*3.2.6. Ecological and precautional thinking in law: Examples from Norway*

realising that nature is servicing us through a number of vital "ecological services". Species diversity represents a large potential for new ways of producing food, medicines or other products, and these options imply that species diversity represent a "gene bank". Healthy ecosystems perform several functions - or services - on which we depend. They enable pollination of agricultural crops, provide clean water, air and soil, allow for decomposition of our wastes and neutralize pollution. The concept of ecological service is to an increasing degree used in national and international fora to promote nature conservation, and to implement the Biodiversity Convention. In order to illustrate the more or less hidden values of ecosystem services, some have tried to transform them into their economical value, including the astronomical costs of manual pollination of agricultural crops, for example, see [25]. The term ecological service is strongly "anthropocentric", which means that we focus on humankind's egoistic interests: *What is in it for me?* A broader perspective is the "biocentric" view: Life in general is in focus, respecting the intrinsic value of all life forms. Wilson [22] used both perspectives, but waiting for the intrinsic value to be seriously respected, he strongly argued for the anthropocentric motivation in nature conservation. Here, we find many duty-based

The vision of an "eco-policy" in which the society is in harmony with nature, has led to several new eco-concepts. The prefix "eco-" is often used in the sustainability debate: eco-friendly products, eco-food, eco-tourism, eco-efficiency practices designed to reduce waste, energy consumption, and resource use, etc. Within eco-effectiveness, the concept "cradle-to-cradle" is creative. Earlier, "cradle-to-grave" was a paradigm illustrating the linear use of resources resulting in waste, while "cradle-to-cradle" regards waste as a resource that can be recycled. Sometimes, the term "green" is used as a substitute: an environmentally friendly economy may be called green economy. The term "ecological footprint" has also become universal: It represents the amount of biologically productive land and sea area necessary to supply the

resources a human population consumes, and to assimilate associated waste, see [26].

Perhaps we should use, more often, the strong moral force in the eco-prefix to *warn*. Political decisions of today are often "eco-unfriendly". The term "ecological crime" is already in use, often shortened as "eco- crime". In Norway, eco-crime is in fact used for both ecological and economical crime, and an official body termed "ecocrime" was erected in 1989. On our journey towards a sustainable future we should probably have a stronger focus on environmentally

Literature has already produced a number of "eco-thrillers", describing how nature strikes back when ecological limitations are crossed. Recently, the term "eco-angst", or "eco-fear", was presented in a Norwegian magazine [27]. Originating from the USA, the concept describes a new mental anguish which seems to be spreading: Bad forecasts for the future, for instance due to climate change, cause depression [28,29]. This is a serious signal to us all, both to politicians and consumers. Eco-fear is due to our eco-crimes. Overconsuming societies led by short-sighted economical profit are not only ecologically unsustainable, but also *mentally*

arguments versus future generations.

134 Environmental Change and Sustainability

unfriendly activities.

*unsustainable.*

*3.2.5. Ecology in our mind: Eco-concepts matter*

Until 1981, most wildlife species in Norway could be hunted without restriction, and hunting of various predators was often encouraged by payment for killing. However, as a better understanding of ecological knowledge showed, predators play an important role in nature and several of these became endagered. Therefore, the Wildlife Act of 1981 (implemented in 1982) stated that all wildlife species should be protected. Hunting became allowed only during specific periods for selected species. This turnover of practice was called "the mirror principle". Wildlife in this connection encompassed all mammals, birds, reptiles and amphibians.

In 2009, a new act was adopted in order to protect biological, geological and landscape diversity and ecological processes through conservation and sustainable use: *Act of 19 June 2009 No. 100 Relating to the Management of Biological, Geological and Landscape Diversity (Nature Diversity Act).* The act reflected an ecological and precautional thinking, as exemplified by the following principles:

*Section 8 (knowledge base):* Official decisions that affect biological, geological and landscape diversity shall, as far as is reasonable, be based on scientific knowledge of the population status of species, the range and ecological status of habitat types, and the impacts of environmental pressures.

*Section 9 (precautionary principle):*If there is a risk of serious or irreversible damage to biological, geological or landscape diversity, lack of knowledge shall not be used as a reason for post‐ poning or not introducing management measures.

*Section 10 (ecosystem approach and cumulative environmental effects):* Any pressure on an ecosys‐ tem shall be assessed on the basis of the cumulative environmental effects on the ecosystem now or in the future.

*Section 11 (user-pays principle):* The costs associated with preventing or limiting any damage caused by a project to biological, geological and landscape diversity shall be borne by the project owner, unless this is unreasonable in the light of the nature of the project and of the damage.

Finally, the act introduced the concepts of *priority species* and *selected habitat types*. These are threatened species or nature types which will be given special concern through specific conservation programs.

In order to implement these various intensions, increased ecological knowledge is often needed about species and habitats, cumulative effects, and risks. We see how ecology feeds politics, and how political intentions again ask for more ecology to be able to practice ecological intentions.

#### **3.3. Global warming: A test on long-term thinking**

Already around 1970, specialists in atmospheric physics warned about a gradual global warming, due to increased concentration of CO2 caused by humans. A "greenhouse effect" due to "climate gases" was gradually considered as a dangerous threat both to man and biodiversity, and in 1992 the Convention on Climate Change was born in Rio de Janeiro to reduce emissions. A much used concept is "climate quota", which means that one country can pay another country for reducing their emissions, instead of reducing its own. Unfortunately, global CO2-concentrations have continued to rise, and at the Rio + 20 meeting in 2012, the international hope was to limit temperature increase to 2o C. The ghost in this situation is the concept of "tipping point" [30]. At a certain temperature, which is unknown, natural mecha‐ nisms may take over and rapidly push temperature still higher by feedback mechanisms, out of human control. For instance, more open water at the poles means that more solar radiation is absorbed, and if the permafrost of the tundra starts to melt, the strong greenhouse gas methan (CH4) will be released to an increasing degree. Besides a massive extinction of species, large groups of people will suffer due to unstable weather with drought, floods, strong cyclones, and rise in sea level. The concept of "climate refugees" is already on the lips.

Our hope is that temperature stabilises below the tipping point. Realistic visions about "carbon-neutral communities" based on "renewable energy" or "carbon-catching and storing" exist, but such options are followed up only slowly. Until now, extra "climate taxes" on CO2 emissions have had little effect. It is still cheap and comfortable to do business as usual. Also, there is a conflict between the two Rio aims: biodiversity and climate. In Norway, for instance, there is a great potential to save energy through better design in buildings etc. Also, existing hydroelectric power stations can be upgraded to become more efficient. Nature conservation organisations want to give energy saving priority, and argue that the "greenest energy is the energy which is not used". However, politicians are thinking along other lines, and the official energy politics in 2012-Norway is to increase the total use of energy. To achieve this, large plans exist for wind parks and new hydroelectric power stations. These plans are marketed as "green", "clean" or "environmentally friendly", although they may destroy nature of inter‐ national value. Furthermore, this extra energy is not needed at present, and it does not substitute a corresponding reduction in the use of fossil energy. This is an example of tensions between possibilities and practice, between NGOs and politicians, which raises temperature also in energy debates. Such debates are to a large degree rhetoric, and "greenwashing" of increased energy use is often a problem.

Solar energy is an interesting option for the future, and some people have the vision of a "solar age". No one owns the solar energy, and it can be downloaded by anyone anywhere. Seen from above, places where people live contain large areas of roofs on which this energy can be collected free.

#### **3.4. Nature as a source of life quality: How to express it?**

Many people claim that contact with nature has a strong recreational effect, and that loss of nature would reduce their life quality (Figure 1). The conceptual framework for these values is still limited, but we shall present some fruitful studies, that have appeared during the past decades. The mental value of nature contact is still undercommunicated and deserves both more concern and further research.

The Way We Think Shapes Our Future: On the Importance of Fruitful Concepts, Well-Founded Attitudes… http://dx.doi.org/10.5772/54702 137

biodiversity, and in 1992 the Convention on Climate Change was born in Rio de Janeiro to reduce emissions. A much used concept is "climate quota", which means that one country can pay another country for reducing their emissions, instead of reducing its own. Unfortunately, global CO2-concentrations have continued to rise, and at the Rio + 20 meeting in 2012, the

concept of "tipping point" [30]. At a certain temperature, which is unknown, natural mecha‐ nisms may take over and rapidly push temperature still higher by feedback mechanisms, out of human control. For instance, more open water at the poles means that more solar radiation is absorbed, and if the permafrost of the tundra starts to melt, the strong greenhouse gas methan (CH4) will be released to an increasing degree. Besides a massive extinction of species, large groups of people will suffer due to unstable weather with drought, floods, strong cyclones, and rise in sea level. The concept of "climate refugees" is already on the lips.

Our hope is that temperature stabilises below the tipping point. Realistic visions about "carbon-neutral communities" based on "renewable energy" or "carbon-catching and storing" exist, but such options are followed up only slowly. Until now, extra "climate taxes" on CO2 emissions have had little effect. It is still cheap and comfortable to do business as usual. Also, there is a conflict between the two Rio aims: biodiversity and climate. In Norway, for instance, there is a great potential to save energy through better design in buildings etc. Also, existing hydroelectric power stations can be upgraded to become more efficient. Nature conservation organisations want to give energy saving priority, and argue that the "greenest energy is the energy which is not used". However, politicians are thinking along other lines, and the official energy politics in 2012-Norway is to increase the total use of energy. To achieve this, large plans exist for wind parks and new hydroelectric power stations. These plans are marketed as "green", "clean" or "environmentally friendly", although they may destroy nature of inter‐ national value. Furthermore, this extra energy is not needed at present, and it does not substitute a corresponding reduction in the use of fossil energy. This is an example of tensions between possibilities and practice, between NGOs and politicians, which raises temperature also in energy debates. Such debates are to a large degree rhetoric, and "greenwashing" of

Solar energy is an interesting option for the future, and some people have the vision of a "solar age". No one owns the solar energy, and it can be downloaded by anyone anywhere. Seen from above, places where people live contain large areas of roofs on which this energy can be

Many people claim that contact with nature has a strong recreational effect, and that loss of nature would reduce their life quality (Figure 1). The conceptual framework for these values is still limited, but we shall present some fruitful studies, that have appeared during the past decades. The mental value of nature contact is still undercommunicated and deserves both

C. The ghost in this situation is the

international hope was to limit temperature increase to 2o

136 Environmental Change and Sustainability

increased energy use is often a problem.

more concern and further research.

