UNIVERSITY OF PISA
Faculty of Mathematical, Physical and Natural Sciences
Master Degree in Marine Biology
“Nanoremediation. A genotoxicity study on
mussel (M. galloprovincialis) gill cells.”
Tutors: Candidate:
Prof. Giada Frenzilli Massimo Genovese
Prof. Marco Nigro
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Table of contents
1. Abstract... 3
2. Introduction ... 5
2.1. Definition of Pollution and sediment management ... 5
2.1.1. Main contaminants related to water pollution ... 8
2.1.2. Heavy metals and PAHs: characteristics and toxic action ... 10
2.2. Nanoparticles in environmental remediation ... 14
2.3. NanoBonD project ... 20
2.4. Bioindicators and biomarkers ... 24
3. Aim of the work ... 26
4. Material and Methods ... 26
4.1. Nanomaterials used ... 26
4.2. Experimental procedure ... 34
4.2.1. In vitro experiments ... 34
4.2.2. In vivo experiment ... 35
4.3. Tests used ... 38
4.3.1. Comet assay ... 38
4.3.2. Cytome assay ... 40
4.3.3. Statistical analysis ... 40
5. Results ... 41
5.1. In vitro experiments ... 41
5.2. In vivo exposure experiment ... 44
6. Discussion ... 46
7. Conclusions ... 50
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1. Abstract
Most countries face serious enviromental problems regarding maintaining and restoring the quality of air, water and soil. In many cases, conventional remediation treatments show limited results in reducing levels of pollutants. Nanotechnology seems to be a promising solution in approaches to remediation (nanoremediation). Nanotechnology is related to interdisciplinary use of particles and structures at the nanoscale (<100nm in any dimension). The aim of the following work is to consider the potential use of cellulose and dextrine-based nanomaterials in nanoremediation, on their possible use to limit toxic substances actions in water environment (substances like polycyclic aromatic hydrocarbons and heavy metals in particular). This work is part of a project (NanoBonD) involving research partners (University of Pisa, Florence, Siena, polytechnic of Milan, Turin) and ‘Acque Industriali’ company. In vitro/in vivo experiments were conducted using Mytilus galloprovincialis gills byopsies samples as an ideal sea enviroment bioindicator. The Comet assay in its alkaline version (pH≥13) was used to measure a reversible genetic alteration on isolated cells. This test allows us to estimate DNA single strand breaks, double strand breaks and alkali-labile sites. DNA strand breakage is a well-known biomarker of genetic toxicity in environmental monitoring and cytome assay, including micronucleated cells evaluation was also performed. This test highlights a permanent, fixed DNA damage, which can be due to the action of toxic agents with clastogen or aneuploidogen properties. It is a widely used genotoxicity biomarker, which provides an accurate measure of DNA damage resulting in chromosomes breakage or their mis-segregation through mithosis. Indeed, it is applied to detect nuclear abnormalities (blebs, buds, nucleoplasmic bridges, ecc…) and micronuclei presence, potentially correlated with the exposure to toxic agents able to interact directly with the DNA or with cellular and nuclear structure, i.e. spindle apparatus, cytoskeleton or nuclear membrane. Experiments have focused on measuring any potential genotoxic action of the nanomaterials synthesized specifically for the NanoBonD project, which should be potentially applied for the nanoremediation.
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Artificial seawater (ASW) treated with nanomaterials was used to expose mussels. Once identified an adequate nanomaterial batch, subsequent experiments focused on exposing mussels to polluted waters treated with nanomaterials were performed in order to measure any protective and/or toxic potential effect of these materials. Results show that elutriates treated with nanomaterials damage biological systems. They show genotoxic action for reasons that have to be further investigated in order to proceed to in situ investigations. These nanomaterials have been synthetized by using biologically compatible materials (cellulose nanofibers and cyclodextrins) so the genotoxic action revealed is probably related to the nanomaterial synthesis processes or maybe to the resulting polymer properties. It is proposed to synthetize new nanosponge batches using different reagents or processes and further investigations are needed in order to perform new in vivo studies.
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2. Introduction
2.1. Definition of Pollution and sediment management
The term ‘Pollution’ is defined as the introduction into the natural environment of contaminants or pollutants that cause several undesired modifications (Merriam, Webster, 2010). It has been always closely linked to the civilization process that, especially from the industrial revolution onwards, inevitably brought environment to critical levels of contamination as we know today. Pollution may operate through chemical substances and/or physical agents (such as light, heat and radioactivity) either from foreign or naturally occurring sources. In former case xenobiotics come in contact with the environment as synthetic compounds not belonging to it (Fossi, 2000). In the latter, instead, natural substances and/or physical agents are present in excess in comparison with natural levels (Park C., 1992). For example, a natural water component, such as naturally occurring chemicals (calcium, sodium, iron, manganese, etc.), may become a contaminant since their concentration increase to harmful levels for aquatic flora and fauna. Indeed critical issues occur since pollutants addition rate is faster that the environment can manage by dispersion, recycle, transformation in harmless form, especially for most persistent contaminants(Encyclopedia Britannica, 2011). Air pollution may be an example: it comes from both natural and anthropogenic sources, pollution occurs since human-caused gas emissions alter the delicate biogeochemical cycle atmosphere-related (Declaration of the United Nations Conference on the Human Environment, 1972). The role of ecotoxicology scientists turns out to be of crucial importance, being focalized to understand potential harmful interaction between xenobiotic or natural contaminants, organisms and ecosystems related highlighting their mechanisms of action through the environment (Bacci, 1994). These contaminants may cause a short- or long- term damage by polluting air, water and soil, affecting, in this way,
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both animal and plant health: it is very important to understand that all the planet is linked by complexes physico-chemical-biological webs of contact, this means that pollutants may be also bounded to such interactions. Pollution sources are oil refineries, chemical plants, coal-fired power plants, motor vehicle emissions, petrochemical plants, heavy industry, plastics factories, agricultural activities, nuclear wastes (Environmental performance report, 2007; Beychok, Milton, 1967) from which about 400 million metric tons of dangerous wastes are generated each year (Pollution, Encarta, 2009-10-21). The major forms of pollution including main contaminants related are listed below:
• Atmospheric pollution: caused by the introduction of chemicals and/or particles into the atmosphere. Among these we could mention common pollutants such as carbon monoxide, chlorofluorocarbons (CFCs), sulfur dioxide, nitrogen oxides and particulate matter (PM);
• Soil pollution: caused by the introduction of pollutants by spilling process. The most significant soil contaminants are hydrocarbons, chlorinated hydrocarbons, heavy metals, herbicides and pesticides;
• Water pollution: caused by the intentionally or by accidental spills discharge of wastewater as industrial waste and untreated sewage into surface water (such as in a river, lake, wetland, or ocean). Chemical compounds such as chlorine, fertilizers and pesticides may be present because of urban and agricultural runoff.
