value and *x*ˆ*i* is actual value. (*N* is the number of total data points).

In this research, all of the SPEI forecasting ARIMA models have been developed using the forecasting packages already available in R-programming language. Packages used are t series, forecast, SPEI and Uurca [50].

#### **3. Results and Discussion**


Contents

Preface

The world is facing many environmental, food, nutritional, social, and economic security challenges. Most alarming is the conservation of natural resources, especially soil and water. Meeting food and nutritional security targets requires the implementation of time demand policies that ensure soil and water retention for sustainable productivity. This book examines ways of improving soil moisture

**Ram Swaroop Meena**

Mendel University in Brno,

India

**Rahul Datta**

Czech Republic

Institute of Agricultural Sciences,

management to support a sustainable ecosystem for the future world.


*Brief Report* **Plasma Flows in Solar Filaments as Electromagnetically Driven Vortical Flows**

## **Yuri E. Litvinenko**

Department of Mathematics, University of Waikato, Hamilton 3105, New Zealand<EMAIL_ADDRESS>
**Abstract:** Electromagnetic expulsion acts on a body suspended in a conducting fluid or plasma, which is subject to the influence of electric and magnetic fields. Physically, the effect is a magnetohydrodynamic analogue of the buoyancy (Archimedean) force, which is caused by the nonequal electric conductivities inside and outside the body. It is suggested that electromagnetic expulsion can drive the observed plasma counter-streaming flows in solar filaments. Exact analytical solutions and scaling arguments for a characteristic plasma flow speed are reviewed, and their applicability in the limit of large magnetic Reynolds numbers, relevant in the solar corona, is discussed.

**Keywords:** space physics; plasma physics


**Acknowledgements**

Portions of this work were supported by NIH research Grant MH64827 (L.D.S).

#### **Conflict of interest**

The views expressed in this article do not represent those of the Department of Veterans Affairs or the US Government


*2.2. Three-Temperature Model*

The three-temperature model estimates vegetation ET by introducing a reference leaf with no ET [34,48].

$$LE = R\_n - R\_{np} \frac{T\_c - T\_a}{T\_p - T\_a} \tag{1}$$

where *LE* is the latent heat consumed by vegetation ET. *Rn* and *Rnp* are the net radiation on the vegetation and reference leaf, respectively (W m−2). *Tc* and *Tp* are the surface temperature of the vegetation and reference leaf (◦C). The surface temperature could be obtained by thermal images, and the maximum *Tc* in the image is regarded as *Tp* [37,38]. *Ta* is air temperature (◦C). *Rn* and *Rnp* could be estimated according to [49].

$$R\_n = (1 - \alpha\_c)R\_s + \Delta R\_l \tag{2}$$

where *Rs* is solar radiation (W m−2). αc is the albedo of the vegetation canopy. To simplify the calculation, the empirical coefficient αc = 0.22 was used in this study [37]. Δ*Rl* is the net long-wave radiation (W m−2), which could be estimated by [50,51]

$$
\Delta R\_l = \left( 0.4 + 0.6 \frac{R\_\varepsilon}{R\_{so}} \right) \left( \varepsilon\_d \sigma T\_a^4 - \varepsilon\_c \sigma T\_c^4 \right) \tag{3}
$$

where *Rso* is the clear day solar radiation (W m−2), which is assumed to equal to *Rs* in this study as all the experiment were conducted in clear sunny days [41]. *εc* the canopy emissivity, and empirical coefficient *εc* = 0.98 was used here [37]. *σ* is the Stefan–Boltzman constant (5.67 × 10−8 W m−2 K−4). *εa* is the atmospheric emissivity and could be estimated according to [52]

$$
\varepsilon\_a = 0.92 \times 10^{-5} T\_a^2 \tag{4}
$$

If αc, *εc*, and *Tc* are replaced by αcp, *εcp* and *Tcp* in Equations (2) and (3), then *Rnp* could be estimated. As we use the leaf with the highest surface temperature in the canopy as the reference leaf in this study, αc, *εc* are assumed to be same to αcp, *εcp*.

The analysis procedures were written into a software named "A system to estimate evapotranspiration by infrared remote sensing and the three-temperature model", which can be downloaded and used freely from https://pan.baidu.com/s/19iuz5PIVjZOR96iVObYBqA.