**3.4. Nature as a source of life quality: How to express it?**

collected free.

**Figure 1.** Nature, with beauty and silence, is a rich source for mental recreation. For many people, nature is an arena for the quality of life. From Jotunheimen mountains, Norway. Photo: S. Hågvar.

Callicott [31] listed the various values of biodiversity, pointing to both *intrinsic values* (diversity for its own sake) and *instrumental values* like various goods, ecological services, and informa‐ tion. Finally, he added the *psycho-spiritual* values, which include aesthetic beauty, religious awe, scientific knowledge, etc. The present author followed up the concept of psycho-spiritual values and pointed at nature as an *arena for the quality of life* [32]. He addressed these values as important among the "third generation" of environmental problems. While pollution prob‐ lems were the main focus at the first UN conference in Stockholm in 1972, a "second genera‐ tion" of environmental problems including climate change and biodiversity loss were discussed in the UN conference in Rio, 1992. He argued that it is due time to address the psychospiritual values in an international context as a "third generation" environmental challenge, broadening the perspective of nature loss to include our mental health.

Edward O. Wilson is a famous biologist who has coined several biological terms, but he has also contributed with an important concept regarding our mental relation to nature. He argues that humankind has a subconscious interest and fascination for other life forms (Figure 2), and calls this "biophilia". The term literally means "love of life or living systems." In his book *Biophilia. The human bond with other species* [22] he reminds us that we have evolved in nature, just like all other species. Genetically, we have not changed significantly since we lived completely surrounded by nature. Since nature is our original home, it may not be surprising that our brain – maybe unconsciously – enjoys contact with nature. Wilson argues strongly for rescuing the species richness of our planet – not only because species have an inherent value, an ecological value, and an applied value – but also because the wonders of life fascinates us and elevates our life quality. Nature presents us for both beauty and mystery, which appeals to our brain. Biodiversity may thus be regarded as an important basis for the human spirit.

**Figure 2.** Wilson's "biophilia" concept indicates that man has a basic interest in, and is fascinated by other life forms. Larva of the Privet Hawk-moth, *Sphinx ligustri.* Photo: S. Hågvar.

Many studies have shown that contact with nature is good for our mental health. A classic book in this context is *The Experience of Nature* by Kaplan & Kaplan [33]. They documented that contact with nature – from gardening and urban parks to the experience of wilderness – increased people's life quality. Regarding nearby nature they concluded:

*People feel more satisfied with their homes, with their jobs, and with their lives when they have sufficient access to nature in the urban environment. People value natural settings for the diverse opportunities they provide – to walk, to see, to think. They are not necessarily aware of the many forms of encounter they have with nature or the variety of benefits that accrue.*

The modern, busy life often makes us feel worn out, and in need for a break or a recovery period. This worn-out state is generally not physical. Rather, these situations involve what we are calling "mental fatigue". A mentally fatigued person finds it burdersome to concentrate, and to pay attention to something uninteresting. Kaplan & Kaplan found that we have two types of attention: An active and voluntary "directed attention" which we use when we concentrate upon a task, and a passive, "involuntary attention" which requires no effort. When we become mentally fatigued so that we need recreation, it is the directed attention which is exhausted. A key issue here involves the concept of "inhibition". In order to maintain one's focus on a particular thought, you have to inhibit the stimuli around you that compete for your attention. To resist the attractions of what you hear, see (or mental associations you get), for instance if you work in a room together with other people, demands mental effort. In short, continuous distraction breaks down your valuable, directed attention. For such "burned-outpeople", nature is a good arena for mental recreation. The directed attention is resting and recovering, at the same time as we can enjoy nature's "soft fascination" by our passive, involuntary attention. Such psychological insight, based upon fruitful concepts, documents the value of having access to nature, as a mental source for recreation and life quality.

an ecological value, and an applied value – but also because the wonders of life fascinates us and elevates our life quality. Nature presents us for both beauty and mystery, which appeals to our brain. Biodiversity may thus be regarded as an important basis for the human spirit.

**Figure 2.** Wilson's "biophilia" concept indicates that man has a basic interest in, and is fascinated by other life forms.

Many studies have shown that contact with nature is good for our mental health. A classic book in this context is *The Experience of Nature* by Kaplan & Kaplan [33]. They documented that contact with nature – from gardening and urban parks to the experience of wilderness –

*People feel more satisfied with their homes, with their jobs, and with their lives when they have sufficient access to nature in the urban environment. People value natural settings for the diverse opportunities they provide – to walk, to see, to think. They are not necessarily aware of the many forms of encounter*

increased people's life quality. Regarding nearby nature they concluded:

Larva of the Privet Hawk-moth, *Sphinx ligustri.* Photo: S. Hågvar.

138 Environmental Change and Sustainability

*they have with nature or the variety of benefits that accrue.*

In the best-seller book *Last child in the woods: Saving our children from nature-deficit disorder* [34], Richard Louv worried about American children growing up without contact with nature. Not only is nature gradually lost where children grow up, but today's access to electronic media results in sedentary children who often prefer to stay indoors. Access to electrical outlets may be more attractive than access to nature! The author concluded that this lifestyle was both mentally and physically negative for children and coined the concept of "nature-deficit disorder". The book and the concept have inspired city planners to include green areas, especially remnants of real nature, as an important element in long-term city planning. For instance, in the author's home city, San Diego, "undeveloped areas" like canyons with a rich plant and animal life were earlier regarded as a problem since they were difficult to urbanize. Instead, a process was started to preserve this varied near-nature by creating "San Diego Urban Canyonlands Park". A new view on nearby nature had been triggered – but someone had to trigger it! There is now a growing understanding in the USA that protection of nearby nature has not only to do with biodiversity conservation, but also with the conservation of people's physical and mental health.

In his book *The big connection. How modern Swedes consider man's place in nature* [35], psychiatrist Nils Uddenberg studied how important contact with nature is for Swedish people. Both large studies with many people, and in-depth-interviews with fewer persons from different social groups documented that contact with nature was important for their life quality. In a large study covering one thousand people, 94 percent agreed with the following statement: "Strol‐ ling in the forest and fields makes me relaxed and harmonious". However, when people were asked to explain why nature mattered, they had problems finding words to explain their statement. They lacked concepts and formulations for this purpose. People often ended up with religious concepts like holy, cathedral, or divinity to try to communicate their experiences, even if they were not religious. In my opinion, here is a field where a further development of concepts should be welcomed. Emotions are valuable for us, but difficult to communicate. The situation might be paralleled by the difficulties of explaining a deep musical experience.

Nature's health-promoting effect in the Nordic countries Norway, Sweden and Denmark, came into focus three years ago by a report from Nordic Council of Ministers [36]. While epidemic diseases represented the main health problem in earlier times, the health of today's people is mainly threatened by *non-epidemic life style diseases*. There are two of them: Over‐ weight due to inactivity, and a depressed mental state, maybe due to a strong materialistic focus. Physical activity is good for both diagnoses, but the effect is best if the physical activity occurs in nature. Nature greets us with the absence of stress, combined with beauty, silence, and other positive experiences. Furthermore, physical and mental improvements support each other through so-called "effect-chains", resulting in improved life quality. In Sweden, and to a certain degree also in Norway, doctors have begun to order outdoor activity as a "green prescription" instead of tablets. Rather than heading to the chemist, people are heading to nature. This is cheap medicine, since nature is free and always open. The new trend reminds us of the importance of having access to near-nature, a resource which is shrinking in many Nordic cities and other densely populated areas. Areal planners are increasingly aware that to preserve green areas and greenbelts are good health politics. Health improvements by green prescription is, of course, also economically favourable for the society by shortening the hospital queues.

Concepts like threatened nature types and threatened species are now universally used. Sometimes we also see the term "threatened nature phenomenons". The Monarch butterfly is famous for its long, yearly migrations between Canada in north and Mexico and California in south. Although the Monarch is not an endangered species the annual migration is considered a threatened phenomenon by IUCN. Since there are very few overwintering sites where the adults aggregate, their populations become vulnerable. These sites are threatened by human activities, mainly logging, development, and agriculture. In 1984, the Monarch Project was created in order to save these sites. The Mexican conservation organization, Monarca, works closely with governmental agencies and local people to establish land protection and enhance alternative economic development in the region.

Also the spectacular, yearly migration of wildebeest and zebras over long distances through Tanzania and Kenya is a threatened nature phenomenon. Plans about a highway through the Serengeti national park, which would halt the necessary migrations, has till now been stopped, due to international protests.

The concept of threatened nature phenomenons deserves to be used more actively. People are not only fascinated by the teeming diversity of other life forms, but perhaps even more about what they are doing: What kind of processes nature reveals for us, and how it all functions. And least of all: Nature's secrets and mystery, about which we like to wonder. In light of the psychological value of nature experiences we should also use the concept of "threatened nature experiences". And furthermore, we should regard nature as a "resource of positive experien‐ ces". My favourite concept, trying to include nature's various contributions to our mental health, is *nature-dependent life quality*.


Author details

Hichem Guedri\* and Hafedh Belmabrouk Electronics and Microelectronics Laboratory, Physics Department, Faculty of Sciences, Monastir University, Monastir, Tunisia

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

Reconstruction of Three-Dimensional Blood Vessel Model Using Fractal Interpolation DOI: http://dx.doi.org/10.5772/intechopen.82247


**A.3 Proper index of arterial stiffness**

β is considered not to depend on pressure, while Ep decreases with a decrease in pressure. Most of the severe MR patients with hypertension were medicated with antihypertensive drugs. Therefore, in our study, systolic pressure in MR was lower than that in the healthy subject group, and there was no difference in Ep between the two groups. However, β was higher in MR. We consider that the increase in β was not caused by MR but caused by the history of hypertension. Actually, 38% of the patients were diagnosed as hypertension and medicated. Thus, β is suitable to evaluate arterial stiffness in low pressure subjects.