Related to water environment, is of particular interest of this work to mention the issues related to management of contaminated sediments. Sediment (matter that settles to the bottom of a liquid) is a material naturally occurred in water by processes of erosion and subsequently transported by the action of wind, water, or ice, and by the force of gravity acting on the particles. For example, sand and silt can be carried in suspension in river water and on reaching the sea be deposited by sedimentation. Sediments are derived of natural physical, chemical and biological
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components and act as a key subject in environmental food cycles and water quality. Organic material in sediments is derived from the decomposed tissues of plants and animals, from aquatic and terrestrial sources, and from various point and nonpoint wastewater discharges. Contaminants from industry, agriculture and urban runoff may reach the sediments in rivers, lakes, and the ocean. Polluted sediments may contains PCBs, dioxin, pesticides, heavy metals, harmful hydrocarbons and other pollutants (especially in many harbors-related environments). Dredging processes that often take place in ports, can re-suspend pollutants in the water from the sediment, further threatening the seafood and tourism industries that depend on healthy fish and clean water. Dredging is an excavation activity usually carried out underwater, in shallow seas or freshwater areas with the purpose of taking up bottom sediments. This process is often used to keep waterways navigable and it is a way to replenish sand on some public beaches, where sand has been lost because of coastal erosion. Dredging is also used as a technique for catching certain species of edible clams and crabs. This process is used to create a new harbor, berth or waterway, to deepen or maintain navigable waterways or channels which are threatened to become silted due to sediment sand and mud accumulation. Gathered sand, clay or rock may reused to construct new land elsewhere by replacing, for example, sand eroded by storms, wave action or human activity on a beach. It is very important to enhance the protective function of the beaches. Dredging reclaim areas affected by chemical spills, storm water surges (with urban runoff), and other soil contaminations. Without dredging operations, much of the world's commerce would be impaired since much of world's goods travel by ship, which need to access harbors or seas.
Dredged contaminated sediments should not be dumped in coastal waters or the lakes since they need adequate treatments. Managed properly, sediments are a resource. Improper sediment management results in the destruction of aquatic habitat that would have otherwise depended on their presence. The United Nations Group of Experts on the Scientific Aspects of Marine Environmental Protection recently recognized that on a global basis, changes in sediment flows are one of the most serious problems affecting the quality of the marine and coastal environment
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(Huber M.E. et al., 1999; Fischetti, Mark, 2001; United Nations Environmental Programme, 2001).
2.1.1. Main contaminants related to water pollution
Water pollution is a global problem which requires ongoing evaluation and revision of water resource policy at all levels: it has been suggested that water pollution is the leading worldwide cause of deaths and diseases (Pink, Daniel H., 2006) since it is accountable for the death of more than 14,000 people every day (West, Larry, 2006). The pollution of water brings to inability to support a human use, such as drinking water, or its constituent biotic communities, such as fishes. As said before, natural phenomena (like volcanoes, algae blooms, storms) and anthropogenic contaminants cause changes in water quality and its environmental stability. Mainly involved are a wide range of pathogens, chemicals and physical modifications (thermal changes and/or discoloration). Pathogens are microorganisms that cause disease. Salmonella, Norovirus and Parasitic worms are just few examples and they’re mainly related to sewage discharges not properly treated (EPA, 2006). Among physical changes, thermal pollution needs to be recalled: water temperature can rise or fall because of human influence. Common causes are the use of water as a coolant by power plants and urban runoff. Thermal pollution is crucial since elevated water temperatures may decrease oxygen levels, which can result in fish death, alterations of trophic web composition, reduced biodiversity and new thermophilic organisms’ invasions (Goel P.K., 2006; Kennish, Michael J., 1992; Laws, Edward A., 2000).
Water contaminants may include organic and inorganic toxic substances:
• Among organic toxic compounds detergents, chlorides, herbicides-insecticides, persistent organic pollutants (POPs) such as polychlorinated biphenyl (PCBs), polycyclic aromatic hydrocarbons (PAHs), dibenzo-p-dioxins and dibenzofurans (PCDF and PCDD), etc. are mainly present;
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• Inorganic substances, such as sulfur dioxide (involved in raising acidity) from industrial discharge, fertilizers nitrates and phosphate-based from agricultural runoff, heavy metals, etc. are the most common.
Some of these substances are dangerous because of their capacity to accumulate in organisms causing potential harmful effects on them and on the environment. In order to understand this degree of danger it is important to explain how processes like biomagnification, bioaccumulation and bioconcentration work:
• Bioconcentration is the mechanism involved into absorption of contaminants directly from water by respiratory organs or skin. It occurs when the uptake from the water is greater than the excretion (Landrum, 1999);
• Bioaccumulation represents the absorption of a substance from food. It increases pollutant concentration in certain tissues of organism involved. This process occurs within a trophic level;
• Biomagnification is the increasing concentration of a substance, such as a toxic chemical, in organism moving up the trophic levels in a food chain. So these substances pass across trophic levels becoming exponentially more concentrated because they are very slowly metabolized or excreted.
One case in which contaminant accumulation takes place in nature is represented by fish related products that revealed to contain various amounts of heavy metals, such as mercury. Long-lived species of fish in particular and high on the food web (such as marlin, tuna, shark, and swordfish) seem to contain higher concentrations of these toxic compounds than others (U.S. Food and Drug Administration, 2012). It is known that fish and shellfish efficiently accumulate mercury in their tissues, but slowly excreted, especially in the form of methylmercury, a mercury highly toxic organic compound (Croteau M., 2005). It clearly rises through the food chain starting from fish consuming small plankton (Cheng et al., 2011). Indeed bioaccumulation in seafood goes on into human populations, where it can result in mercury poisoning. Mercury is dangerous to both natural ecosystems and humans because it is capable to damage the central nervous system (Park K. S., Seo Y.C.,
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Lee, S.J., Lee, J.H., 2008). Heavy metals are not degradable and come in contact with organisms by mechanism of sequestration and excretion of metals, especially those exposed naturally to high levels of these substance. Concern arises when organisms are exposed to levels higher than normal whose excess they cannot efficiently and rapidly excrete to prevent damage.
Another main group of compounds that biomagnify is the persistent organic
pollutants (POPs) group, which includes the so mentioned before PCBs, PAHs, PCDF
and PCDD. Most of them are resistant to environmental degradation through photolytic, chemical and biological processes since organisms have not been exposed before and have not evolved specific mechanisms to manage them. Due to their persistence peculiarity, they bioaccumulate with damaging effects on organisms. Since they cannot be diluted or excreted in urine (which is a water-based solution), these lipophilic substances accumulate in fatty tissues. When eaten by another organism, fats are absorbed in the gut, carrying the substance that accumulates in the fats of the predator. In this way, POPs spread on long distances from the source and interact with organisms through absorption from suspended particulate matter or sediments (Van der Oost et al., 1996). These sediments could act as a long-term toxic compounds releasing point, showing the discharges history of the area (Connor, 1984).