**3. Attachment**

138 Sexual Abuse – Breaking the Silence

There are several significant factors that contribute to the deleterious outcome from childhood sexual abuse. These factors, age at time of abuse, frequency of abuse, duration and severity of the abuse, originally identified by David Finkelhor (1984), formulates the

The age of the child when the abuse occurs signifies the interruption of a normal developmental trajectory. The traumatic sexualization occurs at a time when the child is not developmentally capable of understanding and processing sexual behavior and

Frequency and duration are two issues that create anticipatory fear and hyperarousal in a child. Frequency is how often the abuse occurs during a given time period. Duration is the ongoing nature of the abuse, (e.g., months, years) and can create a sense of futility in the child in that the abuse appears to be never ending. The longer-term consequences of frequency and duration are that they contribute to the socialization of trauma, creating a

The severity of the abuse creates fear, negative feelings regarding sex, (Sprei & Courtois, 1988; Jehu, 1989) and a general physiological hyperaroused state for the child (Teicher, Andersen, Polcari, Anderson, Navalta, 2002; van der Kolk, 2011). In cases where there was

While Finkelhor (1984) discusses the traumatic sexualization of the child as the core issue creating negative outcomes it is not, in and of itself, the primary contributing factor. The closer the relationship of the offender to the child, the stronger the level of betrayal, feelings of mistrust, and a damaged world-view and belief system. Courtois (2005) states, "Abuse by a stranger does not generate the divided loyalty and resultant denial or dismissal of abuse disclosure that is the case when abuse is intrafamilial, especially when it occurs in the

The presence or absence of familial support when disclosing abuse (Paine & Hansen, 2002) is another significant factor in the recovery process. There is significant literature that addresses the notion that when one is supported and believed by family members, the ability to heal and begin the recovery process has a better prognosis (London, Bruck, Ceci, & Shuman, 2005; Priebe & Svedin, 2008). The manner in which the family responds to disclosure is also important. Families who respond with demonstrations of aggressive anger and threats to the perpetrator in front of the victim only serve to instill fear and regret about

Resilience and the interplay of genetics combined with risk and protective factors have been examined with children in high-risk environments for over 15 years. Cicchetti and Blender (2004) define resilience as, "… comprehending the factors contributing to positive outcomes

Previous anecdotal information relied on the conceptualization that social support alone (a significant family member, a teacher, etc.) could provide the at-risk child with the required coping mechanisms to mediate adversity. Social support combined with the child's innate capacity to ward off the negative aspects of adversity are thought to be the primary predictors of the child's ability to deal with and successfully survive maltreatment, and thus

disclosing, due to the nature of the adults seemingly "out of control" behavior.

extreme, on-going abuse, the child may have been physically and genitally harmed.

**2. Factors that contribute to deleterious outcome**

"how and why" children can be so harmed by sexual abuse.

subsequently robs the child of childhood innocence.

nuclear family (involving parent and/or siblings)" (p. 95).

despite the presence of significant adversity…" (p. 17325).

sense of victimhood.

considered resilience.

Bowlby (1980) presented the significance of early caregiving experiences on the ability of a neonate to attach to its caregivers, and thus internalize "working models" or representations of positive caregiving experiences. Van der Kolk (2005) states, "A child's internal working models are defined by the internalization of the affective and cognitive characteristics of their primary relationships" (p. 402).

For humans, brain development is optimally done in a social context, that is with primary caregivers providing not only sustenance but nurturance as well. When the caregiving relationship is less than optimal or in fact abusive, such as in the cases of neglect or physical and sexual child abuse, the child will experience deleterious outcomes.

For the CSA survivor's health, neglect may have reverberations on many levels throughout his or her life. Psychological and physical health, interpersonal functioning, and overall well-being are impacted by levels of neglect. A significant consideration is that the CSA survivor may lack the primary belief that he/she is an individual of worth, deserving to take care of her physical health. Therefore, her ability to be an educated health consumer, to ask questions during medical exams, and her ability to follow-up with annual visits, required immunizations, and diagnostic testing, etc., may all be compromised.

The CSA survivor may not have experienced good role models and thus may not have learned that brushing ones teeth every day is a necessary component of good health care. He or she may not have learned about the necessity of hygiene, immunizations, and annual check-ups and treatment for childhood illnesses, such as ear infections. Not having a good role model may have taught her that these types of behaviors are not a necessary part of life and certainly not a consideration for good health.

#### **Posttraumatic Stress Disorder (PTSD), chronic PTSD, and Developmental Trauma Disorder**

CSA survivors can suffer with posttraumatic stress symptomatology and PTSD. PTSD is defined as exposure to a traumatic event in which the threat of death or serious injury or witnessing threat or serious injury or the threat to physical integrity has occurred. In addition, intense fear, helplessness, or horror responses to a traumatic event may have occurred (DSM IV, TR p. 428). Symptomatology include avoidance of the initial traumatizing event or environment, frequent dreams or nightmares of the event, intrusive thoughts and images, flashbacks – feeling as if the event is occurring again – which is generally triggered by sensory stimuli, and physiological reactivity, e.g. a heightened state of agitation, motor activity, or physical reactions.

Chronic PTSD generally occurs when there is more than one traumatic event and/or if the initial ongoing traumatic event was severe and ongoing, e.g., combat situations. The CSA survivor may have been abused by a family member as a child, experienced dating violence, married an abusive spouse, and/or may have been sexually abused again by a stranger and/or another family member. Very often, these types of lifelong traumatic experiences involve the notion that many survivors carry; that life usually entails traumatic events that

Childhood Sexual Abuse and Adult Physical and Dental Health Outcomes 141

The hypothalamic-pituitary-adrenal (HPA) axis is a mediating pathway of the stress response (Neigh, Gillespie, Nemeroff, 2009, van der Kolk, 2011) and it's the function of the HPA to modulate hormones that address stressful events. To promote survival, a chain reaction of powerful hormones and neurochemicals are produced to assist the individual in dealing with the immediate stressful event (Neigh, Gillespie, Nemeroff, 2009). Deactivation of these hormones occurs through a "negative feedback loop" alerting the individual that

However, if the stress response becomes chronic due to repeated exposure to stressors, defects at different levels of the negative feedback system, or both, the result is a sustained increase in the level of stress hormones and the initiation of pathological changes across multiple physiological systems, resulting in stress related diseases (p. 391; also see McEwen,

The sympathetic nervous system (SNS) is dominant over the parasympathetic nervous system (PNS), and will not yield to the PNS until some form of resolution takes place. Resolution can take the form of fighting, jogging, meditating (Howard, 2006). The adrenal glands release enough adrenaline to get your attention at the first sign of stress. Adrenaline also helps to "imprint" an emotional or traumatizing event. If the stress continues, the hypothalamus secretes corticotropin-releasing factor. In this process cortisol is also released. Cortisol is a steroid hormone that is produced naturally in the body to assist in the adaptive struggle when an individual is facing acute traumatic situations. When the stress or the traumatic situation is chronic, high levels of cortisol become toxic. Prolonged cortisol production impairs the immune system and thus, healing. Vulnerability to stress-related disorders and diseases, such as gastrointestinal disorders (ulcers) and heart disease commences. Chronic trauma and stress induces lower cortisol production and this decrease creates an enhanced autoimmune system. In the absence of any other illnesses, the autoimmune system will attack various systems within the body and create illnesses such as Fibromyalgia, Chronic Fatigue Syndrome, thyroid diseases, and Krohn's disease (Bergmann,

Minor results of this stress-related impairment include colds, flu, backaches, tight chest, migraine headaches, tension headaches, allergy outbreaks, and skin ailments. More chronic and life-threatening results can include hypertension, ulcers, accidentproneness, addictions, asthma, infertility, colon or bowel disorders, diabetes, kidney disease, rheumatoid arthritis, and mental illness. Killers that can result include heart

Chronic trauma, coupled with the severity of the abuse itself, has long-term devastating impact on health and mental health due to the chronicity of these physiological states.

CSA survivors may experience shame and self-blame regarding the sexual abuse and approach both mental health practitioners and health professionals with distrust, fear, and anxiety. CSA survivors may also experience a range of PTSD symptomatology that includes

**5. Mental health features of the CSA survivor that impede their health**

danger is no longer present. Neigh, Gillespie, and Nemeroff (2009) state,

2008).

2011). Howard (2006) states,

**outcomes**

disease, stroke, cancer, and suicide (p. 816).

avoidant behaviors, depression, and dissociation.

one cannot escape. CSA survivors with chronic PTSD often experience hyperarousal and hypervigilience that are continuous physiological states. Chronic PTSD frequently includes the inability to self-soothe and emotionally regulate. Health risk behaviors such as alcohol and substance abuse, cigarette smoking, eating disorders, and self-injurious behavior (cutting) are correlated with childhood sexual abuse. These health risk behaviors can be considered attempts by the CSA survivor, albeit poor ones, in the adaptive struggle in which self-medicating is necessary for survival. They may also be attempts to avoid intrusive memories.

 Because the nature of sexual abuse is chronic, usually perpetrated by a caregiver, and occurs during childhood, it impacts critical periods of neurobiological development (van der Kolk, 2011). When caregivers are abusive, absent or neglectful, and/or helpless in the face of trauma, children cannot develop a sense of safety and stability that emanates from the caregiver nor can they rely on that caregiver to restore a sense of calm and reliability. The child's responses to stress become diffuse and inadequate. The ability to self-soothe and emotionally regulate – managing stress, impulsivity, and anxiety – are markedly impaired. A significant issue is that the child experiences a sense of betrayal and perceives the world as a hostile and attacking environment.

Mental health practitioners and researchers have examined the concept of complex PTSD (Herman, 1992a, 1992b) and the tentative diagnosis of Developmental Trauma Disorder (Cook et al, 2005; van der Kolk, 2005). Cook et al (2005) state, "A comprehensive review of the literature on complex trauma suggest seven primary domains of impairment observed in (traumatically) exposed children: attachment, biology, affect regulation, dissociation, (e.g., alterations in consciousness), behavioral regulation, cognition, and self-concept" (p. 392). Included in these domains are the child's worldview and functional impairments such as academic, interpersonal, legal interactions, and vocation (van der Kolk, 2005). This tentative diagnosis, yet to be wholly accepted by the trauma field, appears to encompass the chronic and devastating nature of trauma to the developing child.


*Article* **Evaluation of Sample Preparation Methods for Non-Target Screening of Organic Micropollutants in Urban Waters Using High-Resolution Mass Spectrometry**

**Nina Huynh 1,2, Emilie Caupos 1,2,3, Caroline Soares Peirera 1,2, Julien Le Roux 1,2,\* , Adèle Bressy 1,2 and Régis Moilleron 1,2**

**Abstract:** Non-target screening (NTS) has gained interest in recent years for environmental monitoring purposes because it enables the analysis of a large number of pollutants without predefined lists of molecules. However, sample preparation methods are diverse, and few have been systematically compared in terms of the amount and relevance of the information obtained by subsequent NTS analysis. The goal of this work was to compare a large number of sample extraction methods for the unknown screening of urban waters. Various phases were tested for the solid-phase extraction of micropollutants from these waters. The evaluation of the different phases was assessed by statistical analysis based on the number of detected molecules, their range, and physicochemical properties (molecular weight, standard recoveries, polarity, and optical properties). Though each cartridge provided its own advantages, a multilayer cartridge combining several phases gathered more information in one single extraction by benefiting from the specificity of each one of its layers.

**Keywords:** emerging contaminants; high-resolution mass spectrometry; micropollutant fingerprint; non-target screening; solid phase extraction; statistical analysis; urban waters

#### **1. Introduction**

The presence of organic micropollutants in the aquatic environment is a major issue in many countries due to their potential ecotoxicological impact on aquatic organisms as well as their potential risks for human health. European directives concerning the quality of surface waters advocate better control of the presence of micropollutants, but they deal with priority organic molecules that are well known by scientists for their high environmental impact [1]. However, a lot of emerging molecules (e.g., substitutes of forbidden molecules, metabolites, and degradation byproducts) enter the environment due to human activities through wastewater effluents. The survey of these unknown molecules using non-target screening (NTS) enables us to anticipate further regulation on water quality [2]. This analytical approach has undergone high development in the last decade thanks to the spreading of high-resolution mass spectrometry, but it requires powerful analytical systems and computational software as well as non-specific sample preparation methods [3].

The increased resolving power of high-resolution mass spectrometry such as time-offlight mass spectrometry (TOF-MS) or Orbitrap-MS allows the accurate mass measurement and isotopic pattern identification [4]. Therefore, these techniques are suitable for the development of three main screening strategies based on accurate-mass databases [5,6]: multi-residue target screening [7], suspect screening [8–10], and NTS [11,12]. Contrary to target and suspect screening which aim to evaluate the presence of molecules based on

**Citation:** Huynh, N.; Caupos, E.; Soares Peirera, C.; Le Roux, J.; Bressy, A.; Moilleron, R. Evaluation of Sample Preparation Methods for Non-Target Screening of Organic Micropollutants in Urban Waters Using High-Resolution Mass Spectrometry. *Molecules* **2021**, *26*, 7064. https://doi.org/10.3390/molecules 26237064

Academic Editor: Thomas Letzel

Received: 2 November 2021 Accepted: 19 November 2021 Published: 23 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

a preliminary list, NTS is the characterization of substances present in a sample without any prior information, based on full scan analysis that enables the acquisition of all peaks comprised within a range of masses. It thus requires the subsequent selection (e.g., by statistical analysis) of features of interest (i.e., specific signal characterized by its retention time and *m*/*z* ratio) followed by their tentative identification with the use of databases and literature information [13,14].

In combination with high-resolution mass spectrometry acquisition methods, the computational technique for data processing is of primary interest. Molecular identification depends on high-resolution data precision [13] but also on the software possibilities to deconvolute, filter, re-align signals [15], associate raw formulas to features of interest and finally compare with existing spectra libraries for identification [16,17]. NTS studies can be performed using proprietary software (e.g., Agilent Masshunter and MPP, Waters Unifi, Thermo Compound Discoverer, ABSCiex MarkerView, SpectralWorks AnalyzerPro, associated with ChemSpider, SciFinder, METLIN, PubChem, or MassBank libraries) as well as free ones (e.g., MZmine, R, EnviMass, and MetFrag) [18] for the identification of molecules, using the information on the fragments. The qualification of a single sample requires a lot of computational treatment, and statistical studies are relevant as a complementary approach to assess organic contamination by comparing different samples. Statistical approaches in NTS have been used to differentiate samples in time [19] and space [20] or to highlight variations during processes [11]. Binary comparison, principal component analysis (PCA), and Venn diagrams are examples of methods that can be applied to a sample set in order to isolate features of interest (e.g., molecules in common between samples or specific to groups of samples) [21]. Discriminated samples can be further characterized, and molecules of interest can then be identified.

Data analysis and statistical evaluation of samples being time-consuming, the quality of acquired data must be guaranteed. Sample preparation is essential to ensure the detection of as many molecules as possible and thus needs to be as unselective as possible. Different strategies and methods can be used in order to reach this objective and also to prevent samples from contamination. Direct injection of water samples can be employed to avoid potential contaminations or losses during sample preparation. However, the low concentration of molecules and interferences with other constituents of the sample (e.g., solid particles, colloids, or organic matter) can prevent the detection of trace organic compounds. Preparation techniques such as filtration [22], concentration [23], and solid-phase extraction [24] are widely used for targeted quantitative analysis and for NTS to decrease interferences and to concentrate the samples.

Most NTS studies employ a sample preparation step that is considered to be as "exhaustive" as possible, and little information is available about the efficiency of SPE for NTS purposes. For target screening purposes, knowing the properties of the targeted molecules makes it easier to select the most appropriate cartridge and phase. Concerning NTS studies, the choice of a cartridge is trickier given that, for example, the choice of a cartridge that is recommended for polar compounds will only retain polar compounds and thus, will leave the non-polar compounds un-retained. As a result, most non-target studies employ *universal* phases such as Oasis HLB to retain as many compounds as possible. Validating the extraction and analysis steps is also a challenge for NTS. The choice of standards to assess cartridge efficiency or to evaluate and correct matrix effects are tricky since the compounds studied in NTS are supposed to be unknown. Therefore, a mix of internal standards composed of a large variety of molecules (in terms of molecular weight, polarity, acidity, functional groups, etc.) is often used to assess the efficiency of the method in recovering and analyzing a large panel of compounds [25–28]. In addition to the injection of internal standards, various methods can be used to check the stability of the instrument or to correct the standard deviation of each feature, such as the injection of a pooled sample (consisting of a mix of all samples to be analyzed), regularly during the sequence. These normalization methods were recently reviewed for NTS applications in lipidomics [29]. Finally, using replicated injections to discard signals that are not repeatable and that could

lead to false-positive identifications is a commonly used step in NTS studies [25,28,30–32]. Still, only a few studies compared the efficiency of different preparation methods for NTS purposes. One study showed that non-target strategies clearly discriminated the signals in terms of number and type of features obtained with several preparation methods (liquidliquid extraction and solid-phase extraction on two cartridges) while the extraction yields of targeted molecules were not significantly different between those techniques [33]. In all cases, the properties of the cartridge, the pH of the sample, as well as elution solvents, and conditioning solvents will play a major role in the selection of molecules finally detected in the analyzed sample. A literature review revealed that numerous phases (e.g., HLB, ENV+, C18, X-CW, X-AW, X-C, and X-A, individually or in series), different sample pH, and different conditions of elution were used in NTS studies for environmental applications (Table 1). Several studies used a multilayer cartridge (composed of HLB, ENV+, X-AW, and X-CW) developed to extract a wide range of micropollutants [27].

**Table 1.** Examples of SPE cartridges, pH of samples, and elution solvents used for sample preparation in environmental studies using non-target screening for the analysis of water samples.

a N.A. = Not Available.

The overall objective of this work was to assess the influence of the extraction method, in particular the type of SPE phase/cartridge, on the non-targeted HRMS analysis of organic contaminants in urban water. To reach this objective, we pursued the following specific aims: (i) to test different nature of phases to extract a large range of micropollutants in urban waters, based on a panel of 9 commercially-available stationary phases and designed for a variety of applications (i.e., from non-polar/moderately polar to more polar compounds and ionic species); (ii) to develop a strategy based on relevant indicators to compare the results, such as the number of detected molecules, their range, and characteristics; and (iii) to apply the optimized SPE method and the developed data analysis strategy to urban water samples.

#### **2. Results and Discussion**

#### *2.1. Comparison of the Cartridge Retention Ability*

In this section, the retention abilities of several cartridges (ENV+, X-A, X-AW, X-CW, HLB pH 2, HLB pH 6, Multilayer, X-C, C18 ENV+, SDBL, and C18) were compared on the basis of different indicators.