2.1.2. Heavy metals and PAHs: characteristics and toxic action
The term ‘heavy metals’ is referred to those metals with a specific density greater than 5g/cm3 and that may damage seriously environment and organisms related
(Järup, 2003). Some of them are essential as taking part in biochemical functions in various organisms. This usually occurs at low concentrations but, as said before, they become toxic once exceeded limit levels of tolerance. In water, the most abundant heavy metals are arsenic, cadmium, chromium, copper, nickel and zinc and an eventual exposure to their high concentrations is a serious risk for both human and animal health (Lambert et al., 2000). Major sources include: soil erosion,
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mining, agricultural activities, urban runoff, sewage discharge and others (Morais et al., 2012). Important characteristic that make heavy metals so toxic and difficult of manage are their properties of chemical coordination and oxidation-reduction: they bind to incorrect proteins sites competing with original metals and causing malfunctioning and damage in cells, while escaping from typical processes of control of homeostasis, transport, etc. (Jaishankar M., 2014). Some studies have found that binding of heavy metals to DNA and some nuclear proteins, cause several problems such as oxidative damage of biological macromolecules (Flora et al., 2008). Some contaminants (such as metals) are capable of catalyze reactions which produce reactive radicals and reactive oxygen species that increase oxidative stress in cells (Pratviel, Genevieve, 2012). This stress occurs in case of imbalance between presence of reactive oxygen species and antioxidant defenses and/or repair mechanisms. An increase of oxidative stress leads to toxic effects producing peroxides and other radicals that damage proteins, lipids and DNA (causing strand breaks). In humans, such dangerous condition is responsible for the development of many diseases such as cancer (Halliwell, Barry, 2007).
Among heavy metals, cadmium is one of the most non-essential toxic heavy metals and bioaccumulates in humans exposed by inhalation or ingestion (biomagnification). It is a common component of electric batteries, pigments, coatings and electroplating (Buxbaum, 2005; Smith C.J., 1999; Scoullos, Michael J., 2001). Once in the environment, it is very persistent, it may be present for decades in sediments or soil, it is usually taken up by plants, gradually accumulates and goes up through the food chain. Cadmium has known adverse properties against cell enzymes and it is involved in oxidative stress increase (Irfan et al., 2013). Due to its molecular mimicry, it interferes with essential metals homeostasis, principally competing with zinc for its binding sites in proteins and enzymes (Martelli A., 2006). Cadmium enters in contact with metallothioneins (MT), cystein-rich proteins, which have the capacity to bind both essential (such as zinc, copper, selenium) and heavy metals (as said cadmium, mercury and silver) (Sigel H., Sigel A., 2009). These proteins are involved in zinc and copper regulation and offer protection against toxic metals and oxidative stress (Felizola S.J., 2014), so they act like a metal storing
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units also involved in transport and detoxification (Peterson C.W., 1996). Once metallothioneins bind cadmium, forming the Cd-MT complex mainly in the liver, they transport this metal through blood to the kidney where is deposited (Klaassen et al., 1999; Nordberg, 2004). As we could imagine low MT expression theoretically would predispose organisms to Cdtoxicity since it would be predominantly present in free form or bonded to other molecules in a harmful way (Nordberg, 2004). Indeed defects on MT function or expression may lead to dangerous transformation of cells and to cancer outbreak (Krizkova S., 2009). On very long exposure time, cadmium deposits finally led to kidney disease, lung damage and fragile bones (Bernard, 2008).
Zinc is the fourth most common metal in use, mainly involved in industry as an
anti-corrosion agent (Greenwood, 1997), by coating iron or steel (galvanization process). It is an essential mineral of both biologic and health importance, in case of deficiency of zinc, in children, can occur causes of retardation in growth, delayed sexual maturation, susceptibility of infections, etc. (Hambidge, Krebs, 2007). There are so many enzymes in biochemistry possessing a reactive center with zinc atom inside, whose properties are fundamental for several physiological reactions (Maret, Wolfgang, 2013). Zinc interacts with numerous organic compounds and is very important in processes that involve RNA and DNA metabolism, transduction of signals and gene expression. Excess of this metal may reach water environment in high concentrations from mining areas by rivers or affect directly the soil causing adverse effects. An excessive absorption of zinc contrasts absorption of copper and iron (Fosmire G. J., 1990), and its presence in solution as free ion is very toxic in plants, invertebrates and several vertebrates such as fishes (Eisler, Ronald, 1993).
Polycyclic aromatic hydrocarbons (PAHs) are organic compounds containing carbon
and hydrogen disposed forming multiple aromatic rings (where electrons are delocalized). They are non-polar and lipophilic compounds found in coal and produced in processes of incomplete combustion of organic matters. Some of these substances are generally insoluble in water (and poorly soluble in organic solvents, like lipids), being the soluble ones known contaminants in drinking water (Xinliang Feng, 2009). The principal source of PAHs is linked to human activity such as burning
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biofuels, wood, releasing PAHs in environment as complex mixtures affecting air, water and soil (Ramesh A., 2011). PAHs mobility is very limited in water environment since, as said before, most of them are insoluble (Choi H., 2010), their persistence in environment is linked to number of aromatic rings forming the structure: for example, two-ring PAHs are more available for biological absorption and degradation (Choi H., 2010), five-ring PAHs (or more) predominate in solid form, have low solubility in water and they are less absorbed and degraded by organisms (Johnsen, Anders, 2005). PAHs carcinogenic action depends on their structure, some of them are genotoxic and provoke cancer, the others are not genotoxic but affect cancer progression or promotion (Baird, 2005). A genotoxic compound has the capacity to modify nucleotides sequence or DNA double strand inside an organism causing permanent, heritable changes that may affect either somatic cells of the organism or germ that may pass into future generations (Kolle, Susanne, 2012). PAHs, after being chemically modified by some enzymes (forming diol epoxides, quinones and radical PAH cations), can react with DNA, provoking mutations, bulky complexes called DNA adducts, that can be stable, causing DNA replication errors, or unstable as they react with DNA strand (Dipple A., 1985; Henkler F., 2012).
Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon formed by a benzene
ring fused to pyrene obtained in wood burning process, coal tar, automobile exhaust fumes and in all smoke obtained from organic material combustion. It is very dangerous as could affect negatively nervous system, immune system, reproductive system and relative metabolites are mutagenic and carcinogenic (Kleiböhmer W., 2001). Once modified into BaP diol epoxide, it intercalates through covalent bonding into DNA, distorting DNA (Volk, 2003). In this way processes such as DNA replication are compromised, mutation and cancer occur. Some studies have shown that BaP diol epoxide targets specifically by inactivating the p53 gene which it is involved in cell cycle regulation acting as a tumor suppressor. Probably BaP diol epoxide provokes a G (guanine) to T (thymidine) transversion within p53 transversions hotspots gene, possibly leading to cancer (Pfeifer, 2002).
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In fig.1 the metabolism of benzo[a]pyrene involving the carcinogenic benzo[a]pyren-7, 8-dihydrodiol-9, 10-epoxide formation is shown:
Fig 1: From Wikipedia, benzo[a]pyrene metabolism.
This BaP diol epoxide needs three enzymatic reactions in order to form (Jiang, Hao, 2007). First of all, BaP is oxidized by CYP1A1/CYP1B1, forming a variety of products, such as (+)benzo[a]pyrene-7,8-epoxide (Shou M., 1996). Cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) are enzymes involved in phase I xenobiotic and drug metabolism induced by aromatic hydrocarbons (Beresford, 1993).
Removing all these contaminants from environment, maintaining and restoring the quality of water, air and soil is one of the great challenges of our time.
Environmental remediation operates for such purpose.
2.2. Nanoparticles in environmental remediation
Dealing with contaminant is difficult as we lack of effective techniques for the treatment of polluted environmental compartments. Indeed, in many cases conventional environmental remediation has shown limited effectiveness in reducing/removing pollutants from compromised environment (Rickerby, Morrison, 2007). The potential usefulness that Nanotechnology may have in remediation is
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the main object of this work. This approach promises more effective and cheaper solution in restoring the quality of polluted environment (Mueller N. C., Nowack B., 2010).
Nanotechnology is defined as:
“Research and technology development at the atomic, molecular, or macromolecular levels using a length scale of approximately one to hundred nanometers in any dimension; the creation and use of structures, devices and system that have novel properties and functions because of their small size; and the ability to control or manipulate matter on an atomic scale” (US EPA, 2007).
These small particles are defined depending on their different structure:
• Nanoparticles are particles which have at least one dimension (out of three) between 1 and 100 nanometers (nm) in size (ISO/TS, 2011);
• Nanomaterial is a natural, incidental or manufactured material containing
particles, in an unbound state or as an aggregate or as an agglomerate and for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm – 100 nm. In specific cases and where warranted by concerns for the environment, health, safety or competitiveness the number size distribution threshold of 50% may be replaced by a threshold between 1% to 50% (European Commission, 2011).
Nanoparticles may be incidentally produced as a byproduct of mechanical or industrial processes. Sources of incidental nanoparticles include vehicle engine exhausts, welding fumes, combustion processes from domestic solid fuel heating and cooking. Incidental atmospheric nanoparticles are often referred to as ultrafine particles, and are a contributor to air pollution (National Council on Radiation Protection and Measurements, 2017). Biological systems often feature natural, functional nanomaterials. The structure of foraminifera (mainly chalk) and viruses (protein, capsid), the wax crystals covering a lotus or nasturtium leaf, spider and spider-mite silk, the blue hue of tarantulas, the "spatulae" on the bottom of gecko feet, some butterfly wing scales, natural colloids (milk, blood), horny materials (skin,
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claws, beaks,feathers, horns, hair), paper, cotton, nacre, corals, and even our own bone matrix are all natural organic nanomaterials. Natural inorganic nanomaterials occur through crystal growth in the diverse chemical conditions of the Earth's crust. For example, clays display complex nanostructures due to anisotropy of their underlying crystal structure, and volcanic activity can give rise to opals, which are an instance of a naturally occurring photonic crystals due to their nanoscale structure. Fires represent particularly complex reactions and can produce pigments, cement, fumed silica etc. Natural sources of nanoparticles include combustion products forest fires, volcanic ash, ocean spray, and the radioactive decay of radon gas. Natural nanomaterials can also be formed through weathering processes of metal- or anion-containing rocks, as well as at acid mine drainage sites (National Council on Radiation Protection and Measurements, 2017).
Engineered nanomaterials (EMNs) may be composed of materials with one or more
external dimensions at nanoscale and have been deliberately engineered and manufactured by humans to have certain required properties (U.S. National Institute for Occupational Safety and Health, 2013;Mueller N. C., Nowack B., 2010). The term engineered nanoparticles (ENPs) is used as referring for all engineered particles with one or more external dimensions at the nanoscale (Som et al., 2010). Nowadays EMNs are used in environmental remediation involving several disciplines such as chemistry, biology, physics and engineering. Their peculiar characteristics may offer a new approach in treating pollutants: nanomaterials may show the same chemical composition of an equivalent material in bulk form, but they demonstrate new characteristics due to their high surface-to-volume ration (Hochella, Madden, 2005), they are indeed significantly more reactive than larger particles (Rickerby, Morrison, 2007). In order to proceed in environmental remediation using nanomaterials, it is important to assess risk factors of exposure to engineered NPs since nanoremediation will inevitably lead to release of nanoparticles into the environment (Karn B. et al., 2009). These particles evolve through the environment interacting with it, they face several modifications which can result in toxic reactions. Specific studies are necessary to estimate potential adverse effects since several parameters may be involved (Brunner T.J., 2006; Choi
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J.Y., 2010; Hu X.K., 2009). In fact, since 1990s the use of ENMs in several remediation applications has been investigated and some of them have entered the market (Mueller, Nowack, 2009; 2010):
• Nanoscale TiO2 is used for degradation of contaminants in air and water, (such as NOx, microorganisms and organic materials) through photocatalytic
process;
• Nanoscale zero-valent iron (ZVI) is used for both soil and groundwater remediation;
• Nanofiltration is used for drinking water and wastewater purification.
Nanoscale TiO2 particles fill an interesting role in oxidative transformation of organic and inorganic contaminants such as heavy metals, pesticides, arsenic and phosphates in water (Obare S.O., Meyer G.J.J., 2004; Hoffman M.R., 1995). They can be immobilized on several supports which are used in water and air detoxifications applications. In this way, through UV excitation, TiO2 gains sufficient oxidizing
potential to degrade pollutants and to kill several types of bacteria. Moreover, TiO2
is very resistant to corrosion so it can be repetitively used without excessively loss of catalytic capacities. Such properties make TiO2 very interesting for environmental
applications (Pandey Bhawana, Fulekar M.H., 2011). However TiO2 is effective for
treatment of transparent wastewater since needs ultraviolet light to work, actually several research groups are evaluating the potential role that solar reactors may have as energy source which is required for TiO2 excitation (Pandey Bhawana,
Fulekar M.H., 2011).
According to many laboratory researches, nanoscale Zero-Valent Iron (nZVI) seems to be very efficient in removing several contaminants such as metals, polychlorinated hydrocarbons, arsenic, pesticides, trihalomethanes, dyes, polycyclic aromatic hydrocarbons (Zhang W.X., 2003). The degradation process is based on redox reactions since, in contact with contaminants, nZVI donates electrons reducing harmful substances to less toxic ones (Nowack, 2008). NZVI is efficient in removing, for example, dissolved metals from solutions by reducing them to zerovalent oxidation state (or lower) or by complexing them with the just formed iron oxides (they are a potential end product from redox reactions of nZVI) (Pandey
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Bhawana, Fulekar M.H., 2011). In groundwater and soil remediation, nZVI is used by directly injection near contaminant source in order to form a reactive barrier of iron particles or by injection in surface-modified form (coated, for example, with surfactants, polyelectrolytes or polysaccharides) which acts as a plume of reactive iron that dissolves contaminants in aqueous phase (a plume is an area occupied by substances that migrate from a source) (Tratnyek, Johnson, 2006; Nowack, 2008). NZVI particles show so much reactivity that if exposed to air they even ignite spontaneously, in fact reaction rates are almost 25-30 times faster than reaction rates of granular iron in micrometer to millimeter range. Due to this reactivity, nZVI must be handled as a slurry which requires specific infrastructures which remix the solution before injection (Mueller, Nowack, 2010; Li et al., 2006).
The high reactivity of nanoparticles may place them in a new group of potentially toxic substances. They be inhaled, ingested or taken through the skin and absorbed by cells even if the equivalent material in larger form is inert (Oberdörster et al., 2007). It is very important to understand how nanoparticles may operate in environment in order to apply eco-friendly nanoremediation processes since most of which add free NPs directly to soil or groundwater. Nanoparticles potential toxicity and behaviour in environment may be influenced by many factors such as composition, structure, molecular weight, water solubility, activity, aggregation/disaggregation potential and the behavioral traits of the organisms exposed (Mueller, Nowack, 2010). Other than produced for specialized materials or processes, nanoparticles are present also as by-products of industrial processes and may naturally occur spreading in water, air or soil, originating from atmospheric, geogenic or biogenic processes (Banfield, Zhang, 2001). Naturally occurring nanoscale Fe oxide particles with metals (such as copper) bound to their surface have been found many kilometers downstream from mining sites, suggesting the ability of these nanoparticles to bind and transport contaminants (Hochella, 2005). So, whereas the nanoparticles may not have toxic peculiarities, the pollutants they carry with them may. Copper, for example, is a well-known toxic substance at high concentrations for algae, plants, fungi and phytoplankton (Sposito, 1989). In some cases, Fe oxide nanoparticles can be absorbed by cells causing cell death and their
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low solubility allows them to persist in biological systems and could potentially induce long-terms effects involving mutagenic influence on organisms. However these studies were conducted at higher dosages than would be encountered normally (Wiesner, 2006; Auffan, 2006; Barbara K., 2009). In conclusion, nanoremediation has the potential to reduce the overall costs of cleaning up large-scale contaminated sites, reduce cleanup time, eliminate the need for treatment and disposal of contaminated dredged soil, and reduced some contaminant concentrations to near zero, and it can be done in situ. In order to prevent any potential adverse environmental impacts, proper evaluation of these nanoparticles needs to be addressed before this technique is used on a mass scale (Barbara K., 2009).
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2.3. NanoBonD project
The following work is part of NanoBonD* project which considers the potential use of cellulose and dextrine-based nanomaterials in nanoremediation to limit toxic substance
actions in water environment (substances like polycyclic aromatic hydrocarbons and heavy metals in particular) coupled to tubular elements made in geotextiles to be used in dewatering processes of sediments dredged from contamined marine areas such as harbors. This project involves research partners specialized in sediments
remediation and dewatering processes (Acque Ind.li, Labromare), control techniques and safety analysis (Biochemie, Ergo, ISPRA), raw materials of plant origin production (such as starch and cellulose) from renewable sources (BARTOLI), research on nanomaterials regarding their synthesis and environmental safety (INSTM including UNIPI, UNISI, UNIFI and Polytechnic of Milan, Turin), and regulatory framework (ERGO). The related aim is to propose an eco-friendly nanoremediation of marine, fresh-water and brackish water sediments gathered up in dredging processes (and subsequently dewatered) which are used to solve soil-related problems such as harbors silting and hydrogeological disorder. Eco-friendly materials are used across the processes in respect of human/environmental health, main point of this project. The main objectives of this project are:
• To produce new adsorbent materials from renewable sources of polysaccharide origin, synthetized by reticulation process of starch (dextrins and cyclodextrins) and cellulosic derivates (cellulosic nanofibers). These nanostructured materials are tested in order to assess their capability to decontaminate environmental compartments (marine, brackish, freshwater) from organic pollutants and heavy metals;
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• To respect the environment parameters especially from producing processes to in situ applications, principally taking care of the most crucial points (for example: in cellulose oxidation process, oxygen is principally used instead of the sodium hypochlorite, in order to use zero environmental impact reagents);
• To couple the decontamination process of the dredged sediments to the dewatering one by using tubular elements in geotextiles;
• Adding photo degradation action through ceramic coating in order to allow nanostructured materials to completely mineralize organic contaminants;
• To make these dehydrated sediments a resource to be used in restoring dikes or beaches;
NanoBonD will operate in order to propose in situ applications for marine, freshwater and brackish sediments remediation. One of the first processes of this project regards the chemical-physical, analytic and eco toxicological characterization of environmental compartments: marine, freshwater and brackish sediments. In order to select the sites for sampling of sediment to be use in the project, core drilling was used in order to minimize potential disturbances and maintain the chemical-physical characteristics of the sample stable. Sampled sediments are analyzed for heavy metals and PAHs, in this way sediment and their elutriate toxicity can be evaluated. About the nanostructured materials synthesis, the aim is to produce eco-friendly nanomaterials intended for remediation, starting from recycled raw materials and to use zero environmental impact processes of production. Organic nanoporous materials are synthetized through cross-linking process acting on starch and cellulose derivate (dextrin, cyclodextrin and cellulose nanofibers). Two types of nanostructured materials are obtained: cellulose-based
NSCel and cyclodextrins-based NSDes, which were demonstrated to be efficient in
contaminants adsorbing, although in H2O solution. Ceramic nanosponges (NSCer) are synthetized by nanosponges coating process, their detoxication capacity works through a photocatalytic reaction that efficiently mineralize adsorbed organic toxic compounds. During nanomaterial synthesis, cross-linking process occur; these are facilitated by specific agents introducing functional groups: such chemical
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modifications are needed for making these substances eligible for several environmental compartments (marine in particular as the more complex one). An important point is the analysis of the releasing potential and bioavailability of these agents from material to water compartment. In other words, interacting with environmental compartments the nanomaterials might release chemicals (used for their synthesis) which might be adsorbed by aquatic organisms and/or affect them. Very few studies have been focused on the potential use of ecofriendly nanomaterial in environmental remediation until now.
Thus, firstly it is necessary to test remediation performance in controlled laboratory conditions, and, only with promising results, it is possible to proceed to in situ testing (Corsi et al., 2014). In order to evaluate nanostructured materials decontamination efficiency, chemical analysis regarding the presence of heavy metals and organic toxic compounds are performed before and after in laboratory treatment (Corsi et. al., 2014). The results show an efficient adsorbent capacity of these materials that has been demonstrated for several contaminants (Trotta et al., 2003; Melone et al., 2015; Melone et al., 2013). The next step consists in eco-toxicological characterization of NSDes, NSCel, NSCer by applying specific assays also considering the compartments of interest (marine, freshwater and brackish). These tests include sub-lethal response studies, measuring biomarkers of effect and exposure using bivalve mollusks (Mytilus galloprovincialis) exposed to waters treated with nanostructured materials in laboratory conditions. The next step consists in simulating geotextiles (coupled with nanostructured materials) dewatering of contamined sediments at laboratory scale. Sediments selected for this step (from marine, freshwater and brackish sources) were analyzed and demonstrated to contain high levels of the main pollutants such as concentrations of Zn, Cd and benzo(a)pyrene. The effective detoxification of these substances and the environmental safety of the processes will be evaluated through eco-toxicological analyses. But how does geotextile application work? Geotextile are used worldwide in dredging processes. Main issues regarding this application is represented by dredged materials disposal, which influences project management and planning. Generally, the dredged sediments are transported in accumulation
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areas near harbors or dehydrated mechanically using centrifuge, filter-press process. The use of tubular elements in geotextiles is a valid alternative in order to dehydrate sediments because it permits to lowering the water content of sediments in close proximity of dredging site and to use dehydrated sediments for realizing or rebuilding harbor quays. The efficiency of this process using geotextiles depends on permeability, porosity, typology of pattern and fiber components. Geotextiles seem to offer resistance capacity during the filling process, which provokes great pressure on the structure while the mud is pumped in. During this process, the high grade of permeability of the geotextiles allows the liquid to come out from the sediments. The porosity, instead, acts as a barrier for the solid materials, which remain inside the tubular element while the water is expelled. Once the process is completed, the now filled geotextile can be easily cut and the dehydrated mud inside may be used as structural elements in morphological remodeling applications. These tubular elements are closed systems, which prevent spillage and/or exposure of toxic substances or pathogen agents, they do not release bad smells, this dewatering process does not need the use of fixed structures, the environment restoration is very simple and these elements can be reused. The equipment necessary for the applications are easily transported and in a very inexpensive way, the energy required is at low levels and the process needs low manual labor. Choosing specific cross-linking agents during nanomaterials synthesis phase might also facilitate
flocculation of suspended organic matter in tubular geotextiles; in this way, it is
unnecessary to add specific flocculation agents (in chemistry, flocculation is a process where colloids come out of suspension in the form of flake either spontaneously or due to the addition of a specific agent). Such process acts as an adjuvant of filtration increasing the expulsion of water out of the geotextiles.
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2.4. Bioindicators and biomarkers
In eco-toxicology, it is fundamental the use of specific organisms which can provide information about contaminant variations through space and time in environment. These organisms are defined bioindicators. Moreover, the biological responses to environmental disturbance, namely the biomarkers, are also very important in ecotoxicology (Hamza, Chaffai, 2014). A biomarker is a biochemical, cellular, physiological or behavioral variation that is measurable in a tissue, biological fluid or in an organism entirely, which suggests evidence of an exposure to one or more pollutants (Depledge, 1989). Biomarkers can be used to identify first exposure or effect signals, obtaining precious information regarding toxic action of contaminants of interest that could harm populations and ecosystems. Biomarkers can be divided in three main classes: biomarkers of exposure, susceptibility and
effect (International Programme on Chemical Safety, 1993). The biomarkers of
exposure show variations related to a first interaction between contaminant and biologic receptor, confirming the exposure. The biomarkers of susceptibility indicate the constitutive or inducible ability of an organism to react to a contaminant exposure by modifying receptors, which alter the susceptibility of the organism to such substances. Finally, the biomarkers of effect indicate an exposure to a toxic compounds and the toxic effect involved (Fossi, 1998). The metabolism of genotoxic substances is a complex phenomenon which can result in high reactive compounds releasing. Such metabolites may damage cell macromolecules such as DNA, lipids and proteins. In such way, the genotoxic exposure can be related to cancer occurrence, harmful effects on germ line, etc., deeply affecting populations (Mitchelmore and Chipman, 1998). In this work, specific end points of environmental-geno-toxicity have been used, namely DNA damage (assessed by the
Comet assay) and chromosomal damage (assessed by the Cytome assay). The Comet assay in its alkaline version (pH≥13) was used to measure a reversible
genetic alteration on isolated cells. This test allows to estimate DNA single strand breaks, double strand breaks and alkali-labile sites. DNA strand breakage is a well-known biomarker of genetic toxicity in environmental monitoring. The Cytome
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assay, including micronucleated cells evaluation was also performed. This test
highlights a permanent, fixed DNA damage, which can be due to the action of toxic agents with clastogen or aneuploidogen properties. It is a widely used genotoxicity biomarker, which provides an accurate measure of DNA damage and/or mitotic machinery alteration resulting in chromosomes breakage or their mis-segregation during anaphase. Indeed, it is applied to detect nuclear abnormalities (blebs, buds, nucleoplasmic bridges, ecc…) and micronuclei presence, potentially related with the exposure to toxic agents able to interact directly with the DNA or with cellular and nuclear structure, i.e. spindle apparatus, cytoskeleton or nuclear membrane. In this thesis, experiments have focused on measuring any potential genotoxic action of the nanomaterials specifically synthesized for the NanoBonD project, which should be potentially applied for the nanoremediation. Bivalves, especially the Mytilus galloprovincialis, are widely used as bioindicators organisms in eco-toxicological studies regarding contaminated coastal areas (Saavedra et al., 2004; Santovito et al., 2005; Box et al., 2007). They are organisms of simple management, identification and gathering and are widely distributed along Mediterranean Sea coasts. They are sedentary filter-feeding animals that can filter about 200 liters of water per-day (Zouiten et al., 2016), so several contaminants may be adsorbed and accumulate inside their tissues (Catsiki and Florou, 2006). Bivalve gills are principally considered in eco-toxicological studies since are mainly involved in feeding-respiratory processes and they constantly interface with the water environment and with contaminants. These mollusks can adapt in several environmental parameters variations such as temperature, oxygen levels, salinity, food availability and their biochemical and physiological features are widely known, those are the reasons why bivalves (like Mytilus galloprovincialis) are mainly used to evaluate marine environmental quality (Andral et al., 2004; Romeo et al., 2003).
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3. Aim of the work
In the framework of the NanoBonD project, the present thesis is aimed to investigate the interactions between nanomaterials (cellulose and dextrins based) and the biological systems. In particular, genotoxic evaluation has been performed in order to assess if the selected nanomaterials within the NanoBonD project (and/or the process of their synthesis) are safe for the Mediterranean mussel (Mytilus galloprovincialis) in term of DNA and chromosomal damage.
4. Material and Methods
4.1. Nanomaterials used
Cellulose-based nanomaterials
Cellulose is a promising starting point for the development of new materials to be used in water remediation. This naturally abundant biopolymer shows several interesting properties and its structure can be modified in order to change its physical and chemical behavior making it an interesting solution in water remediation applications (E. Vismara 2009;S.-L. Chen 2011). In details, substances such as cellulose nanofibers (CNFs) can be used for the preparation of nanostructured materials. CNFs obtained from 2,2,6,6-tetramethylpiperidinyloxyl (TEMPO)-mediated oxidation (TOCNFs) shows heavy-metals absorption properties thanks to the presence of carboxylic groups on cellulose surface (T. Saito 2005; H. Sehaqui 2014). During the synthesis process, several properties for increasing remediation efficiency and offering a peculiar stability in water environment can be added. For example, by introducing amino groups on the cellulose polymer chains,
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the adsorption properties of organic pollutants and heavy metals resulted to be increased. For such purpose, branched polyethyleneimine (bPEI), a substrate containing amino groups, is used for material synthesis (H. Tian 2007).
According to this work, ‘’TEMPO-Oxidized Cellulose Cross-Linked with Branched Polyethyleneimine: Nanostructured Adsorbent Sponges’’ (Melone L. et al. 2015), efficient adsorbent sponge-like materials stable in water can be prepared by the amidation of the TOCNF carboxylic groups with bPEI, without using a cross-linker. This material is successfully able to adsorb organic pollutants, including p-nitrophenol (pNPh), 2, 4, 5-trichlorophenol (tCPh), and the antibiotic amoxicillin (AM), and for heavy-metal ions like CuII, NiII, CdII, and CoII. Different concentration of
bPEI and the amount of dry TOCNF could influence the overall stability of the substance. It is very important to modify this concentration ratio correctly in order to obtain a stable material, which can be used in water environmental applications since water flow plays an important role in both the design and operation of the adsorbent unit (G. Crini 2005; G. Crini 2010). In water, bPEI-TOCNF(2:1) (2:1 is referred to the reagents ratio) showed good mechanical stability and was chosen as model material used in further investigations. In decontamination efficiency investigation of these sponges involving wastewater treatment, p-nitrophenol (pNPh) was initially used as pollutant model. This substance is an industrial precursor in different drugs, fungicide and dyes synthesis. The efficiency of these materials increases directly with N/C (used to identify the bPEI/TOCNF ratio, due to the increasing of concentration of amino groups) as shown in fig. 3, since the adsorption process presents an acid-base interaction between bPEI amino-groups and an acidic substrate.
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Fig 2: BPEI-TOCNF adsorbent sponge synthesis process (obtained from Melone et al., 2015).
Cellulose-based sponges show interesting adsorption performances also regarding to heavy metals, because of the presence of chelating amino groups. The fig. 4 shows the amounts of metal adsorbed by the sponges (in contact with copper, cobalt, cadmium and nickel). The red filled bars show adsorption level obtained using about 300mg of bPEI-TOCNF (2:1) sponge, which was put in contact with 10ml of an aqueous solution of metal salt (0.1 M). Cd and Cu were adsorbed in higher amount than the others were. Blue striped bars refer to a competitive adsorption with all metals in the same solution (the concentration of each metal was 0.1M). Cellulose-based adsorbent material can be easily obtained using TEMPO-oxidized cellulose nanofibers (TOCNFs) and branched polyethyleneimine (bPEI), resulting in a sponge-like structure which presents a good stability in water and shape-memory capacity. These materials show very significant adsorption effects in contact with some typical organic pollutants and heavy metals.
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Fig 3: Adsorption of pNPh onto bPEI-TOCNF sponges having different N/C ratios. The inset shows the
relationship between the N/C ratio and the bPEI/TOCNF ratio based on the preparation of the
materials (obtained from Melone L. et al. 2015).
Fig 4: Adsorption of metals ontobPEI-TOCNF (2:1) T: 258C; contact time: 12 h; initial concentration:
0.1m; volume of solutions: 10 mL; mass of adsorbent sponge: 300 mg. Red bars: independent adsorption experiment; blue striped bars: competitive adsorption experiment (obtained from Melone L. et al. 2015).
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Dextrine-based nanomaterials
Cyclodextrines are a group of compounds made of sugar molecules bound in a ring
structure. They are produced starting from starch and are used in food, pharmaceutical, drug delivery, chemical industries and environmental engineering (Menuel et al., 2007; Thatiparti et al., 2010). Cyclodextrins are composed of 5 or more α-D-glucopyranoside units linked 1->4, as in amylose (a fragment of starch). Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape:
• α (alpha)-cyclodextrin: 6-membered sugar ring molecule; • β (beta)-cyclodextrin: 7-membered sugar ring molecule; • γ (gamma)-cyclodextrin: 8-membered sugar ring molecule.
Fig 5: Chemical structure of the three main types of cyclodextrins. From https://commons.wikimedia.org/wiki/File:Cyclodextrin.svg
Cyclodextrins are capable to form host-guest complexes with hydrophobic molecules (Brocos, Pilar, 2010), as a result, they have found applications in a wide range of fields. They are used in pharmaceutical applications as they can solubilize hydrophobic drugs and crosslink to form polymers used in drug delivery (Thatiparti et al., 2010; Barreto et al., 2008). However, they also found application in environmental remediation: cyclodextrins can immobilize toxic substances including heavy metals, trichloroethane or can form complexes with other substances enhancing, in this way, their decomposition.
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Between May and August in 2016, the Chemistry Department of the University of Turin, have synthetized four nanostructured materials composed by cross-linked cyclodextrin and maltodextrin. The related studies were performed in order to investigate their behavior in water and their absorption capacity of metal cations from solutions (Copper in detail).
Fig 6: Space filling model of β-cyclodextrin. (From
https://commons.wikimedia.org/wiki/File:Beta-cyclodextrin3D.png).
The synthesis process of nanostructured materials was performed by inducing cross-link reaction of β-cyclodextrin (β-CD) and Kleptose Linecaps maltodextrin
(LC) (provided by Roquette). The reaction takes place by adding cross-linking
agents: PDMA (pyromellitic dianhydride) and CA (citric acid). These substances provide carboxylic groups, which react with hydroxyl group of dextrin compounds.
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Fig 7: Structures of used reagents. (*) from
http://www.roquette-pharma.com/taste-masking-solution-kleptose-linecaps-maltodextrin/ .
The products are named βNS-PYRO 1/8, LC-PYRO 1/8, βNS-CITR 1/8 e LC-CITR 1/8 (the term 1/8 is referred to the used reagents ratio). The nanostructured material remediation capacity was verified by adsorption test performed using Cu2+ as metal
cation model, (Cu2+ can be simply detected by ultraviolet–visible spectroscopy
technique). The required amount of material was milled before the experiments. The best Cu2+ adsorbing capacity was performed by βNS-PYRO 1/8 (150mg dissolved
in 50ml CuSO4 solution with 100 ppm of Cu2+): in this specific case almost 70% of
copper was successfully adsorbed by the material. It seems (as shown in fig.8) that this amount does not differs so much during time, since the amount of copper adsorbed after 30 minutes of exposure is not so different than the amount adsorbed after 6 hours. The adsorption proceeds rapidly and is complete within 30 minutes of exposure.
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Fig 8: We can see the Cu2+ adsorption levels during time at increasing levels of ns-pyro.
The cross-link processes of β-CD and LC are simply and rapid and the nanostructured materials can efficiently adsorb water inside the constituent porous, increasing their volume of about 330%. The four polymers have shown acidification properties when in contact with water solution, especially for saturated NaCl solution since cation exchange occurs between Na+ from solution
and H+ of polymers. This pH modification issue needs more investigations since its
level is not compatible with a typical water-related biological system. In conclusion, these adsorption tests show very encouraging results in removing pollutants from water solution, and the synthesis processes involved are rapid and cheap. For these reasons, these nanostructured materials (nanosponges and cyclodextrins) have been chosen as within the Nanobond project.
Cu
2+ad
sorbe
d
6.5 hours
30 min
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4.2. Experimental procedure
4.2.1. In vitro experiments
Before the in vivo exposure experiment, preliminary investigations were conducted on different nanostructured material batches in order to evaluate their potential toxic effect on marine organisms (nanosponge batches n°1-2 and cyclodextrins). In particular, genotoxicity of selected nanomaterials was assessed on gill biopsies of M. galloprovincialis (n=3), exposed to artificial seawater (ASW) previously treated with neo-synthetized cellulose-based and dextrin-based nanostructured materials. The resulting ASW was tested without dilution and after diluting with ASW (50% and 25%). Well plates (24) were used for the exposures of gill biopsy. Control wells (ASW) and positive control wells (H2O2 100mM) were used in the investigation.
After the treatment, gill tissues were taken from the wells and inserted in 2ml eppendorfs in which were added 500 μl of dispase (1,5 mg/ml), a digestive enzyme used to dissociate tissues. The eppendorfs were located in a thermostatically controlled water bath set up at +37°C for 20 minutes (in order to allow the enzyme to work correctly). Subsequently, dispase activity was arrested by adding 500 μl of cold HBSS, the resulting cell suspension was collected by filtering through a 100 μm mesh nylon filter and transferred in 2 ml eppendorfs. The cell suspension obtained was centrifuged at 2000 rpm for 5 min and the pellet used for both the Comet assay and the Cytome assay.
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4.2.2. In vivo experiment
80 Mytilus galloprovincialis specimens have been taken from mussel farm located in relatively unpolluted marine area (Arborea, Oristano, Sardinia). Bivalves were located in 5 polyethylene/fiberglass tanks (12 organisms per tank), using each one for a specific treatment (the tanks present seven liters of capacity and 20x20x30 cm in dimensions). The organisms were mantained in natural seawater for 24 hours (collected offshore in front of from Talamone, GR, shown in fig. 12). The in vivo exposure has been set up as follows:
• CTRL-1, natural (control) seawater collected offshore in front of Talamone; • CTRL-2, control seawater collected from Livorno area (shown in fig.); • E, elutriate obtained from Livorno harbor sediments (using CTRL water); • ETNS, elutriate treated with nanosponges (using CTRL water);
• ETCD, elutriate treated with cyclodextrins (using CTRL water).
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Fig 10: The image above is a summarizing scheme of the experimental procedure and the average
length of organisms for each tank. Regarding E, ETNS and ETCD tanks, the number of bivalves died during the exposure are reported.
The water used in each treatment has been filtered (0.45 µm) and renewed every
24 hours, the duration of exposure was of 48 hours. Physico-chemical parameters of
the tanks were measured every 24 hours at T0 and T24 and are presented in fig. 11. Fig 11
37 Once taken the organisms, hemolymph was collected from posterior adductor muscle by a 1 ml syringe, while gills and digestive glands were collected during dissection process.
Fig 12
Samples were used for the evaluation of the following parameters: • DNA damage by the Comet assay;
• Chromosomal damage by the micronucleus/cytome assay.
In addition, many other biological responses at biochemical/cellular levels were analysed within the Nanobond project, namely:
• Induction of Methallothionein (MT) genes, involved in metal homeostasis (performed by University of Siena);
• Evaluation of antioxidant and detoxifing enzymes including CAT (catalase), GST (glutathion-S-transferase);
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• Levels of Malondialdehyde (MDA), a product of lipid peroxidation, (performed by University of Siena);
• Lysosomal membrane stability, measured by NRRT test (Neutral Red Retention Time) on hemocytes (performed by University of Siena).
Once collected gill samples, they were located in petri dish, in which 500 μl of 20‰ saline solution (HBSS, Hank’s balanced salt solution) were added to preserve the tissue needed for Comet and Cytome assays.
4.3. Tests used
4.3.1. Comet assay
The pellet was suspended in HBSS and procedures for slide preparation and electrophoresis were carried out according to Frenzilli et al. (2001). The eppendorfs were put in thermostatically controlled water bath set up at +37°C and were added, in each eppendorfs, 150 μl of LMA (low melting agarose) 0.5% dissolved in PBS a 37°C. Glass slides were prepared in number of two for each sample in order to replicate it and refine the statistical results. Subsequently, 75 μl of the mixed solution (obtained from pellet and added gel) were put on each glass slides (two per sample) previously treated with a NMA (normal melting agarose) 1% layer. Glass slides were put for 10 minutes at +4°C and 85 μl of LMA were added on the surface to form the last layer. The slides were put again for 10 minutes in the fridge at +4°C and finally were put in lysing solution (composed by NaCl 2.5 M, Na2EDTA 100 mM, TRIZMA BASE 10 mM, NaOH, DMSO 10% and TRITON X100 1%) and stored in the fridge at +4°C for a minimum of an hour. Every step was conducted under yellow illumination since direct light can damage DNA of the samples. The protocol continues with electrophoresis after the immersion of the slides into an alkaline buffer solution (NaOH 10 N, EDTA 200 mM, pH >13 and distilled water) for ten minutes, in order to denaturate the DNA. The Comet assay in its alkaline version
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(pH≥13) was used to measure a reversible genetic alteration on isolated cells. This test allows us to estimate DNA single strand breaks, double strand breaks and alkali-labile sites. The electrophoresis runs for 5 minutes at 25 V and 300 mA parameters for allowing eventual DNA fragments to migrate. The slides were subsequently neutralized (using 0,4 M di Tris-HCl, pH 7,5 solution), once the pH was restored and the slides dehydrated, these samples were stored in appropriate boxes.
Slides were stained with ethidium bromide and observed under a fluorescence microscope (200×). Damaged nuclei were comet shaped due to DNA migration towards the anode. The amount of DNA damage was quantified as the percentage of DNA migrated into the comet tail (tailDNA) using an image analyser (Kinetic Imaging Ltd., Komet, Version 4). At least 50 nuclei per slide and 2 slides per sample were scored, for a total of 100 nuclei and the mean calculated.
Fig 13: Comet assay, comets picture: nucleus containing no-damaged DNA (I); nuclei with damaged
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4.3.2. Cytome assay
Fig 14: Picture of micronucleated cell observed through optical microscope after Giemsa staining.
Aliquots of the mussel gill cell pellets were processed according to Scarpato et al. (1990), prefixed for 20 min in a solution 5% acetic acid, 3% methanol, 92% HBSS 20‰, and centrifuged for 5 min at 2000 rpm. The supernatant was removed and 5ml fixative solution (from 5:1 to 3:1, depending on the humidity) was added to the suspended pellet, this process was repeated twice. After the last fixative and centrifugation, suspended cells were spread on slides (two slides per mussel), the air dried and stained with 5% Giemsa solution for 10 min. One thousand cells with preserved cytoplasm per specimen were scored (500 per slide) to determine the frequency of micronuclei and other nuclear abnormalities according to the following criteria. Micronuclei were defined as round structures, smaller than 1/3 of the main diameter; micronuclei had to be on the same optical plan as the main nucleus but possess distinguishable boundaries from it (Schmid, 1976).
4.3.3. Statistical analysis
The results were investigated by multifactor analysis of variance (MANOVA) and the multiple range test (MTR) by using percentage of migrated DNA (Comet assay) and micronuclei frequencies and other nuclear anomalies (Cytome assay). Differences between means were considered statistically significant when p-value was <0.05.