#### 2.1.1. Discrimination of Cartridges Based on Optical Properties

To get a preliminary overview of the diversity of retained organic materials, optical properties (UV absorbance at 254 nm and 3D-spectrofluorescence spectra) of one sample from a river located in an urbanized area (Marne River) were measured before and after extraction (i.e., on the water phase during the SPE loading step). These optical properties are commonly used to characterize dissolved organic matter (DOM). As DOM is composed of molecules of various sizes and polarities with which micropollutants can interact, the behavior of DOM can be related to the behavior of micropollutants. Based on these interactions (e.g., hydrogen bonding, hydrophobic, van der Waals, or dipole-dipole interactions), fluorescence can be used for a better understanding of the fate of micropollutants, for example, in advanced wastewater treatment processes such as adsorption onto activated carbon [40]. DOM retention on SPE cartridges was evaluated to characterize their ability to retain a large variety of organic materials but also to determine if it can hinder the detection of micropollutants. DOM is indeed known to sometimes reduce the adsorption of target compounds on SPE cartridges (competition effect) and to limit the ionization of molecules for their detection in mass spectrometry [41,42]. The good retention of DOM on SPE cartridges could thus be detrimental to the detection of organic molecules in non-target analyses. Figure 1 presents the retention of DOM on each SPE cartridge based on the percentage change of UV absorbance at 254 nm before and after extraction. The highest recoveries were obtained for X-A, X-AW, X-C, and the Multilayer phases (recoveries ≥ 90%). Other cartridges exhibited significantly lower retention of organic materials (<60%). Especially, extractions with the HLB cartridge retained fewer organic materials (~60%). SDBL, X-CW, and HLB (pH 6) do not seem to be appropriate phases for a large screening of organic material. *Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 20

**Figure 1.** Retention of DOM based on UV absorbance at 254 nm (UV254) for different SPE cartridges. UV254 retention (%) was calculated as the percentage change between UV254 of the Marne River sample and UV254 measured at the outlet of each cartridge (during the loading phase). **Figure 1.** Retention of DOM based on UV absorbance at 254 nm (UV254) for different SPE cartridges. UV254 retention (%) was calculated as the percentage change between UV254 of the Marne River sample and UV254 measured at the outlet of each cartridge (during the loading phase).

For a better evaluation of the quality of DOM retained on the cartridges, 3D-fluorescence spectra were acquired from the Marne River sample before and after extractions. Fluorescence regional integration (FRI) was performed [40,43], calculating the regional For a better evaluation of the quality of DOM retained on the cartridges, 3D-fluorescence spectra were acquired from the Marne River sample before and after extractions. Fluorescence regional integration (FRI) was performed [40,43], calculating the regional fluorescence

fluorescence intensities for each excitation-emission matrix (EEM) spectrum. The EEM

V. They correspond to large molecules like polysaccharides and humic-like substances [23,44,45]. Other fluorophores (regions I and II) were also observed, representing more hydrophilic and smaller molecules, proteins, and aromatic amino acids [46]. Table 2 presents fluorophores retention depending on the phase of SPE used. Similar results were

**Table 2.** DOM retention (%) based on fluorophores for different SPE cartridges (indexes FRI derived

FRI 84 85 64 70 58 71 58 94 89 55 49

FRI 76 69 67 68 58 86 48 96 90 59 47

III FRI 72 46 46 55 55 73 27 92 86 50 28

IV FRI 68 62 64 65 45 80 41 93 86 53 45

FRI 70 47 50 56 53 55 28 93 86 48 28

As expected, the cartridges retained aromatic proteins and humic-like materials (regions I, III, and V [37,40]). Higher retention was observed for XA and XAW (≥85%). Like the results obtained with UV absorbance, the extractions on SDBL and XCW phases led to poor DOM retention (retentions generally lower than 50%). It is interesting to note that regions II and IV were well retained by XA, XAW, and Multilayer (≥80%). They can be associated with the retention of simpler, more polar, and nitrogenous compounds [47].

**layer SDBL XA XAW XC XCW**

**pH 2 HLB Multi-**

obtained with other indexes derived from [43] (data not shown).

**ENV+ ENV+ HLB**

from [43]).

Region I

Region II

Region

Region

Region V

**Index C18 C18**

intensities for each excitation-emission matrix (EEM) spectrum. The EEM spectrum of the Marne River sample before extraction (Supplementary Materials Figure S1) exhibited mainly two fluorophores: one in the region III of FRI and the other in region V. They correspond to large molecules like polysaccharides and humic-like substances [23,44,45]. Other fluorophores (regions I and II) were also observed, representing more hydrophilic and smaller molecules, proteins, and aromatic amino acids [46]. Table 2 presents fluorophores retention depending on the phase of SPE used. Similar results were obtained with other indexes derived from [43] (data not shown).

**Table 2.** DOM retention (%) based on fluorophores for different SPE cartridges (indexes FRI derived from [43]).

As expected, the cartridges retained aromatic proteins and humic-like materials (regions I, III, and V [37,40]). Higher retention was observed for XA and XAW (≥85%). Like the results obtained with UV absorbance, the extractions on SDBL and XCW phases led to poor DOM retention (retentions generally lower than 50%). It is interesting to note that regions II and IV were well retained by XA, XAW, and Multilayer (≥80%). They can be associated with the retention of simpler, more polar, and nitrogenous compounds [47]. Based on EEM spectra, these three cartridges seem to be the most efficient to retain hydrophobic materials as well as smaller and more polar compounds for global screening of the molecules present in the sample.

#### 2.1.2. Discrimination of Cartridges Based on HRMS Features

Samples were then analyzed by HRMS, and differences between SPE cartridges were investigated through criteria classically used in omics studies. A PCA of the HRMS features found in the different extracts of the Marne River sample was performed to quickly identify similarities and differences between the feature sets obtained from each cartridge (Figure 2).

All cartridges were clearly discriminated by the first two components of the PCA (covering 45.3% of the total variance), which indicated that each type of SPE phase extracted specific sets of HRMS features. The first component (PC 1) was mostly correlated with the features derived from the Multilayer cartridge and explained 30% of the dataset variability. The second component (PC 2) explained 15% of the variability and discriminated all individual cartridges. Polymeric cartridges (ENV+, SDBL, and C18/ENV+) were all clustered near the center of the PCA, meaning that they all extracted similar features. Cartridges for which sample loading was performed at pH 2 (HLB pH 2 and XA) also exhibited similar behavior. Features from anionic exchange cartridges (XA and XAW) were almost identical.

To better understand the repartition of cartridges obtained by the PCA (Figure 2), fingerprints (i.e., bubble plots of all detected features) were compared between the most different cartridges (XCW, ENV+, HLB, and the Multilayer cartridge) for the Marne river sample (Figure 3).

2.1.2. Discrimination of Cartridges Based on HRMS Features

the molecules present in the sample.

(Figure 2).

Based on EEM spectra, these three cartridges seem to be the most efficient to retain hydrophobic materials as well as smaller and more polar compounds for global screening of

Samples were then analyzed by HRMS, and differences between SPE cartridges were investigated through criteria classically used in omics studies. A PCA of the HRMS features found in the different extracts of the Marne River sample was performed to quickly identify similarities and differences between the feature sets obtained from each cartridge

**Figure 2.** PCA (graph of individuals) of Marne sample extracts on different SPE cartridges. **Figure 2.** PCA (graph of individuals) of Marne sample extracts on different SPE cartridges.

**Figure 3.** Fingerprints of detected features from the Marne River sample after SPE on ENV+ (blue), HLB (orange), Multilayer (green), and XCW (red) cartridges. The size of bubbles is proportional to the area of the feature. **Figure 3.** Fingerprints of detected features from the Marne River sample after SPE on ENV+ (blue), HLB (orange), Multilayer (green), and XCW (red) cartridges. The size of bubbles is proportional to the area of the feature.

The Multilayer cartridge displayed the most intense features on the entire *m*/*z* range, whereas ENV+ displayed more intense features for *m*/*z* < 400, and HLB retained more intense features with *m*/*z* > 400. XCW showed more intense signals at retention times greater than 15 min (i.e., less polar compounds), whereas most intense signals on the two other individual cartridges were obtained at retention times before 15 min. Thus, the PC 2 component of the PCA may describe the polarity of the retained features on each cartridge. This hypothesis is supported by the fact that the C18 cartridge, which is designed for less polar compounds, was close to the XCW one on the PCA. Finally, a comparison of common features retained by the cartridges was performed (Figure 4). The Multilayer cartridge displayed the most intense features on the entire *m*/*z* range, whereas ENV+ displayed more intense features for *m*/*z* < 400, and HLB retained more intense features with *m*/*z* > 400. XCW showed more intense signals at retention times greater than 15 min (i.e., less polar compounds), whereas most intense signals on the two other individual cartridges were obtained at retention times before 15 min. Thus, the PC 2 component of the PCA may describe the polarity of the retained features on each cartridge. This hypothesis is supported by the fact that the C18 cartridge, which is designed for less polar compounds, was close to the XCW one on the PCA. Finally, a comparison of common features retained by the cartridges was performed (Figure 4).

**Figure 4.** Upset diagram, representing the intersection of the different cartridges in terms of retained features. The "set size" represents the total number of features from each cartridge, the "Intersection size" represents the size of the intersection (i.e., number of features in the intersection designated by the dots), the dots represent the cartridges intersected (i.e., number of intersections). **Figure 4.** Upset diagram, representing the intersection of the different cartridges in terms of retained features. The "set size"represents the total number of features from each cartridge, the "Intersection size" represents the size of the intersection (i.e., number of features in the intersection designated by the dots), the dots represent the cartridges intersected (i.e., number of intersections).

The first thing to notice is that the Multilayer cartridge had the biggest set size among the studied cartridges, meaning that it retained the highest number of features. The two closest cartridges in terms of retained features were the ENV+ and C18/ENV+ cartridges with 49 common features, which is not surprising given the similar nature of the phases. The cartridge involved in the highest number of intersections was the SDBL (23 intersections for 210 features intersected); however, the cartridge with the highest number of features intersected was the Multilayer one (20 intersections for 212 features intersected). Therefore, the Multilayer was the cartridge with the highest number of specific features (323 features only found in this cartridge), but also the one with the highest number of intersected features, making it the most interesting cartridge for non-target studies by retaining the most extensive set of features. The first thing to notice is that the Multilayer cartridge had the biggest set size among the studied cartridges, meaning that it retained the highest number of features. The two closest cartridges in terms of retained features were the ENV+ and C18/ENV+ cartridges with 49 common features, which is not surprising given the similar nature of the phases. The cartridge involved in the highest number of intersections was the SDBL (23 intersections for 210 features intersected); however, the cartridge with the highest number of features intersected was the Multilayer one (20 intersections for 212 features intersected). Therefore,the Multilayer was the cartridge with the highest number of specific features (323 features only found in this cartridge), but also the one with the highest number of intersected features, making it the most interesting cartridge for non-target studies by retaining the most extensive set of features.

Compared to the results obtained when considering the optical properties (Section 2.1.1), the cartridges that retained more fluorescent organic materials exhibited a lower number of HRMS features (e.g.*,* XA, XAW). On the contrary, SDBL and XCW showed low retention of fluorescing materials but a high number of HRMS features. This could be due to the competition of DOM with organic compounds for the adsorption on the SPE cartridge [41,48] or to matrix effects in the HRMS analysis. Interestingly, this behavior can also be observed for the same cartridge used at different pH. Extraction on HLB at pH 6 gave a high number of features in HRMS and low retention of fluorescing organic materials (fluorophores), contrary to the extraction on HLB at pH 2. Notably, the Multilayer cartridge exhibited both retention of fluorophores and a large number of HRMS features; this cartridge thus seemed less affected by organic content. Compared to the results obtained when considering the optical properties (Section 2.1.1), the cartridges that retained more fluorescent organic materials exhibited a lower number of HRMS features (e.g., XA, XAW). On the contrary, SDBL and XCW showed low retention of fluorescing materials but a high number of HRMS features. This could be due to the competition of DOM with organic compounds for the adsorption on the SPE cartridge [41,48] or to matrix effects in the HRMS analysis. Interestingly, this behavior can also be observed for the same cartridge used at different pH. Extraction on HLB at pH 6 gave a high number of features in HRMS and low retention of fluorescing organic materials (fluorophores), contrary to the extraction on HLB at pH 2. Notably, the Multilayer cartridge exhibited both retention of fluorophores and a large number of HRMS features; this cartridge thus seemed less affected by organic content.

#### *2.2. Characterization of the Cartridge Retention Ability with Different Matrices 2.2. Characterization of the Cartridge Retention Ability with Different Matrices*

Based on the previous observations (HRMS fingerprints and DOM retention), five cartridges were retained for further investigation with different matrices: the Multilayer cartridge (highest number of retained features and highest intensities), HLB at pH 6 (universal cartridge, used in many studies), ENV+ (universal cartridge, representing the polymeric cartridges clustered in the PCA), XAW (high retention of fluorophores) and XCW (retaining more apolar compounds). Three types of samples were extracted and compared, with the increasing complexity of matrix (i.e., presence of organic and inorganic constituents): Ultrapure water (Milli-Q), surface water (Marne River), and wastewater effluent (WWe). Based on the previous observations (HRMS fingerprints and DOM retention), five cartridges were retained for further investigation with different matrices: the Multilayer cartridge (highest number of retained features and highest intensities), HLB at pH 6 (universal cartridge, used in many studies), ENV+ (universal cartridge, representing the polymeric cartridges clustered in the PCA), XAW (high retention of fluorophores) and XCW (retaining more apolar compounds). Three types of samples were extracted and compared, with the increasing complexity of matrix (i.e., presence of organic and inorganic constituents): Ultrapure water (Milli-Q), surface water (Marne River), and wastewater effluent (WWe).

#### 2.2.1. Recoveries of Internal Standards 2.2.1. Recoveries of Internal Standards

To compare the different SPE cartridges and evaluate the corresponding matrix effects, the recovery of internal standards was first calculated (Figure 5). To compare the different SPE cartridges and evaluate the corresponding matrix effects, the recovery of internal standards was first calculated (Figure 5).

**Figure 5.** Recovery of internal standards after extraction by the cartridges for different matrices. **Figure 5.** Recovery of internal standards after extraction by the cartridges for different matrices.

> In Milli-Q, recoveries ranged from 4% to 63% for atenolol-d7, from 35% to 112% for caffeine-d9, from 113% to 154% for carbamazepine-d8 and from 40% to 155% for sulfamethoxazole-d4. Carbamazepine-d8 exhibited an overall great recovery with all cartridges and showed the least differences between cartridges. On the contrary, atenolol-d7 gave lower recoveries, and sulfamethoxazole-d4 and caffeine-d9 exhibited large differences in recoveries between cartridges. Carbamazepine-d8 has the highest log Kow among the four molecules; its better recovery on all cartridges could be explained by its lower hydrophilicity. Extractions were all carried out at pH = 6.5–7, meaning that, for atenolol-d7, caffeine-d9 and carbamazepine-d8, the acidic form was predominant, In Milli-Q, recoveries ranged from 4% to 63% for atenolol-d7, from 35% to 112% for caffeine-d9, from 113% to 154% for carbamazepine-d8 and from 40% to 155% for sulfamethoxazole-d4. Carbamazepine-d8 exhibited an overall great recovery with all cartridges and showed the least differences between cartridges. On the contrary, atenolol-d7 gave lower recoveries, and sulfamethoxazole-d4 and caffeine-d9 exhibited large differences in recoveries between cartridges. Carbamazepine-d8 has the highest log Kow among the four molecules; its better recovery on all cartridges could be explained by its lower hydrophilicity. Extractions were all carried out at pH = 6.5–7, meaning that, for atenolold7, caffeine-d9 and carbamazepine-d8, the acidic form was predominant, whereas, for

sulfamethoxazole-d4, its basic form is predominant. The only molecule showing a significant difference between XAW and XCW cartridges (designed for compounds with pKa < 5 and pKa > 8, respectively) was atenolol-d7 (pKa = 9.6), with a better recovery on XCW as expected. The strong acidity (pKa of sulfamethoxazole-d4 = 1.6) or basicity (pKa of caffeine-d9 and carbamazepine-d8 are 14 and 13.9, respectively) of the three other compounds may explain their less contrasted retention of those two cartridges. Overall, the Multilayer cartridge gave the highest recoveries of those standards (despite a quite poor recovery of caffeine-d9).

Lower recoveries of caffeine-d9 were recorded with the Marne River and WWe samples for all five cartridges as compared to the Milli-Q water sample. The three universal cartridges (Multilayer, HLB, and ENV+) were more affected by complex matrices. The ionic XCW cartridge showed the least difference in recoveries for the three matrices. However, the XAW cartridge exhibited a very low recovery of most internal standards from the Marne River sample, while the two other samples (Milli-Q and WWe) showed similar recoveries. Better retention of caffeine-d9 was expected on the XCW cartridge because of its high pKa value of 14. Compared to other cartridges, recoveries of caffeine-d9 on XCW was low but more consistent across the different types of water. Its lower recovery can be explained by the employed elution step that was recommended by the cartridge supplier for specific compounds (Alprenolol, Acetaminophen, and Clomipramine). The elution step could be optimized with this cartridge to obtain better recoveries for internal standards such as caffeine-d9 as well as for the total number of features retained.

An unexpected high signal was obtained for carbamazepine-d8 and sulfamethoxazoled4 in the Marne River sample after SPE, leading to excessive recovery values (>1000%), which were therefore not included in Figure 5. The extraction of those two components on WWe, led to signal suppression as compared to the Milli-Q sample. Such matrix effects were previously reported in wastewater effluent with ion suppression of 34% for sulfamethoxazole, 5.4% for caffeine, and 23% for carbamazepine on Strata-X cartridges [49]. Atenolol-d7 exhibited various behaviors, with a signal enhancement for HLB and XAW, signal suppression for ENV+, and both signal suppression (WWe sample) and signal enhancement (Marne River sample) for the Multilayer cartridge and XCW.

Matrix effects (i.e., signal suppression or enhancement) or adsorption competition on the cartridge were observed for all detected internal standards. A high suppression was especially observed for the XAW cartridge, in accordance with the impact of adsorbed organic materials already described in Section 2.1.2.

#### 2.2.2. Range and Properties of Retained Features

The number of features retained on each cartridge was compared for the WWe and the Marne River sample (Table 3). As mentioned previously, the Multilayer cartridge displayed the highest number of features compared to the other cartridges, which explains its widespread use in non-target screening studies [27,35,36]. HLB and ENV+, commonly considered as "universal cartridges", were also quite efficient for the Marne River sample, retaining 32% and 47% fewer features than the Multilayer cartridge, respectively. They also retained the highest number of features from the WWe sample, ENV+ being the most efficient. XCW retained 11% more features from the Marne River sample than ENV+, which suggests the presence of cationic substances in this sample. Table 3 displays the properties of the features retained on each cartridge for both samples.

The Multilayer cartridge retained globally bigger molecules than other cartridges, as seen from both average *m*/*z* and weighted average *m*/*z*. For the two types of samples, HLB also retained features with larger molecular sizes and ENV+ smaller ones. When those values were not weighted by the feature area, no clear trend could be observed. The polarity of retained features (as described by the average retention) was also not significantly different between the cartridges. However, it can be noted that the Multilayer cartridge average retention of features from the Marne River sample (46% ACN) was equal to the mean of the average retention of all four other cartridges (i.e., the four phases used in the

Multilayer cartridge). For the WWe sample, the total area of the detected features followed the same ranking order as the total number of features (i.e., ENV > HLB > XAW > XCW). However, this was not the case for the Marne River sample, for which ENV+ exhibited 15% less detected features compared to HLB but a slightly more intense total signal. This observation indicates that the molecules retained on ENV+ were more ionizable, therefore different from HLB, or better retained, as it was demonstrated by the difference in their fingerprints (Figure 3). The Multilayer cartridge showed the highest total intensity among all cartridges, which demonstrates its ability to retain a large number of compounds with a high signal. This property is clearly beneficial for NTS studies to obtain clean mass spectra and thus to allow easier identifications.

**Table 3.** Properties of features retained on various SPE cartridges. The average retention is given as the percentage of acetonitrile in the mobile phase (ACN) needed to elute a given feature. The weighted values correspond to the average parameter, weighted by the intensity of each individual marker.

The distribution of *m*/*z* values was visualized for both types of the sample matrix and for each cartridge (Figure 6). To the authors' knowledge, this representation has not yet been used for the characterization of HRMS data, although it is useful for identifying differences between samples. Comparison of the cartridges shows that ENV+ was less effective in retaining molecules with *m*/*z* > 600, whereas HLB covered a larger range of *m*/*z*. It is interesting to note that the Multilayer cartridge combined the ranges of *m*/*z* distribution of ENV+, HLB, XAW, and XCW.

#### *2.3. Application: Evaluating a Disinfection Treatment by Performic Acid*

The above-mentioned indicators showed that the Multilayer cartridge was the most efficient among the different tested cartridges and that it is the most suitable to be used as a universal cartridge. It was therefore tested in a real-case application to characterize the evolution of organic compounds during a disinfection treatment of wastewater effluents by performic acid (PFA). Raw wastewater effluent and the same effluent treated with 30 ppm of PFA (contact time of 10 min) were extracted with the Multilayer cartridge and analyzed by HRMS (Figure 7).

The Multilayer cartridge allowed the detection of a wide number of compounds covering the whole range of *m*/*z* values at low and high retention times. Sample after treatment showed a significant decrease in the area of the markers with a retention time between 17 and 21 min and an increase for the markers between 5 and 7 min compared to the non-treated wastewater.

layer cartridge.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 12 of 20

**Figure 6.** *m*/*z* distribution of the retained features on the different cartridges. \* Number of features for Multilayer WWe were divided by eight. **Figure 6.** *m*/*z* distribution of the retained features on the different cartridges. \* Number of features for Multilayer WWe were divided by eight. analyzed by HRMS (Figure 7).

**Figure 7.** Bubble plot of WWe (blue) and WWe after 30 ppm PFA treatment during 10 min (orange) extracted on a Multilayer cartridge. **Figure 7.** Bubble plot of WWe (blue) and WWe after 30 ppm PFA treatment during 10 min (orange) extracted on a Multilayer cartridge.

**Figure 7.** Bubble plot of WWe (blue) and WWe after 30 ppm PFA treatment during 10 min (orange) extracted on a Multi-This observation suggests that compounds with lower polarity (i.e., higher retention time) were transformed into more polar ones by the treatment. Table 4 displays the distribution of the number of markers before and after treatment in different zones defined by the *m*/*z* and retention time ranges. Zone 2 (bigger and more polar molecules) exhibited a significant increase in both the number of markers and their total area. Zone 1 and zone 3 (molecules with *m*/*z* < 500) showed a decrease by almost a third of the number of markers after treatment, and a decrease in their total intensity as well. Finally, PFA treatment slightly increased the number of markers in zone 4 (bigger and less polar molecules), but the overall intensity of signals decreased in that zone. Globally, these results show that PFA treatment formed more polar molecules and removed a larger proportion of small molecules.

**Table 4.** Distribution of markers before and after PFA treatment.

#### **3. Materials and Methods**

#### *3.1. Chemicals and Standards*

All standards were obtained from Sigma-Aldrich (Dr. Ehrenstorfer, Augsburg, Germany, purity > 99%). Methanol (MeOH), ammonia (35%), hydrochloric acid (37%), and formic acid (98%) were LCMS grade and purchased from Fischer Scientific (Illkirch Cedex, France). Ethyl acetate (EtAc), dichloromethane (DCM), and acetone were obtained from Merck (Darmstadt, Germany), and acetonitrile (ACN) was purchased from Biosolve (Dieuze, France), at analytical grade. HPLC water (Milli-Q) was produced from deionized water using a Millipore Milli-Q system (IQ 7000, Merck, Darmstadt, Germany) equipped with an LC-pak polisher (Merck, Darmstadt, Germany).

Deuterated standards were used to evaluate matrix effects from different samples, to evaluate the extraction efficiency on the various SPE phases, and to correct the retention time of the chromatograms. A mixed solution of 4 deuterated compounds (atenolold7, caffeine-d9, carbamazepine-d8, sulfamethoxazole-d4) was prepared in MeOH at a concentration of 10 mg/L for further injection in samples.

Before sampling and analysis, glassware was washed with TFD4 (Franklab, Montignyle-Bretonneux, France), rinsed with deionized water, and calcined at 500 ◦C to remove any trace of organic contamination.

#### *3.2. Sample Collection and Preliminary Preparation*

Various types of samples were used: Milli-Q water produced in the laboratory, surface water under anthropic pressure (Marne river, France), and wastewater effluent (WWe).

Thirty liters of water from the Marne River were collected from a bridge at Chennevièressur-Marne (GPS coordinates: 48.79071188130754, 2.5215935793518462) downstream the wastewater treatment plant of *Marne Aval* (Noisy-le-Grand, France). The sample was collected in 3 × 10 L amber glass bottles and filtered the same day, using 0.7 µm glass fiber filters (GF/F Whatman). After homogenization, aliquots of 500 mL were prepared and completed to 1 L with Milli-Q water in order to avoid potential matrix effects occurring during the SPE loading step. The initial pH and dissolved organic carbon (DOC) were 8.5 and 1.89 mg C/L, respectively. Some samples were acidified, either to pH 6.5 with 50 µL of formic acid or to pH 2–3 with 400 µL of formic acid according to recommendations for specific SPE cartridges.

Ten liters of treated effluent were collected in June 2019 from the wastewater treatment plant of *Seine Amont* (Valenton, France) in 10 L amber glass bottles and filtered on 0.7 µm glass fiber filters (GF/F Whatman). The initial pH and DOC were 7.9 and 7.1 mg C/L, respectively. One liter subsamples were acidified to pH 6.5 with 50 µL of formic acid.

#### *3.3. SPE Cartridges*

#### 3.3.1. Selection of Cartridges

Strata-X cartridges (500 mg, 6 mL) (Phenomenex, Le Pecq, France) are ionic-based polymeric sorbents, chosen to retain compounds according to their pKa: Strata X-AW (for pKa < 5), Strata X-CW (for pKa > 8), Strata X-A (for pKa < 2), Strata X-C (for pKa > 10.5). Oasis HLB (200 mg, 6 mL) (Waters, Milford, MA, USA) is a reversed-phase cartridge used to select a wide range of substances, including neutral, basic, acidic, and polar ones. Silicabased cartridges Strata C18 (200 mg, 6 mL) and Strata SDBL (500 mg, 6 mL) (Phenomenex, Le Pecq, France) are employed to retain neutral hydrophobic and non-polar molecules. ENV+ (500 mg, 6 mL) (Biotage, Glamorgen, UK) is a polymeric phase appropriate for the retention of polar analytes. C18/ENV+ (400 mg, 6 mL) (Biotage, Glamorgen, UK) is a layered cartridge composed of the previous ENV+ phase as the bottom layer and a C18 phase as the top layer to improve the range of analytes possibly extractable. Finally, a homemade Multilayer cartridge made of Oasis HLB (200 mg), Isolute ENV+ (150 mg), Strata X-AW (100 mg), and Strata X-CW (100 mg) was prepared according to the method developed by Kern et al. [27].

#### 3.3.2. Extraction Protocol

Samples were spiked with the mix of internal standards at 100 ng/L, 24 h prior to extraction and stored at 4 ◦C. Experimental conditions for conditioning, loading, and elution on each cartridge followed recommendations of suppliers, or the method developed by Kern et al. for the Multilayer cartridge [27], and are described in Table 5. All extractions were carried out on Visiprep (Sigma-Aldrich, Augsburg, Germany) and Autotrace (AT280, Caliper) SPE systems. All cartridges were loaded with 1 L of sample at pH 6–7, except for Strata X-A (pH 2–3 recommended) and Oasis HLB, for which both pHs were tested. Before elution, the cartridges were dried for 30 min. After elution, the extracts were stored in the dark at 4 ◦C prior to their analysis. SPE extracts were evaporated under a stream of nitrogen, then reconstituted in 1 mL of Milli-Q water and MeOH (80/20, *v*/*v*), and filtered through 0.2 µm PTFE filters before a 10 µL injection on the analytical system.

**Table 5.** Description of the methods used for water sample extraction on the different SPE cartridges.

#### *3.4. UV-Visible and 3D Fluorescence Analyses*

Spectroscopic measurements were performed on samples before and after filtration on the SPE cartridges (loading phase). UV-Visible analyses were conducted on a spectrophotometer (UviLine 9400, Secomam, Aqualabo, Champigny-sur-Marne, France), and 3D-fluorescence spectra were obtained by a spectrofluorometer (FP-8300, Jasco, Pfungstadt, Germany). Those apparatuses were both equipped with a Xenon lamp and a 1 cm-quartz cell. For the fluorescence spectra, EEM were generated by scanning excitation wavelengths from 240 to 450 nm (every 5 nm), and the emission wavelengths were detected between 250 to 600 nm (every 2 nm), at a scan speed of 1000 nm/min and response at 0.1 s. To avoid any effect of the intern filter, samples with an absorbance at 254 nm higher than 0.1 were diluted.

3D-fluoresence indexes where chose according to previous studies [40,43]. Fluorescence regional integration (FRI) was performed, regions I to V were calculated and compared.

#### *3.5. LC-HRMS Analyses*

LC-HRMS analyses of the Marne River samples were conducted on SYNAPT HDMS QTOF (Waters) coupled with a Nano ACQUITY UPLC system (Waters). The column was an ACQUITY UPLC Peptide BEH C18, 130 Å (100 µm × 100 mm, 1.7 µm). The mobile phase was Milli-Q water with 0.1% formic acid (A) and ACN (B) following the gradient described in Supplementary Materials Table S1, for a total run time of 40 min. Analyses were performed in positive mode (ESI+) with screening between 50 and 1000 *m*/*z*. LC-HRMS analyses of Milli-Q and WWe samples were performed with a Vion–UPLC-IMS-QTOF (Waters) equipped with an ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 µm) column and the corresponding pre-column. The mobile phase was Milli-Q water + 0.1% formic acid (A) and ACN + 0.1% formic acid (B) following the gradient described in Supplementary Materials Table S1, for a total run time of 34 min. Analyses were performed in positive mode (ESI+) with screening between 50 and 1000 *m*/*z*. To ensure data quality, several steps were implemented.

Quality insurance procedure: Before each analysis, a quality reference standard, consisting of 9 components (Acetaminophen, Caffeine, Leucine enkephalin, Reserpine, Sulfadimethoxine, Sulfaguanidine, Terfenadine, Val-tyr-val, Verapamil), was injected five times to check the system performance by calculating the mass error deviation, the average peak width and, when available, the CCS (collision cross section) error for those compounds. If the mass error deviation was higher than 2 ppm or the peak width was longer than 3.0 sec, a system calibration was conducted. The same procedure was repeated until the expected conditions were met. Each sampling sequence began with three blank injections to wash the column and five pool injections to stabilize the column. The pool sample consists in a mix of equal volume of each sample and is used as a quality control. Each sample was then injected in randomized triplicate to minimize the effect of instrumental deviation. For data treatment, only the features detected in every replicate of a given sample were considered. Every 10 injections, a pool was re-injected to monitor the system. The clustering of these pool samples in the PCA was checked to assess the reproducibility of the system.

Raw HRMS data were exported directly after acquisition and converted in \*.mzML format via MSConvert (Version 3) [50,51] for further treatment in R software (Version 3.6.2). Pre-treatment of raw data was performed with the *Patroon* package [52], using OpenMS for peak picking and alignment. Features that were not present in all replicates of a given sample were discarded. PCA was performed with the *FactoMineR* [53] and *factoextra* [54] packages, and Upset diagrams were plotted with the *UpSetR* package [55].

The extraction efficiency of an SPE cartridge was determined based on the area of detected internal standards in the samples compared to the area of a standard mix injected. The number of detected features and the total area of the signal (i.e., sum of the areas of each feature) were compared for each cartridge. The average *m*/*z* and average retention of a sample were calculated from the *m*/*z* values and retention times of each feature detected

in the sample. Retention was expressed as the percentage of ACN needed to elute a given feature at the corresponding retention time. Values of average *m*/*z* and average retention were also weighed by the area of each feature.

#### **4. Conclusions**

The goal of this work was to compare the efficiency of several commercially available SPE cartridges and of a homemade Multilayer cartridge (as developed by Kern et al. [27]) for non-target screening purposes. Various parameters were monitored, such as the recovery of a limited number of analytical standards, the number and properties of detected features in HRMS, or the global retention of DOM (through 3D fluorescence measurements) on each cartridge. The Multilayer cartridge was the most effective at retaining internal standards and a large range of HRMS features, and it was not as affected by the adsorption of DOM as other cartridges (i.e., adsorption competition effects and/or matrix effects). It seemed to take advantage of the four phases involved in its composition (HLB, ENV+, XCW, and XAW) in terms of diversity of compounds (i.e., polarity and molecular size).

Although the other cartridges had some specificity missed by this Multilayer cartridge (e.g., an important number of specific features was only retained on the ENV+ cartridge), its efficiency in extracting a great number of compounds was clearly demonstrated. This observation could be further confirmed by using different analytical tools (e.g., gas chromatography) or different ionization modes and sources to increase the range of molecules detected. Moreover, a further investigation involving the tentative identification of some retained features could be performed. The optimization of extraction conditions (e.g., masses and combinations of sorbents, types, and volumes of eluting solvents) could also be investigated to retain an even greater number of features. It would finally be useful to better characterize the chemical space (log Kow, pKa, chemical functions, etc.) covered by each cartridge. A larger number of internal standards covering a broader range of chemical properties could thus be studied. This could help determine the relevance of each type of cartridge towards specific purposes (e.g., the detection of a larger number of polar or ionic compounds or some specific families of molecules).

**Supplementary Materials:** The following are available online. Figure S1: EEM matrices of Marne river (A) before extraction, (B) after extraction on X-A, (C) after extraction on HLB, (D) after extraction on C18/ENV+, (E) after extraction on SDBL, (F) after extraction on the Multilayer cartridge, Table S1: Description of the gradient used for non-target analysis.

**Author Contributions:** Conceptualization, A.B., J.L.R. and R.M.; methodology, A.B., C.S.P., E.C. and J.L.R.; software, J.L.R. and N.H.; validation, A.B., E.C., J.L.R. and N.H.; formal analysis, E.C. and N.H.; investigation, C.S.P. and N.H.; resources, A.B., E.C., J.L.R. and R.M; data curation, E.C. and N.H.; writing—original draft preparation, E.C. and N.H.; writing—review and editing, A.B., E.C., J.L.R., N.H. and R.M.; visualization, N.H.; supervision, A.B., J.L.R. and R.M.; project administration, A.B.; funding acquisition, A.B., E.C. and J.L.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study has been conducted within the projects Cosmet'eau (OFB/AESN),Screenatm'eau (OSU-EFLUVE) and WaterOmics (ANR-17-CE34-0009). The authors gratefully acknowledge the French Biodiversity Agency (OFB, formerly ONEMA), Agence de l'Eau Seine-Normandie (AESN), the French National Agency for Research (ANR) and the Observatoire des Sciences de l'Univers— Enveloppes FLUides de la Ville à l'Exobiologie, OSU-EFLUVE) for financial support.

**Data Availability Statement:** Raw HRMS data of Marne river samples are available at https://doi. org/10.5281/zenodo.5589621, accessed on 18 November 2021.

**Acknowledgments:** The authors would like to thank Damien Habert (CRRET) and Emmanuelle Mebold (PRAMMICS) for their technical support on HRMS instruments.

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

**Sample Availability:** Samples of the compounds are available from the authors.

#### **References**

*Article*


*3.3. Proposed Biosynthetic Pathway of Albucidin*

Structurally, albucidin is closely related to oxetanocin A (Figure 2b), which has been isolated from the culture of *Bacillus megaterium* NK84-0218 [22]. Both compounds are the only known naturally occurring nucleosides featuring four membered oxetane rings in their structure. From a structural view, albucidin is 2 -dehydroxymethyl oxetanocin A. Two genes, *oxsA* and *oxsB,* encoding a putative HD domain phosphohydrolase and a cobalamin-dependent S-adenosylmethionine radical enzyme

have been reported to be responsible for oxetanocin biosynthesis [23]. dAMP, dADP and dATP were identified as direct oxetanocin precursors [24]. The product of *oxsB* catalyses the contraction of the deoxyribose ring, while the product of *oxsA* is responsible for the removal of one or multiple phosphates from a phosphorylated 2 -deoxyadenosine derivative [24,25]. Through the simultaneous actions of OxsA and OxsB, the phosphorylated 2 -deoxyadenosine is converted to the oxetanocin A precursor, its aldehyde form, which must be reduced to complete biosynthesis [24]. This reaction is not encoded by the genes within the oxetanocin A cluster and is likely to be carried by an unidentified enzyme of *B. megaterium* NK84-0218.

Gene inactivation studies have given evidence that two genes, *albA* and *albB*, are required for the production of albucidin. Both genes encode putative SAM radical proteins. At the protein level, the *albA* gene shows homology to biotin synthases and the *albB* gene shows homology to the product of the oxetanocin biosynthetic gene *oxsB*. Despite the high structural similarity of albucidin and oxetanocin A, the homologue of the second oxetanocin biosynthetic gene *oxsA* cannot be found within the albucidin cluster or in the genome of albucidin producer *S. albus* subsp. *chlorinus* NRRL B-24108. The homology of the *albB* gene to *oxsB* implies that the product of *albB* might also be responsible for the ring contraction reaction in albucidin biosynthesis. However, the structural differences between albucidin and oxetanocin and the absence of an *oxsA* homologue imply substantial differences in biosynthetic routes leading to the biosynthesis of the nucleosides. Due to the lack of an *oxsA* homologue that is responsible for the dephosphorylation of adenine deoxyribonucleotides during oxetanocin biosynthesis, we propose that deoxyadenosine is used instead of dAMP, dADP or dATP as a precursor for albucidin production. The product of *albB* is likely responsible for the contraction of the deoxyribose ring of deoxyadenosine (Figure 3) in a similar manner as its homologue OxsB catalyses oxetane ring formation in oxetanocin A biosynthesis [24]. As a result of this reaction, the aldehyde form of oxetanocine A is formed. The conversion of the latter into albucidin is likely to be catalysed by the product of *albA,* which removes the aldehyde group from the 2'-position (Figure 3).

**Figure 3.** The proposed scheme of albucidin biosynthesis. 2 -Deoxyadenosine (**1**) is converted into the aldehyde form of oxetanocin A (**2**) by the product of *albB*. The latter is then converted into albucidin (**3**) by the product of *albA*.

In this paper, we report the identification, cloning, and heterologous expression of the albucidin biosynthetic gene cluster from *Streptomyces albus* subsp. *chlorinus* NRRL B-24108. Albucidin is a nucleoside phytotoxin featuring a rare oxetane ring in its structure. This metabolite shows herbicidal activity against a broad spectrum of grass and broadleaf weeds. In treated plants, albucidin induces metabolic perturbation, chlorosis, and bleaching [10]. The exact MOA of the compound remains unknown. The identification of the albucidin cluster presented in this paper enables biosynthetic studies of albucidin, optimization of its production as well as albucidin supply for the determination of its MOA.


**8. Customer-based reliability indices**

The most widely used reliability indices are averages that weight each customer equally. Customer-based indices are popular with electric companies [14] since a small residential customer has just as much importance as a large industrial customer. Regardless of the limitations they have, these are generally considered acceptable techniques showing adequate measures of reliability. Indeed, they are often used as reliability benchmarks and improvement targets. The formulae for customer-based indices include: