CNR Environment and Health Inter-departmental Project

Consiglio Nazionale delle Ricerche

Dipartimento Terra e Ambiente

CNR Environment and Health Inter-departmental Project: present knowledge and prospects for future research

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CNR Environment and Health Inter-departmental Project

Editor

Fabrizio Bianchi, Liliana Cori, Pier Francesco Moretti Editorial Office

Anna Russo, Tiziana Siciliano Composition and Copy Editing

Luigi Mazari Villanova Web release

Daniela Beatrici (www.dta.cnr.it) Print: Edizioni Nuova Cultura Publisher: Consiglio Nazionale delle Ricerche - Roma © 2010, Consiglio Nazionale delle Ricerche All rights reserved ISBN 978-88-8080-113-9 Cover image courtesy of Alfonso Mascia, “Flamingo Nursery at Saline Contivecchi”, Stagno di Santa Gilla, Cagliari, Italy PIAS logo designer: Luca Duè, Pisa, Italy ii

Presentation

The National Research Council (CNR) is the major public research organisation in Italy; its duty is to carry out, promote, spread, transfer and improve research activities in the main sectors of knowledge growth and of its applications for the scientific, technological, economic and social development of the Country.

To this end, CNR activities are organised into macro areas of interdisciplinary scientific and technological research covering several sectors: biotechnology, medicine, materials, environment andthe territory, information and communications, advanced production systems, judicial and socio-economic sciences, classical studies and arts.

Increasing knowledge about the complex interaction between environment and health is a major objective and is recognized as a worldwide challenge that the Italian National Research Council (CNR) wishes to take up.

Our effort aims at transferring the knowledge developed by our research institutes on environment and health issues to our society, providing local, national and international governments with priorities to be applied in social policies.

In the EH sector, CNR carries out institutional R&D activities in coordination with the Ministry of University and Research and, apart from these institutional activities, CNR and the Italian Ministry of the Environment have signed numerous agreements and contracts of project implementation, consultancy and service activities. CNR is also involved in programmes and projects of the Ministry of Health.

The Department of Earth and Environment (DTA) includes thirteen research Institutes involved in research, surveys, technologies and knowledge transfer in a wide range of Earth sciences addressing atmospheric, terrestrial and aquatic issues. The mission of DTA is to gather knowledge and predict the behaviour of the earth system and its resources to contribute to a sustainable future for the planet and mankind.

The Department of Medicine includes twelve research Institutes performing basic, clinical and epidemiological research on many types of diseases as well as on disease prevention also through the interaction with National Health System structures. Tasks of the Department of Medicine include also innovation and technology transfer in medicine as well as education and training of personnel on health-related themes.

A total of thirty-seven Institutes, including some belonging at the Departments of Agriculture and Food, Materials and Devices, Energy and Transportation and ICT, are involved in research on EH interface.

In 2006 the CNR started a multi-disciplinary program aiming at promoting and coordinate collaborative research and joint actions on environment and health. In mid 2007, following priorities set also by the European Union, the CNR activated an Inter-departmental Project called Environment and Health (Italian acronym: PIAS CNR). iii

CNR Environment and Health Inter-departmental Project The Environment and Health Project has the overall objective of promoting an integrated research in these two scientific sectors, and the specific objective of developing the knowledge on sources of pollution and the consequent negative effects on health, the instruments and methods to analyse the interaction between environment and health, the instruments and methods to be used in managing complex situations.

As operative instruments, activities were identified in collaboration with CNR researchers, to strengthen competences and to facilitate the participation in international networks.

CNR is working to promote and transfer the results of research to the production system and to decision makers; to promote the experience of Health Impact Assessment on programmes, plans and policies; to contribute to developing Environment and Health monitoring systems; to test and validate environment and health indicators suggested by the EU and the WHO, supported by suitable information systems; to test and validate the use of geographic Information System (GIS) for a joint evaluation of territorial health and environmental data and to improve instruments and methods for risk reporting through recommendations and guidelines.

The PIAS CNR project is based upon the multidisciplinary activity of researchers of CNR and other public bodies, including the WHO, the Italian National Institute of Health, Italian Universities, the National Health System, the Ministry of the Environment and Territory, the Ministry of Health, the Regional Agency for Environmental Protection and Epidemiological Observatories.

The present publication includes overviews, results and suggestions produced by the PIAS working groups on effect of pollutants in the soil, water and soil monitoring for the protection of the environment and human health, the role of atmospheric pollution in terms of its harmful effects on health, Human bio-monitoring, Environment and Health surveillance systems, Monitoring contaminants in the food chain and their impact on human health. Two Pilot Studies on “Endocrine disruptors and their effects on health” and “Ultrafine particles and cardiopulmonary effects” are in progress.

A PIAS conclusive international workshop is planned for late 2010; this will represent a chance for the CNR Institutes as well as for collaborative Institutions for sharing experiences and conclusions between research groups and plan future research.

Giuseppe Cavarretta

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& Gianluigi Condorelli

CNR Department of Earth and Environment

CNR Department of Medicine

Director

Director

Summary

Summary Presentation Introduction

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Fabrizio Bianchi, Liliana Cori, Pier Francesco Moretti

The fate of pollutants in soil G. Petruzzelli, F. Gorini, B. Pezzarossa, F. Pedron 1. Introduction 1 2. State of the art and interactions between basic and applied research 1 3. Pilot Study: Influence of soil characteristics on the mobility of contaminants in the industrial area of Gela (Sicily) 12 4. CNR Specific expertise: qualified teams and external collaborations 18 5. Future perspectives and developments 28 References 31

Water and Soil Monitoring for the Protection of Environment and Human Health M. Rusconi, S. Polesello 1. Introduction 39 2. Working methodology in working group 2 of the CNR Environment and Health InterDepartmental Project (PIAS) 40 3. State of the art of CNR activities 41 4. Emerging issues 51 5. Future perspectives and developments 57 References 59

Role of Atmospheric Pollution on Harmful Health Effects A. Pietrodangelo, M. Bencardino, A. Cecinato, S. Decesari, C. Perrino, F. Sprovieri, N. Pirrone and M.C. Facchini 1. Introduction 69 2. Air Pollution and Health 71 3. Inorganic air pollutants 76 4. Organic air pollutants 89 5. Particles in the ultrafine (UFP) and nano-size fractions 94 6. Future perspectives and developments in the framework of the “pilot study for the assesment of health effects of the chemical composition of ultrafine and fine particles in Italy� project 99 References 101

Human Biomonitoring E. Leoni, A. I. Scovassi

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CNR Environment and Health Inter-departmental Project 1. Introduction 2. From basic to applied research 3. The CNR specific expertise: qualified teams and external collaborations 4. Relevant findings 5. Future perspectives and developments References

111 115 116 120 122 126

Environmental Health Surveillance Systems E.A.L. Gianicolo, A. Bruni, M. Serinelli 1. Introduction 129 2. Environmental and health surveillance: state of the art 130 3. Environmental and health surveillance experiences 133 4. Epidemiological and environmental characterization of Brindisi (experimental site) 137 5. Proposal for an enviromental health surveillance system 142 6. Conclusions 142 References 143

Monitoring Contaminants in Food Chain and their Impact on Human Health A. Mupo, F. Boscaino, G. Cavazzini, A. Giaretta, V. Longo, P. Russo, A. Siani, R. Siciliano, I. Tedesco, E. Tosti, G.L. Russo 1. Background 145 2. State of the art and interaction between basic and applied research 151 2.1 Food quality control 151 3. CNR specific expertise: qualified teams, external collaborations and funding 154 4. Highlights 156 5. Future Perspectives: the effects of low-dose exposure to dietary contaminants in children and young adults: a working hypothesis 163 6. Conclusion 168 References 169

The pilot study on Endocrine Disruptors D.G. Mita 1 Introduction 2. State of the art: international and national initiatives 3. Ground and content of the pilot study 4. Expected results References

181 182 183 185 185

Pilot study for the assessment of health effects of the chemical composition of ultrafine and fine particles in Italy M.C. Facchini, F. Cibella, S. Baldacci, F. Sprovieri 1. Background 2. Objectives References

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189 191 191

Introduction

CNR Environment and Health Inter-departmental Project: present knowledge and prospects for future research INTRODUCTION The World Health Organization states that the environment influences our health in many ways through the exposure to physical, chemical and biological risks, and through the changes that our behavior operates in response to those factors. This is what is commonly addressed as the “Environment and Health issue” (EH), which has gained an increasing attention at local, national and international level.

There is a growing political support to the implementation of concrete actions, as well as an ever increasing knowledge of these issues. The need has been recognized to jointly tackle these issues with the aim of producing more detailed and focused studies and planning actions to reduce the negative impacts of some human activities on the environment.

Several international initiatives addressed this issue in the last years.

The environment and health issue in the European Union Since the turn of the century, when the Lisbon Strategy was launched “to stimulate growth and create more and better jobs, while making the economy greener and more innovative”, the European Union has been playing a leading role in addressing the Environment and Health issue. Following the Lisbon Strategy, the 2001 Gothenburg Council established sustainable development as an overall objective for the EU development, taking responsibility to ensure that future generations will not be forced to live in worse conditions that the present one. An European Environment and Health Strategy has been promoted in 2004, paving the way to the coordinated initiatives and joint efforts of the enlarged European Union, and an Action Plan was produced to implement it.

In 2004, the European Commission issued the Environment and Health Action Plan designed to combine information on environment, the ecosystem and human health so to assess with greater effectiveness the overall impact of environment on human health.

The ultimate goal of the EU strategy is to set a cause-effect framework on environment and health and provide the necessary information for the drafting of a EU policy on pollution sources and the impact pathways of stress factors on health. The Action Plan is based on some fundamental aspects: 1) to understand the relation between pollution sources and health effects through the development of environment and health indicators and integrated monitoring systems to assess human exposure and bio-monitoring systems; 2) to enhance research and thus identify potential hazards to human health and develop methodologies to analyse the environment-health relation; 3) to better inform the population and improve risk communication systems.

In the European Union, several other initiatives have been active in the environment and health sector: the Sixth Environmental Action Programme 2002-2010 contains specific indicators measuring the reduction of health risks posed by the environment; the Public Health Programme 2008-2013 adopts specific measures to reduce environment-related health risks; the 6th and 7th Research and Development Framework Programme contains a section on environment and health research initiatives. A number of other laws regarding environment, energy production, infrastructures, territorial management, agriculture, vii

CNR Environment and Health Inter-departmental Project have a direct impact on health: for this reason a unified Strategy and a surveillance system is necessary and welcomed at the EU level.

The Environment and health issue for the United Nations and the World Health Organisation One of the initiatives that can be considered as a milestone in the environment and health sector is the list of the eight benchmarks provided by the United Nations Development Programme in 2000, to be fulfilled within 2015: the Millennium Development Goals.(1) All these Goals have in fact crucial implications for both the environment and health.

The setting up of a monitoring skill, with relevant indicators to be checked over the time by each country Member of the UN, shall also be considered as an added value to this initiative.

On environment and health, however, the World Health Organisation (WHO) is the leading international actor, whose paramount mission has always been to raise awareness on health issues in all international Fora. In this context, it is important to point out that the WHO uses a definition of the environment that is somewhat broader that the one utilised by the European Commission in its Environment and Health Strategy; the WHO definition includes socio-economic factors such as poverty and the lack of infrastructures, whereas the Commission focuses on chemical and biological pollution. It seems desirable that the EU new Constitutional Treaty could fill this gap and change the current scope of its Articles 152 and 174.

In 1989, the WHO Office for Europe promoted the Environment and Health Process, ‘to raise awareness and start collaboration between sectors, particularly the health and environment sectors’. The First Ministerial Conference on Environment and Health was held in Frankfurt in 1989, gathering together the relevant public institutions of the 52 countries of the WHO European Region. The Second Conference was held in Helsinki in 1994, the Third in London in 1999.

The most recent Conference, held in Budapest in June 2004, launched the Children’s Environment and Health Action Plan for Europe to improve the protection of future generations. During this Conference, the European Commission presented the EU Environment and Health Strategy and a specific Action Plan to implement it.

The Fourth Ministerial Conference on Environment and Health in Budapest 2004 The National Research Council of Italy (Italian acronym: CNR) was part of the Italian delegation, led by the Ministry of the Environment, at the Budapest Conference. The Italian representatives, in partnership with the Regional Environmental Centre for Central and Eastern Europe (REC), proposed and organised awareness-raising initiatives - called ‘breathing days’ and focused on air pollution - in some Budapest schools, to underline the active role that younger citizens could play in driving positive change.

CNR also participated in the workshop “Environment and Health in EU Structural Funds Technical Assistance”, organised as a side event during the WHO Conference. The workshop was a first opportunity to discuss the recent Italian developments with partners from the large WHO-EU Region and to present all the coordination efforts made by Italy to an international audience. Experiences developed in the 52 countries are undoubtedly different and their analysis can be useful to plan future developments and actions. viii

Introduction The Authorities responsible of the use of the EU Structural Funds Technical Assistance in Objective-1 Italian Regions (areas lagging behind in their development), gave to the environment and health issue a relevance in the 2000-2006 planning period. CNR worked together with the Ministries of the Environment and Health and the National Health Institute, to establish an network of experts to analyse the specific issues of each territory and address crucial environmental and health factors.

In Italy, the core competencies in environment and health are given to Regional Authorities, and during the mentioned 2000-2006 planning period the European Union allocated funds to the Ministry of the Environment for the establishment of Regional Environmental Authorities to perform surveillance and monitoring activities; and to the Ministry of Health to assist Regional Epidemiological Observatories.

In the framework of their technical assistance activities in seven Italian regions, the Ministry of the Environment and the Ministry of Health joined their efforts to support Regional Agencies for the Protection of the Environment and Regional Epidemiological Observatories. The research on highly polluted sites has been selected as the first issue to be developed. Polluted sites are in fact considered a priority in the agendas of regional bodies and for the public opinion, and therefore these bodies had to be trained to develop their environmental epidemiology competences, methodologies and tools. The technical assistance of the two Ministries helped to implement activities and promoted a culture of collaboration among the different regional bodies in charge of environment and health protection, where the environmental epidemiology had the role of supplying a common framework to experiment and develop such cooperation.

The Italian National Research Council (CNR) The Italian National Research Council (CNR) competence on both the environment and health is well established. The activities of the Department of Earth and Environment (Italian acronym: DTA) are carried out by 13 research Institutes. DTA delivers independent research, survey, technologies and knowledge transfer in the earth and environmental sciences to advance knowledge on the planet as a complex and interacting system.

Research activities cover the full range of the Earth science, including atmospheric, terrestrial and aquatic issues. The mission of DTA is to gather knowledge and predict the behaviour of the earth system and its resources to help design a sustainable future for the planet and mankind.

The Department of Medicine includes twelve institutes performing clinical and epidemiological researches, both autonomously and by interacting with the National Health System structures. Research and health activities include clinical and epidemiological studies in cardiology, pneumology, oncology, neurology, immunology, infectious diseases, genetics and molecular medicine. Innovation and technology transfer in medicine and Education and the training of personnel and NHS are the other main activity areas.

The CNR report on areas presenting environmental hazards for health (2006) In late 2006, the President of CNR and two Directors of Departments were asked to refer to a Chamber of Deputies Public Hearing in the framework of the “discovery investigation to evaluate the environmental impact of urban pollution, waste disposal and high risk areas�. On that occasion, CNR delivered a complete report called “State of knowledge ix

CNR Environment and Health Inter-departmental Project review on environment and health in high risk areas in Italy�.

The report includes the research activities carried out by the CNR Institutes in the 54 areas identified as Reclamation Sites of National Interest,(Italian acronym: RSNI) directly managed by the Ministry of the Environment for the remediation activities. The research activities were developed by 17 CNR Institutes, belonging to the following Departments: Earth and Environment, Medicine, Materials and Devices, Molecular Design, Information Communication Technology, Agrofood.

To give an idea of the size of this problem, it suffices to say that the population affected by the impact of polluted areas is estimated in 6.4 million (but this number increases up to 8.6 million if all the residents in the Municipalities of Milan and Turin, whose territories are however only partly affected by RSNI, are included), living in the 54 RSNI that stretch themselves over the territory of 311 municipalities. These blunt figures are important to assess the size (or – to help make a guess of the impact) of health adverse effects that can be caused by substances recognised as hazardous and released in the environment.

In fact, even modest risk increases, when acting on such large figures, can determine very serious impacts in terms of the development of diseases, symptoms of indisposition, mortality rates. Besides, since the residents of polluted areas are not equally exposed and include socio-economically vulnerable people, or more susceptible people due to a genetic predisposition or co-morbidities, the impact assessments are crucial. The size of the exposed population and the risk intensity are the main criteria to assess the health impact and, more in general, the impact on the public health service: in terms of possible scenarios, the two extremes are represented by a situation in which very aggressive factors act on limited groups of people (particularly exposed workers or small communities), or by situations where risk factors have a weak action but act on a large number of exposed people.

The areas included in the RSNI sites are former industrial areas, caves, landfills; in those cases the remediation procedures are long lasting and often very difficult to undertake from a technical viewpoint; in the case of productive areas, remediation can be implemented when an economic advantage exists, a change in production is required or strong pressure from the public and administrators is exerted.

CNR activities covered a wide range of research sectors: monitoring, pollution characterisation, remediation, epidemiology, planning.

The identification of pollution is the first step, carried out through different monitoring devices in soil, sub-soil, fresh and sea water, indoor and outdoor air.

The pollution characterisation requires the use of devices such as a radioactive tracer and mass spectrometry. In sea water environment, the characterisation is based upon the assessment of the presence and conditions of the fish population, micro-organisms, phytoplankton, sediments.

As concerns emissions into the atmosphere, the research includes studies on climate and modelling, and studies on the relationship between indoor and outdoor pollution.

The removal of pollutants urges on the analyses of the remediation potential for water and soil pollution, including bioremediation, phytoremediation and soil washing.

Emerging fields of research are represented by electromagnetic remote sensing, electromagnetic diagnostics, passive remote sensing in optics, and methodologies for x

Introduction automatic interpretation and integration in Geographic Information Systems (GIS), Global Positioning System (GPS) and georeferencing systems.

Indicators to monitor environmental health effects need a wide range of data searching, analysis and testing; epidemiological research can help to identify the existence of environmental pressures, also using environmental data and GIS. Personal exposure can be monitored using portable personal devices.

Health Impact Assessment procedures and experiences are finally presented in the Report as a tool to be used for evaluation and public participation building in highly polluted areas.

The Environment and Health Inter-departmental Project of CNR-PIAS The CNR experience described above paved the way to build a multi-disciplinary programme, to promote and coordinate collaborative research and joint actions on environment and health.

Following the said report, the Earth and Environment and the Medicine Departments launched a call for project ideas and proposals on environment and health research activities to the whole network of CNR Institutes, taking as reference international priorities and recent European developments.

130 proposals from 37 Institutes (from the Departments of Earth and Environment, Medicine, Materials and Devices, Molecular Design, Information Communication Technology, Agrofood, Energy and Transport) were submitted in few months, with a cost estimation of approximately 30 million Euro.

In mid 2007, a project named Environment and Health Inter-departmental Project (Italian acronym: PIAS-CNR) was presented to the CNR Board.

Objectives The actions and objectives planned in the PIAS have been selected taking into account the EU and WHO Action Plans, the work presently carried out by European experts groups, the indications found in national strategic documents and plans, the excellence of CNR and the scientific and institutional collaborations that could be involved. The deliverables to be accomplished in the framework of PIAS have also been planned accordingly to an evolving real financial support.

The Environment and Health Project has the overall objective of promoting an integrated research between these two scientific sectors, and of developing, in particular: - knowledge of pollution sources and their consequent negative effects on health - instruments and methods to analyse environment and health interactions - instruments and methods to be used in managing complex situations.

On the base of the allocation of available funds to PIAS, the activities have been divided into: start-up phase (phase 1) and research development phase (phase 2).

They will fulfil the following specific objectives: 1. To promote collaboration activities on environment and health among CNR researchers; 1a. to establish a database of the running projects promoted or implemented by CNR Departments or in collaboration with other Institutions; 1b. to promote the creation of think thanks and project development groups; xi

CNR Environment and Health Inter-departmental Project 1c. to promote training workshops.

2. To facilitate the participation of CNR researchers in international experts’ Working Groups on environment and health: 2a. to help disseminate information and to support the CNR participation in EU Commission Working Tables on Environment and Health Strategy of the 7th Research Framework Programme; 2b. to help disseminate information and to support the CNR participation in the WHO work on Environment and Health, preparatory to the realisation of the Fifth Interministerial Conference on Environment and Health to be held in Italy in 2009.

3. To support the access to the funding schemes of the EU 7th Research Framework Programme on Environment and Health: 3a. to help disseminate information and project drafting to get access to the EU 7th Research Framework Programme on Environment and Health; 3b. to help disseminate information and project drafting to get access to other international and national funding schemes, including Structural Funds 20072013.

4. To develop the present CNR competences on Environment and Health Research: 4a. To strengthen the research by selecting pilot projects on issues where collaborations and competences of the various Institutes are more advanced and consolidated, and whose results are considered as feasible with the available financial resources.

In the research development phase, the following scientific objectives must be achieved: • to promote the transfer of research results to the production system; • to promote the transfer of research results to the decision makers; • to promote the experience of Health Impact Assessment, HIA, on present planning instruments (including the Strategic Environmental Assessment, SEA) and the production of methodological handbooks; • to produce transferable results for the development of Environment and Health supervision and monitoring systems; • to test and validate environment and health indicators suggested by EU and the WHO, supported by suitable information systems; • to test and validate the use of georeferentiation tools (GIS) for a joint evaluation of territorial health and environmental data; • to test and validate instruments and methods of risk reporting in different frameworks and to produce guidelines.

Project development PIAS will be implemented through accurate and exhaustive examinations, development of methods and field applications. It will take advantage of the network of scientific partner institutes and a network of sites or areas in which studies and field research have been carried out or are presently underway. The sample areas are selected as those which show the most relevant health and environment impact issues (urban, rural, waste disposal, reclamation areas, etc.).

This project is based upon multidisciplinary Working Packages (WP) and Organisation Units (OU) in which CNR researchers, mainly from the Medicine and the Earth and Environment Departments participate. Researchers from other Institutes with which CNR xii

Introduction collaborates (for instance, the WHO, the National Institute of Health (ISS), the National Institute for Occupational Safety and Prevention (ISPELS), the Institute for Environmental Protection and Research (ISPRA), the ENEA; Universities, the National Health System, the Ministry of the Environment and Territory, the Ministry of Health, some Regional Agencies for Environment Protection, ARPAs and Regional Epidemiological Observatories are also involved in the PIAS. The project is formed by two methodological modules for knowledge development (WP1, WP2), an operative module representing the “engine” of the project (WP3), an interface module to transfer technology innovation and industrial development results (WP4) and an interface module to transfer communication, information and training results (WP5).

The OUs can operate in just one WP or in two or more WPs. Structure

(Research areas, WP division, work orders, etc.)

Overall project structure and working packages (WP) The relevance of the issue and the multidisciplinary research involved in the study of mechanisms and methods as well as in technology application and development, make this a project of primary interest not only for a national research council, but also for the Ministries, such as the Research, the Health and the Environment ones, as well as for other public and private Institutions.

Research Areas 1. Studies on environmental fate, biological perturbation mechanisms and health (WP1); it is the basic module for the development of the necessary knowledge to produce results to be tested in WP3.

1.1. Environmental quality assessment • Lifecycle and the environmental fate of pollutants • Chemical, physical and toxicological characterization of sub-systems • Risks for ecosystems and human and animal health • Detection of risk thresholds and effective health control levels xiii

CNR Environment and Health Inter-departmental Project • Exposure assessment and identification of exposed people 2. Analytical and methodological tools to meet the new environment and health challenges (WP2); this is the basic module to develop the necessary methodologies and tools to carry out the tests foreseen in WP3.

2.1. Monitoring and assessment tools 2.1.1 Environment-health indicators • Bio-indicators of environmental quality, with reference to health risks; • Bio-markers of exposure, physiological reaction, early damage of people’s biological tissues, substances indicating health risk; • Indicators of health consequences, diseases or conditions indicating adverse effects associated to known or suspected environmental risk exposure • Assessment indicators of actions/programmes/measures impacting the primary interest effect classes.

2.1.2. Health impact assessment, cost-benefit analysis and other evaluation and decision-making tools.

2.2. Measuring, supervision and recovery tools 2.2.1 Assessment and mitigation of health-impacting environmental effects; 2.2.2 Monitoring on environmental health indicators, ex-ante and ex-post evaluations; 2.2.3 Instruments to measure, control and recover/improve the environment and health. With particular reference to instruments measuring the exposure dose (individual and environmental dosimetry) especially in the case of combined exposure to different potentially hazardous agents.

3. Field surveys (WP3) This is the core module, the “engine” of the project, oriented towards the field experimentation of the outputs of the previous two working packages (WP1 and WP2), focused on the development of knowledge and methodologies, and the preparation of useful results for technological development and communication management as foreseen in the subsequent packages (WP4 and WP5).

Another objective of the WP3 is the feed-back through knowledge development packages, in particular the WP2, to produce materials, methods and tools for the development of application research and to provide inputs to the WP4 and WP5.

The WP3 main objective is the assessment of the exposure, risks and effects on areas and sites of primary interest for environmental health impact (urban, rural, industrial, waste processing, reclamation areas, etc.). This package is structured in two different, parallel and interconnected activity sectors dealing with knowledge implementation and the experimentation of specific methods on the environment-health relation, starting from the study of hazardous environmental factors or from the study of environmentally sensitive diseases.

3.1 Environmental contamination that produces health effects • inhaled fine particulate matter (PM10), (PM2.5) and ultrafine or nano particles (PM0.1) • heavy metals • asbestos • radon xiv

Introduction • • • •

dioxins and PCB (polychlorinated biphenyl) endocrine disruptors organic pollutants electromagnetic fields, with particular reference to extremely low frequency electromagnetic fields (ELF); mobile telephone systems and occupational exposure • relations between external and internal air pollutants 3.2 Environmentally sensitive diseases • respiratory diseases, in vulnerable groups (children, senior citizens) and in urban population • bronchial asthma associated to the interaction of climatic and oxidizing pollutant factors (NOX, O3) • neurological development diseases • adverse reproductive events and child and paediatric tumours • effects of endocrine disruptors • cardiovascular diseases as a consequence of air pollution exposure • tumours and other diseases in adults as a consequence of environmental exposure in uterus and early life • genetic sensibility and gene-environment interaction 4. Technological and industrial development (WP4) This is a cross-interface module for all project activities, used to provide technological development for the outputs from previous modules.

4.1 Environmental monitoring 4.2 Reclamations 4.3 Biotechnologies 5. Information, communication and training (WP5) This is a cross-interface module for project activities, used to communicate the outputs from previous modules 5.1 Risk perception methods and tools 5.2 Risk communication methods and tools 5.3 Governance strengthening instruments (including regulatory suggestions) 5.4 Instruments to promote public accountability 5.5 Information and training methods and tools PIAS implementation PIAS implementation is closely linked to the funds available at each stage: the first phase, now completed, allowed to explore the potentialities of a collaborative work, and to conceive project developments, as described in the following chapters.

The implementation is based on a voluntary project agreement among the CNR Institutes interested to work together on the topics identified as relevant. Working Groups and Pilot Studies Groups have been identified after the call for project ideas and proposals.

Six Working Groups (WGs) were defined to study this complex chain, starting from the environmental pollution to the emergence of illness. Several CNR Institutes are involved in each WG, together with University and Public Bodies, where relevant.

WG1 – The fate of pollutants in soil (the chain of pollution from emission to human xv

CNR Environment and Health Inter-departmental Project exposure) WG2 - Water and soil monitoring for the protection of environment and human health (specific monitoring for the pollutants identified as dangerous for health) WG3 - Role of atmospheric pollution on harmful health effects (the relation of outdoor with indoor pollution, the molecular mechanisms of disease promotion) WG4 - Human biomonitoring (biomarkers of exposure and early damage, the relations among epidemiological studies, research in toxicology, in vivo and in vitro experiments) WG5 – Environment and health surveillance systems (to develop a protocol for high risk hot spots and selected areas) WG6 – Monitoring contaminants in food chain and their impact on human health (the pollution chain from the emission to the food chain, the pollution of animal feed) Two Pilot Studies(PSs) are in progress. They focus on well-established issues, and are in charge of drafting a feasibility project to apply for EU-FP7 or other call for proposals.

Several CNR Institutes are involved in each PS, together with the University and Public Bodies, where relevant.

The Pilot Studies deals with the following challenging fields: PS1 – Endocrine Disruptors and health effects PS2 – Ultrafine particles and cardiopulmonary effects WGs deliverables include: a workshop to put together experts and building capacities; intermediate documents and working reports to share and discuss; a middle term national workshop for WGs and PSGs together; a summary document including all the activities and prospects for future research (this publication); a conclusive document including the operational proposals and the pre-feasibility projects addressed to the CNR Board of Directors; an international workshop, to be organised in late 2010.

One young researcher was hired as assistant for each WG, whereas PSG are operating to advance research and define pre-feasibility projects.

The national PIAS workshop to compare and discuss the advancements after one year of activity was held in June 2009 and it is now available on line, with video and audio streaming at http://media.src.cnr.it/Filmati.php?evento=72&scheda= National projects and international collaborations The National Institute of Health (ISS) coordinates a Strategic Environment and Health Programme, now in its second year of activity. The Programme is divided into six projects for a total of 41 units and covers the health impact associated with living in polluted sites, in areas affected by waste disposal/incineration facilities, and the exposure to air pollution in urban areas. CNR researchers, together with other Institutions, participate in the Programme implementation. The Programme included the following Projects: The role of ultrafine particles in the pathogenic mechanisms of cardio-respiratory effects produced by an urban pollution; Possible health effects of waste disposal in populations living near disposal/incineration plants and comparative evaluation of the applied technologies; Short-term effects of air pollution in urban areas: effects of gases and fine and ultrafine particles, pollution-temperature interaction, individual susceptibility; long term effects of air pollution: cohort studies in adults and children; Meteo-climatic conditions and health: definition and identification of risk, effects measurement, the evaluation of intervention xvi

Introduction effectiveness on epidemiologically relevant pathologies; Health risk in polluted sites: exposure estimation, bio-monitoring, epidemiological characterisation.

The Institute for Environmental Protection and Research (ISPRA) is involved in several activities concerning the environment and health, such as the dissemination of information on environmental monitoring, novel risks and the promotion of Health in All Policies.

ISPRA is also part of the ERA-ENVHEALTH network.

The ERA-ENVHEALTH is one of the European Union network initiatives of the ERANET ‘family’. The project started in September 2008, and put together managers from 16 environment and health research programmes from 10 countries with the coordination of AFSSET, the French Agency for Environmental and Occupational Health Safety. ERAENVHEALTH strategic objectives are: to establish a network of programme managers and financers to share information and expertise on research; to define opportunities for research cooperation and coordination; to identify priority areas for multinational research; to develop coherent joint activities at the EU level; to implement joint multinational calls for specific research proposals on environment and health; to provide policy support for the implementation of the Environment and Health Action Plan (2004-2010) and other EU policies concerning the environment and health issue. The originality of the ERA-ENVHEALTH resides in the promotion of a trans-national joint call in order to experiment joint funding and to fully assess the implementation. The ERA-ENVHEALTH fosters the use of environment and health research results to support policy development, and supports the early identification of critical issues having a public impact.

The NRC and the Institute for Environmental Protection and Research are the Italian partners of the Project, whereas the Environmental Protection Agency of the Tuscany region (ARPAT) and the Regional Agency for Prevention and the Environment of the Emilia-Romagna region (ARPA-ER) are Consultative organisations and collaborate to spread information and to seek the participation of the scientific community.

The Environment and Health Inter-departmental Project of CNR (PIAS) promotes interdisciplinary research on the interaction between environment and health.

PIAS established a network for collaboration and join activities among researchers coming from different research institutes, that traditionally operate separately in environment sciences or in health disciplines, and to bridge the gap towards an interdisciplinary approach.

The project implementation confirmed the soundness of the original design. PIAS, with its advances in knowledge and its results, represents now a valuable and durable platform to start a broader research programme focused on field investigations and basic research on environment and health, including studies on mechanisms, research on methods and tools, risk communication and innovation for technology transfer.

The successful establishment of the PIAS Working Groups can be now considered as the framework to evaluate several proposals and action plans to be implemented.

Its documented high scientific level, coordination ability and resources attraction, competitive attitude, capacity building and networking have made it possible to complete the priority-setting stage.

The consolidation and strengthening of the existing collaborations in the European and international networks is one of the main goals of the PIAS Project to guarantee a top level research and to offer advanced competences and instruments at the national level. xvii

CNR Environment and Health Inter-departmental Project THE CONTRIBUTION OF PIAS WORKING GROUPS (WGS) AND PILOT STUDY (PSS): SUMMARY OF ACTIVITIES. The fate of pollutants in soil (WG1) Since the soil has several functions directly related to human health, such as the production of food as well as a filter action for groundwater, the preservation of its functionality from any possible threat caused by human and natural events is clearly important. The fate of contaminants in soil has to be addressed in order to evaluate the potential exposure of people, taking into account the complexity of pathways, the interactions with soil surfaces, the changes in the chemical and biological conditions of soil environment. The study of soil environment can thus provide a basis for the assessment of human exposure and health adverse effects. Research activities such as the dietary uptake of vegetables grown in polluted soils, accidental soil ingestion, bio-accessibility and bioavailability must be analysed. The case study of Gela (Sicily) industrial allowed a progress in our knowledge of specific contamination sources (such as landfills or industrial sites) and vulnerable groups, the latter studied on the basis of their place of residence, work activity or dietary habits. Pollution pathways are strongly influenced by the chemical and physical nature of soil, by its equilibrium in a thermodynamically open multiphase system. The identification and understanding of the mechanisms linking soil quality and health is proposed through an integrated approach.

Seven CNR Institutes plus one University Department are directly involved in the PIAS Working Group.

Water and soil monitoring for the protection of the environment and human health (WG2) Current water and soil monitoring programmes are based on the sampling and laboratory analysis of chemical and microbiological variables. Different methods to measure effects, directly applied on living organisms, both at individual and at community level, have been integrated into monitoring plans. Emerging problems are related to new classes of pollutants, not yet regulated by legislation. The CNR Institutes carry out research on several emerging environmental issues, such as engineered nanoparticles and perfluorinated compounds in water environments, that have been chosen as case studies. An innovative monitoring approach, ranging from the measurement of the effects to the identification of causal molecular agents, is discussed. “Toxicity Identification and Evaluation” (TIE) and “Effect Directed Analysis” (EDA) monitoring procedures were reviewed. In parallel to “traditional” in-vitro tests, the development of the “omics” disciplines is fundamental to study the relationships between the genome or protein structure and the activity and biological effects of exogenous agents. Methods to identify the activity of dioxin-like compounds as the cause of a specific adverse effect on river organisms are presented and discussed. Applications and perspectives of investigative monitoring were examined (in the case of an unknown agent or source for toxic or other biological effects) and of screening (for risk assessment of specific pollution sources). All the above mentioned activities are crucial for an effective management of the territory to safeguard human and environmental health.

Seven CNR Institutes are directly involved in the PIAS Working Group. xviii

Introduction The role of atmospheric pollution on harmful health effects (WG3) Gaseous and particulate species in outdoor and indoor air play a key role in increasing the morbidity or mortality observed in many clinical studies. The knowledge of the main toxicity patterns of atmospheric pollutants needs to be improved, especially as concerns particulate species. This is mainly due to the varying size-distribution, chemical composition and different mechanisms of toxicity of fine and ultrafine particles (UFPs).

Recent findings on toxicity routes attributable to gases and particulate matter (PM) species (i.e. the water-soluble organic fraction (WSOC) studied for the strong oxidative potential to biological tissues) are reviewed. Toxicity routes are discussed to hypothesize the relationships among sources, diffusion pathways, receptor sites and susceptible populations. Strategic aspects are underlined, to be further developed in the ‘Feasibility study for the assessment of the health effects of the chemical composition of ultrafine particles’, presently in progress. The nature and role of aerosol particles and gaseous mixtures are major research issues, due to their potential hazard for human health; the connection of the toxicological and epidemiological impacts of atmospheric particulate matter to its chemical composition is of paramount importance to assess effective pollution abatement strategies. During a number of field experiments, state-of-the-art instruments have been used for aerosol characterization.

Two CNR Institutes are directly involved in the PIAS Working Group.

Human Biomonitoring (WG4) Human Biomonitoring (HBM) aims at identifying biomarkers useful to measure environmental exposure, at monitoring its biological effects and the causal relationship with pathological conditions, and at defining, where possible, the genetic susceptibility of the overall population. The search of reliable biomarkers, i.e. objectively measured and validated as health or disease indicators, requires the expertise of scientists with different specializations, able to tackle increasingly complex problems through a multidisciplinary approach. The PIAS work-package aims at promoting a scientific strategy to develop and validate effect, exposure and susceptibility biomarkers. The chapter presents a knowledge review, both in the basic and applied research, as well as future perspectives and developments. Two main HBM objectives have been examined: i) the determination of the levels of toxicants in biological fluids in the overall population; ii) the search for new exposure, effect and susceptibility biomarkers. Four crucial points have been tackled: the management of environment and health issues through a multidisciplinary approach, the combination of medical tools with a biological approach based on biochemistry, biophysics, cell and molecular biology, bioinformatics, molecular genetics and genomics; the validation of conventional/new exposure biomarkers; the validation of conventional/new effect biomarkers; the identification of genetic susceptibility markers in the Italian population. The purposes of modern HBM have expanded beyond their origin in occupational medicine to cover a wide variety of diagnostic procedures and assessments of environmental pollution, leading to the identification of potentially hazardous exposure before the evidence of adverse health effects. The definition of exposure limits to minimize the likelihood of significant health outcome appears as the final goal.

Four CNR Institutes plus one National Public Research Institute (ISPESL), one Scientific xix

CNR Environment and Health Inter-departmental Project Foundation and one Hospital are directly involved in the PIAS Working Group.

Environment and health surveillance systems (WG5) An integrated environmental and health surveillance system is the systematic, ongoing collection and analysis of information related to disease and the environment (indicators) and its dissemination to individuals and institutions. It is a scientific tool for the implementation and evaluation of policies aimed at preventing, controlling and protecting health and the environment. Different analytical approaches to classify environmental and health indicators have been examined and discussed. A protocol to be tested in areas with different environmental risks has been developed, in order to monitor environment and health indicators and to provide useful tools for primary prevention programmes and communication. The goal is to select a set of environmental and health indicators to be assessed for their utility and availability in time and space.

Five CNR Institutes plus two Departments of the National Institute of Health, two Regional Environment Protection Agencies and one Local Health Unit are directly involved in the PIAS Working Group.

Monitoring the contaminants in the food chain and their impact on human health (WG6) A growing attention is paid in Europe to food safety and to the relation between diet and consumer’s health. Changes in lifestyle, modification in food production and distribution determine the eating habits of Western populations. Data from the annual report of the European Commission Rapid Alert System for Food and Feed (RASFF), summarizing notifications on food contaminations occurred in different countries, are useful to plan efficient food control programmes. In this context, the PIAS working group studied how specific classes of environmental contaminants (e.g. pesticides, metals, dioxins) may affect human health through the food chain. Issues of major interest in this sector are, for instance, the determination of heavy metals and dioxins in food matrixes and biological samples; the existing experimental models to assess the harmful effects of contaminants on human reproduction; the role played by the cytochrome P450 in the xenobiotics metabolism. Finally, a research programme based on a holistic approach has been defined.

The proposal, whose target is the young population, aims at identifying the cause-effect relationship between the presence of contaminants in the diet, their accumulation in humans and the risk of developing chronic diseases. A discussion on the bioavailability and adaptive response is presented, to suggest a possible functional link (at molecular level) between the onset of specific diseases and the concentrations of contaminants measured in food. An integrated approach to assess the impact of food contamination on human health can increase our scientific knowledge and build consumers’ trust and confidence.

Three CNR Institutes plus one Public Research Agency are directly involved in the PIAS Working Group.

The Pilot Study on ‘Endocrine Disruptors and health effects’ (PS1) The pilot study focuses on the relationships among exposure to endocrine disruptors and some selected diseases and the identification of how the environment and the diet xx

Introduction synergistically operate in promoting some severe pathologies in wildlife and humans.

Several experimental studies reported that also very low doses of endocrine disruptors can affect the endocrine system, causing diseases and altering the development of mammalian (humans included) and non-mammalian species. Cancer, cardiovascular risk, modulation of adrenal, gonad and thyroid functions, and endometriosis are some of the diseases that cause alarm in the citizens, associated to the exposure to endocrine disruptors. Research activity focuses on three lines: i) the possible relationship among the levels of toxic pollutants in biological fluids and the risk or occurrence of cardiovascular diseases, as well as the alteration of thyroid, gonad and adrenal functions in the population of Gela (an high environmental risk area); ii) in vivo experimental studies on endometriosis using mice exposed to Bisphenol A, iii) measurements of endocrine disruptors concentration in fish used for human consumption in the selected area.

Three CNR Institutes in collaboration with the Italian Endometriosis Foundation are directly involved in the PIAS Working Group.

Pilot Study on ‘Ultrafine air particle and cardiopulmonary effects’ (PS2) It aims at combining the results of two advanced activities in the identification of the composition of atmospheric ultrafine particles and those of the health studies exploring short-term effects of air pollutants exposure in subjects with selected diseases. Five workpackages focus on the improvement of knowledge in: i) the chemical composition of ultrafine particles and their variability in urban and rural sites in Italy, based on available multi-stage impact data and on initial measurements using Aerosol Mass Spectrometers, ii) the methodologies to measure the oxidative potential of the water-soluble organic fraction (WSOC) of the aerosol, iii) the short-term effects of exposure to air pollutants in subjects with pre-existent arrhythmia, iv) the short-term effects of exposure to air pollutants in subjects with pre-existent lung diseases, finally v) results are to be evaluated to design an integrated Italian research activity for projects to be presented in the framework of the available EU projects call for proposal.

Four CNR Institutes plus one University Department, one Regional Environment Protection Agency and one Local Health Unit are directly involved in the PIAS Working Group.

On the whole, eighty-six researchers belonging to nineteen CNR Institutes and other twelve Public Research Bodies are directly involved in the PIAS project and have been collaborating at the preparation of the present publication.

Fabrizio Bianchi PIAS Coordinator, CNR - Institute of Clinical Physiology fabriepi@ifc.cnr.it Liliana Cori PIAS Scientific Support, CNR - Institute of Clinical Physiology liliana.cori@ifc.cnr.it Pier Francesco Moretti CNR - Department of Earth and Environment

pierfrancesco.moretti@cnr.it xxi

CNR Environment and Health Inter-departmental Project

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The fate of pollutants in soil G. Petruzzelli, F. Gorini, B. Pezzarossa, F. Pedron CNR, Institute of the Ecosystem Studies (ISE), Pisa (Italy) petruzzelli@ise.cnr.it

ABSTRACT Different disciplines must be urgently put together to understand environmentally related diseases, and to develop strategies capable of reducing the negative effects of pollution on human health. Within this framework, the importance of soils and their characteristics on human health is receiving a growing interest as the essentiality of soil for human life becomes increasingly clearer.

Understanding the transport and transformation of pollutants from the source of origin to the final receptors (environmental ecosystems and humans), through different soil typologies, is of paramount importance.

On the basis of the duration, frequency and intensity of exposure, it is also important to evaluate which concentrations are necessary to produce biological alterations in living organisms, leading to the onset of a pathology.

One of the main objectives of the Environment and Health Inter-departmental Project, PIAS-CNR, was to highlight the close links between environmental matrices and human health. Within this framework, Working Group 1 (WG1) focused attention on the fate of contaminants in the environment, particularly on the soil ecosystem. The WG1 proposal aims to go beyond total diet studies and to understand mechanisms and processes by which contaminants enter the food chain and influence to various extents nutrition and the health of humans.

1. INTRODUCTION The integration of different disciplines can help to overcome the compartmentalization of increasingly specialized scientific knowledge. This is urgently needed in order to understand environmentally related diseases, and to develop strategies capable of reducing the negative effects of pollution on human health. Within this framework, the importance of soils and their characteristics on human health has been growing in interest as the essentiality of soil for human life becomes increasingly clearer (38, 46, 75).

Soil functions, such as food production, are directly related to human health,. The action of filters on groundwater highlights the need to preserve soil given that its

efficiency can be reduced by human activity and natural events (1, 108).

The fate of contaminants in soil is important in terms of evaluating their possible exposure to humans. Another significant element is the complexity of pathways determined by emission sources, interactions with soil surfaces, and changes over time in the chemical and biological conditions in the environment where the soil is located (Fig. 1).

2. STATE

OF THE ART AND INTERACTIONS

BETWEEN BASIC AND APPLIED RESEARCH

Soil is defined as the top layer of the earth’s crust and is made up of mineral particles, organic matter, water, air and living organisms. Soil is a multiphasic and

CNR Environment and Health Inter-departmental Project

Figure 1. Soil – health relationships extremely dynamic system, with numerous functions: it is the main producer of biomass and raw material, it supports biodiversity development (habitat, species, etc.), it provides the main source of carbon, and plays a fundamental role in human activities and in the survival of the ecosystem.

The formation and regeneration of soil are extremely slow and thus soil is considered as a non renewable source.

Some soil degradation processes derive from anthropic activities (compaction, salinization, contamination, impermeabilization, decrease in organic matter, reduction in biodiversity), and natural phenomena (erosion, flooding, landslides). The resulting effects, however, can be aggravated by human activities, such as agricultural practices, industrial activities, tourism, urban and industrial development, and town and country planning (1). These processes can lead to a decrease in soil fertility, a loss of carbon and biodiversity, a reduced ability to retain water, an alteration in the gas and nutrient cycles, and a less efficient degradation of contaminants. Soil degradation has a direct effect on water and air quality and on climate changes. It can also influence human health and present a danger in terms of food safety (9, 33, 58).

2

Data analysis shows that soil degradation in Europe may cost 38 billion euros per year.

The Thematic Strategy of the European Union for soil protection (2006) proposes guidelines aimed at protecting soil and maintaining its ecological, economic, social and cultural role (30, 31). The Strategy is contained in the Plan of Environmental Action of the European Community, adopted July 2002 and valid until 2012. Its priorities include climate change, nature and biodiversity, the environment and health, natural resources, and waste (32).

Co-ordinated action at a European level is necessary in terms of the consequences of soil degradation on other issues related to the environment or food safety. Before this directive was approved, soil had never been the focus of European protection measures, and soil protection was related only to regulations concerning environmental protection or other strategic fields, such as agriculture and rural development.

The Strategy is a legislative bill which permits the sustainable protection and use of soil, integrates soil protection within national and European politics, and raises public awareness. According to the directive, member states have to take measures to avoid soil contamination with dangerous substances and plan an inventory of contaminated sites.

The fate of pollutants in soil When chemical concentrations present a risk for human health or the environment, the directive calls on member states to remediate the polluted lands, with the aim of removing, controlling, edging or reducing the pollutants. Similar strategies are also followed at an international level (25, 53, 74, 98, 99, 110, 132, 133, 134, 135, 139, 140, 141, 142, 143).

2.1 Soil and human health Soil has always been vital to humans and fundamental to human health since it is the main resource for food production. The link between the continuously increasing world population and the ability of soil to sustain that growth was the topic of Thomas Maltus’s 1798 essay. The maintenance of suitable nutritional food sources is an old problem that is still present today. Soil is not the only element that affects the food supply, but it is an extremely important resource needed in overcoming this complex issue. In developing countries characterized by a high rate of soil degradation, the lack of safety regarding an adequate dietary intake is a relevant problem. Worldwide food production and demand is likely to increase, making it crucial to manage and conserve soil. One of the the E.U objectives is to protect soils against erosion and pollution since longterm productivity is likely to be affected by soil degradation resulting in a relevant reduction in yields on agricultural land, not only in developing countries (77).

Soils may influence human health also in other different ways. Ingestion of soil may result in significant exposure to toxic substances. Children are the object of special interest, since soil adhered to fingers may be inadvertently swallowed by bringing the hands to the mouth, especially during outdoor activities (68).

One of the most common effects of soil

ingestion is the alteration of the mineral content and nutrient balance in individuals.

Ingested clays, due to the acidic environment of the stomach, release the elements contained within them through the mechanism of cation exchange (82).

Concerning the toxicity of the contaminants to which humans can be exposed through the ingestion of soil, lead is a major concern and focus of study. Children are subject to a greater risk because lead acts as a neurotoxin, with particularly serious effects on the development of the nervous system during childhood (96).

Soil ingestion plays an important role in the risk assessment of contaminated sites, where also soil inhalation is considered of particular concern.

In the early 1980s studies revealed that most of the soil dust inhaled by humans is trapped and then swallowed, passing through the gastro-intestinal tract.

However, a portion of this dust is trapped inside the lungs, where it can progressively lead to bronchitis, pneumoconiosis and cancer of the lungs. The reaction of the lungs to dust obviously depends on the kind and the amounts of the dust inhaled (137). Particles with a size comparable to those of clay and originating from wind erosion of the soil, once inhaled, can settle in the pulmonary alveoli, causing progressive inflammation in the lungs.

Further damages arise following inhalation of particles coated with toxic substances and also of the biotic components of soil (20, 135). The fungus aspergillus present in the soil is the biggest killer along with the human AIDS virus, causing lung infections following immunosuppression (113). An infectious disease known as “desert fever� is caused by inhaling spores of the fungus Coccidioides immitis. In the U.S, it is estimated that each year between 50,000 and 100,000 people are affected 3

CNR Environment and Health Inter-departmental Project by symptoms of Coccidioidomycosis (7).

Tetanus is the most common desease to potentially affect people who come into contact with soil. This disease is due to a toxin produced by Clostridium tetani spores of anaerobic microorganisms. The bacteria, present in the surface layer of soils as well as in human and animal secretions, are especially abundant in cultivated and fertilized fields. Hookworm, characterized by multiple clinical manifestations including anemia, is a disease also caused by skin contact with the soil and whose hexogene agent is detectable in the nematodes Ancylostoma duodenale and Necator americanus. The survival of the hookworm larvae in the soil is favored in moist, sandy, crumbly environments and at temperatures between 24 and 32 °C. Infection can occur by oral ingestion of contaminated food and, presumably, direct ingestion of soil. The disease has a high incidence in the rural areas of the tropics, with higher occurrences in children (63).

Other diseases are ascribable to soil characteristics. Podoconiosis has been correlated with soils containing particles of colloidal size derived from wethering of basaltic rocks that are able to penetrate the epidermis intact (119).

Soil quality largely determines ground and surface water quality. In Bangladesh drinking water is contaminated with arsenic at concentrations of up to 1000 Îźg/l.

The consumption of contaminated water led to the spread of disease and death, with typical epidermal lesions (105). Considering the sources of As and the mechanisms that result in groundwater pollution, it is possible that Fe hydroxides present in the sediments are reduced by the activity of microorganisms, favoring the release of As absorbed in groundwater (92).

When the soil ability to retain organic compounds by sorption processes is 4

reduced, groundwater pollution by organics of industrial origin is a widespread problem.

The same happens for pesticides that can penetrate into the soil through different routes, such as root systems, leaves and the decomposition of plant and animal tissue.

Other pesiticides are directly applied to the soil and can be released from soil to surface water and groundwater (120).

Many of these compounds are considered endocrine disruptors and have severe health implications.

Further contamination derived from agriculture practices is the release of the NO3- anion from soil. This ion has a high solubility in aqueous environments, and being negatively charged is poorly absorbed by most soil surfaces. The excessive presence of nutrients causes algal bloom with serious consequences on the whole aquatic ecosystem. Toxins produced by rapidly growing cyanobacteria can cause numerous human disorders, including gastroenteritis, atypical pneumonia, allergic reactions, and liver diseases including cancer (12). Among the consequences of ingestion of nitrate ions is childhood methemoglobinemia, which severely damages the ability of hemoglobin to carry oxygen in the blood (73).

The use of antibiotics applied to agricultural crops for the control of plant diseases, and their addition to animal feed have given rise to several problems of immediate and practical importance. The inactivation of antibiotics in soil may be determined by: intrinsic chemical instability of the antibiotic molecule; adsorption on soil clay minerals and organic matter; microbiological degradation.

Since antibiotics are a heterogeneous group of compounds, varying greatly in their chemical structure and reactivity, and soils are not homogeneous, no generalizations regarding the stability and biological

The fate of pollutants in soil effects of antibiotics in soil are possible.

Although some antibiotics in soil are unstable chemically, and many are degraded microbiologically, it appears that several antibiotics persist in some soils for a time sufficient to produce harmful effect.

Soil bacteria are considered to be a source of new resistance mechanisms to clinically used antibiotics. In Europe, the livestock industry consumes thousands of tons of antibiotics per year. The application of cattle manure to soil might be a relevant source of antibiotics. The bacteria populations resistant to antibiotics in soils are lower in unmanured soils than in feedlot soils.

In the U.S, the presence in groundwater of antibiotics such as tetracycline, added to feed to promote livestock growth, may present a possible means of determining antibiotic resistance in humans. The analysis of soil and ground water samples from reserves close to farms have shown that the bacteria are identical to those in the gastrointestinal tract of animals, and contain genes that are resistant to antibiotics (27). This study suggests that genes are transferred from bacteria of the gastrointestinal tract of cattle to other ecosystems. Since in the U.S. about 40% of the water used for civilian consumption comes from groundwater (and this value has been gradually increasing), the presence of antibiotics can lead to serious consequences for human health (136). In the EU, livestock consumes approximately 5000 tones of antibiotics each year.

Though there is no set limit on the use of medicines in agriculture, veterinary authorities have ruled that any compound that can be accumulated at concentrations higher than 7.5 g per hectare must undergo environmental impact studies (114).

The deposition of feces in the soil from humans and animals may potentially contaminate fresh water sources with

bacteria, protozoa and viruses (122).

For example, Escherichia coli 0157 is a virulent pathogen that in humans gives rise to a broad spectrum of symptoms, including hemorrhagic colitis (83). Cattle are the main reservoir of the bacterium, which, once reaching the ground, remains there for several months. The most common causes of infection from E. coli 0157 are associated with the consumption of contaminated meat and dairy products, although infection in humans may also occur due to the contamination of soil and drinking water.

A noteworthy amount of metals has been released into the environment by anthropogenic activities, in particular by industrial processes and persist in the soil due to their non biodegradability. Heavy metal pollution is responsible for many negative consequences both for human health and the environment (17, 59, 61, 72).

Most heavy metals are considered essential micronutrients and each of them requires an adequate daily intake. However trace elements are toxic if there are excessive amounts of them in the human body, and they have adverse physiological effects at relatively low concentrations. Soil ingestion represents a direct route for the elements to humans. The transfer of many elements from soil through the food chain is an important although indirect mean of exposure. Consequently, deficiencies, excesses or imbalances of inorganic elements from food sources may have important consequences. An inadequate intake of microelements is recognized as an important contributor to the global burden of disease through increased rates of illness and death from infectious diseases, and of disability such as mental impairment (16). An increase in the concentrations of microelements in soil derived from weathering processes 5

CNR Environment and Health Inter-departmental Project of the parent rock material or by human activities such as industrialization, mining, agricultural practices, and urbanization, can cause an excessive release of elements in the food chain and can have implications on human health. The itai-itai syndrome is probably the best known example of metal contamination in the soil that has some implications for human health through the ingestion of contaminated food. It developed in Japan in the 1950s, and it is caused by food, especially rice, and drinking water contaminated by Cd (35, 97, 111).

As a consequence, all legislations concerning soil strictly regulate the soil cadmium content to avoid its accumulation in agricultural crops. However, Cd accumulation in plants is determined by the available fractions of metals in soil rather than their total content. Although the soils may contain high concentrations of metals or organic contaminants, factors such as pH, clay content, and organic matter impact on speciation, mobility and bioavailability of pollutants, influencing. the amount absorbed by animals and humans (131).

2.2 The fate of contaminants in soil Soil contamination occurs through either point source or diffuse pollution; the main difference between the two types of contamination lies in how the contaminants are transferred to the soil.

Point sources, such as manufacturers, landfills, incinerators, use soil as a support and are linked to the activities that necessarily transfer pollutants into the soil (64). Diffuse sources are associated with natural phenomena (long range transport, atmospheric deposition, sedimentation by surface water), with agricultural practices, with recycling and inadequate waste treatments. The most dangerous 6

contaminants in soil are, in general, persistent organic pollutants (POPs) and inorganic pollutants, above all heavy metals. Persistent organic pollutants have an anthropic origin and are characterized by high lipoaffinity, semivolatility and resistance to degradation. In the case of heavy metals, that cannot be degraded or destroyed, the presence in the soil could be due to natural processes, for example the formation of soil, and to anthropogenic activities. Some are important essential elements (Cu, Fe, Mn, Zn, Co), if present in optimal concentration ranges, while others (Hg, Pb, Cd) are potentially toxic elements (19, 78, 80, 81, 84, 109, 130).

2.2.1 The nature and behavior of inorganic contaminants Heavy metals are one of the numerous classes of substances that can reach critical levels in terms of human health, food safety, soil fertility and ecological risks (80, 126).

Heavy metals are common contaminants in the soil and bioaccumulate, thus their concentration in the organism increases over time compared to the level measured in the environment. This is because the absorption rate is higher than the excretion rate in the organism (128).

The distribution of heavy metals between the solid phase and the soil solution is considered to be the key factor when assessing the environmental consequences of the accumulation of metals in the soil (2, 69).

A physical and chemical analysis along with an analysis of the soil profile is essential for assessing the soil as a barrier against inorganic contaminants, particularly heavy metals (121). The retention of heavy metals in the solid phase of the soil is dependent primarily on the pH, and is linked to clay minerals, humic substances, iron oxides and hydroxides, and manganese found in

The fate of pollutants in soil the soil, which all control the attenuation effect even on anionic forms (116).

The retention and release process of heavy metals includes precipitation and decomposition, ionic exchange, and adsorption and desorption.

The precipitation/release reactions may involve discrete solid phases or solid phases, which are absorbed onto the soil surface. The ion-exchange reactions derive from an exchange between an ionic species in the soil solution and an ionic species retained in sites with permanent charge on the soil surface. The absorption and desorption processes can affect all ionic or molecular species and generally concern absorbent sites with a pH-dependent charge. These surfaces are iron, aluminum and manganese oxides and hydroxides, clay minerals and humic substances. pH pH is the most important parameter governing concentrations of metals in soil solutions that regulate precipitation– dissolution phenomena. Metal solubility tends to decrease at a higher pH. In alkaline conditions the precipitation of solid phases diminishes the concentration of metal ions in solutions and the reverse happens with a lower pH. pH values also regulate specific adsorption and complexation processes. The sorption of metals is often directly proportional to soil pH due to the competition of H+ (and Al3+) ions for adsorption sites, however this competition may be reduced by specific adsorption. Metal hydrolysis at higher pH values also promotes the adsorption of the resulting metal hydroxo complexes, which beyond a threshold pH level (which is specific for each metal) drastically reduce the concentration of metal ions in the soil solution. At low pH levels, on the other hand, sorption processes are reduced due

to the acid catalysed dissolution of oxides and their sorption sites, whereas the complexation by organic matter tends to decrease with increasing acidity.

Clay content Ion exchange and specific adsorption are the mechanisms by which clay minerals adsorb metal ions. This is done through the adsorption of hydroxyl ions followed by the attachment of the metal ion to the clay by linking to the adsorbed hydroxyl ions or directly to sites created by proton removal. Highly selective sorption occurs at the mineral edges. However notable differences exist among clay minerals in their ability to retain heavy metals which are more strongly adsorbed by kaolinite than montmorillonite. This is probably due to a higher amount of weakly acidic edge sites on kaolinite surfaces. In expandable clays (vermiculite and smectite) the sorption processes essentially involve the inter-layer spaces, and are greater than in non-expandable clays such as kaolinite.

The importance of clay minerals, and of soil texture in determining the distribution of heavy metals between the solid and the liquid phases of soil has direct consequences on the metal bioavailability of plants. For the same total concentration it is well known that heavy metals are more soluble and plant available in sandy soil than in clay soil.

Organic matter content The organic matter content of soils is often small compared to clay. However, the organic fraction has a great influence on metal mobility and bioavailability due to the tendency of metals to bind with humic compounds in both the solid and solution phases in soil. The formation of soluble complexes with organic matter, in particular the fulvic fraction, is responsible 7

CNR Environment and Health Inter-departmental Project for increasing the metal content of soil solutions. However higher molecular weight humic acids can greatly reduce heavy metal bioavailability due to the strength of the linkages. Both complexation and adsorption mechanisms are involved in the linking of metals by organic matter thus including inner sphere reactions and ion exchange. Negatively-charged functional groups (phenol, carboxyl, amino groups etc.) are essential in metals retained by organic matter. The increase in these functional groups during humification produces an increase in the stability of metal organic complexes, which also show a greater stability at higher pH values.

Cation exchange capacity The density of negative charges on the surfaces of soil colloids defines the CEC of soil. This capacity is governed by the type of clay and amount of organic colloids present in the soil. Montmorillonitic type clays have a higher net electrical charge than kaolinitic type clays; consequently, they have a higher cation exchange capacity. Soils containing a high percentage of organic matter also tend to have high cation exchange capacities.

The surface negative charges may be pH dependent or permanent, and to maintain electroneutrality they are reversibly balanced by equal amounts of cations from the soil solution. Weak electrostatic bonds link cations to soil surfaces, and heavy metals can easily substitute alkaline cations on these surfaces by exchange reactions. Moreover, specific adsorption promotes the retention of heavy metals, also by partially covalent bonds, although major alkaline cations are present in soil solutions at much greater concentrations.

Redox potential Reduction-oxidation reactions in soils are 8

controlled by the aqueous free electron activity pE often expressed as Eh redox potential. High levels of Eh are encountered in dry, well aerated soils, while soils with a high content of organic matter or subject to waterlogging tend to have low Eh values. Low Eh values generally promote the solubility of heavy metals. This can be ascribed to the dissolution of Fe–Mn oxyhydroxides under reducing conditions resulting in the release of adsorbed metals.

However under anaerobic conditions, the solubility of metals could decrease when sulphides are formed from sulphates.

Differences in individual metal behaviour and soil characteristics result in conflicting reports regarding the effects of redox conditions on metal solubility.

Iron and manganese oxides Hydrous Fe and Mn oxides, are particularly effective in influencing metal solubility in relatively oxidising conditions. They are important in reducing metal concentrations in soil solution by both specific adsorption reactions and precipitation. Although Mn oxides are typically less abundant in soils than Fe oxides, they are particularly involved in sorption reactions with heavy metals. Mn oxides also adsorb heavy metals more strongly, thus reducing their mobility. This action is particularly important in contaminated soils. Specific adsorption of metals by hydrous oxides follows the preferential order: Pb > Cu >> Zn > Cd. Other factors There are a number of other factors which may affect the solubility of metals in soils. Temperature, which influences the decomposition of organic matter, can modify the mobilisation of organo-metal complexes and consequently plant uptake.

An increase in the ionic strength of soil

The fate of pollutants in soil solutions reduces the sorption of heavy metals by soil surfaces due to the increased competition from alkaline metals. Similar effects also derive from the simultaneous presence in soil solutions of many heavy metals which compete for the same sorption sites. This results in an increase in mobility in contaminated soils due to the saturation of adsorption sites. The living phase of soil is also of great importance in determining metal solubility, which is dependent to some extent both on microbial and root activity. In the rhizosphere, plants can increase metal mobility by increasing their solubility. This happens following the release in the exudates both of protons which increase the acidity, and organic substances which act as complexing agents. Microbial biomass may promote the removal of heavy metals from soil solutions by precipitation as sulphides and by sorption processes on new available surfaces characterized by organic functional groups.

2.2.2 Organic pollutants Among the many organic compounds present in soil, the most dangerous are the “persistent organic pollutants” that derive, in general, from anthropic activity, are extremely persistent in the environment and are transported for long distances (5, 28, 55, 66, 71). In specific environmental conditions they bioaccumulate and biomagnify, reaching considerable concentrations that represent a threat for human health and ecosystems. Of the twelve groups of persistent organic pollutants, the following three are acknowledged internationally: polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs).

PCBs are high hydrophobic extremely stable compounds and have very good dielectric and thermostability properties;

these characteristics led to the diffusion of PCB for industrial and civil use (8).

After accidental ingestion or due to their presence in food compounds, PCBs are absorbed through the gastrointestinal tract, and then accumulate in body fats as a consequence of their hydrophobicity (85).

The International Agency for Research on Cancer (IARC) has classified PCBs as potential carcinogenic agents for humans: experimental tests suggest, in fact, that these compounds may increase the risk of skin, liver and brain cancer (24). In order to protect human health and the preservation of the environment, the European Community banned the commercial use of PCBs in 1990. However, these persistent compounds are still present both in natural soils, owing to long-distance transport, and in soils that have been contaminated by specific industrial activities (13).

PCDDs and PCDFs, which are generally known as “dioxins” (118), are the undesired by-products of chemical and combustion processes and are also produced from natural events, such as accidental fires and volcanic eruptions. The dioxins are a group of 210 chlorine-containing chemicals, 17 of which have a toxicological interest owing to their carcinogenic potential and their effects on reproductive, endocrine and immune systems (48). Owing to their high persistence in the environment, they remain in soil, which become pollutant reservoirs (117). In humans, the main route of exposure to dioxins is through food, which represents 90% of the total exposure (51, 87).

EDCs. Over the last few years there has been an increasing interest in identifying the long-term damage to reproduction and development; xenobiotics with potential endocrine activities or endocrine disrupter chemicals (EDCs) have been identified as the main possible risk factors (47).

9

CNR Environment and Health Inter-departmental Project Endocrine disrupters are a heterogeneous group of persistent organic and inorganic pollutants including dioxins, PCBs, pesticides, and industrial compounds.

They are characterized by their potential to affect the correct functions of the endocrine system, especially the homeostasis of sexual and thyroid hormones (10, 29, 125). These molecules may enter the soil environment by agricultural practices or industrial waste disposal. The risks derived from EDCs are determined by the distribution of these compounds among the soil phases.

Depending on the chemical properties of the molecules, EDCs can be either strongly retained by solid soil phases, or leached to deeper layers. Their mobility is largely determined by adsorption – desorption processes on solid soil phases.

A probable role of endocrine disrupters is attributable to polybrominated byphenyls (PBDEs), a class of manufactured chemicals structurally similar to PCBs, which were used in the past as flame retardants (15, 40). Even though most PBDEs were banned within the European Union in 2006, studies have revealed that PBDE levels have increased both in the environment and in human tissues and body fluids (34, 37, 50, 60).

Pesticides are a class of compounds used to kill harmful organisms, especially in agriculture. However many are also toxic for other organisms, including humans (93). The presence and bioavailability of pesticides in soil can adversely impact soil quality with related consequences on water and air quality. Soil characteristics regulate the processes that affect the behavior of pesticides such as adsorption, degradation, volatilization adsorption by crops. Pesticide adsorption to soil depends on both the chemical properties of the pesticide and properties of the soil, in particular organic matter. Organochlorinated pesticides have 10

been used for many decades and one of their main features is their high persistence in soil and transfer into the food chain, with the consequence of well known toxic effects in biota (67).

Behavior of organic contaminants Organic molecules in soil are a carbon source for microorganisms. Therefore, the conditions that influence the breakdown of organics by microflora should be considered. Microflora are not always able to attack organic molecules and digest them completely, but often only partially break them down. This results in compounds that are even more toxic than the initial ones.

The intrinsic toxicity and health risks following the ingestion of organic compounds are well known, both natural compounds and those deriving from productive processes. On the other hand, there is less information about the potential contamination, caused by organic compounds present in the soil, on the food chain (plants-animals-humans). Organic compounds should be evaluated in terms of their chemical properties and their relative absorption potential by plants, but also in terms of the influence that the soil has on them. In fact, these compounds can be volatized, absorbed and therefore immobilized, or transported along the soil profile even to underground water.

The most important chemical properties of organic molecules are those dealing with their absorption in the food chain: the distribution coefficient (octanol/water (Kow), the Henry constant, solubility, half-life, and the bioconcentration factor (BCF).

The behavior of an organic contaminant in the soil depends on the interactions that are established with the solid, liquid and gas phases of the soil, and with the living phase. These relations give rise to the major

The fate of pollutants in soil phenomena that rule the fate of the organic contaminants concerning adsorption, biotic and abiotic decay, leaching and volatilization.

Adsorption and desorption The adsorption processes of organic compounds on the active surfaces of the soil are particularly important because they delay mobilization and leaching of organic contaminants.

The distribution of the contaminants between the liquid and solid phase of the soil can be synthetically described by the distribution coefficient Kd, which in turn can be expressed as a function of organic carbon (Koc) and of Kow. The compounds that have high levels of Kow and low solubility will be mostly retained by the soil surfaces and be less available to environmental processes.

Biodegradation Biodegradation is the most important mechanism for the removal of organic compounds in the soil. Degradation by the microbial flora can increase the solubility and therefore the availability of recalcitrant compounds for microorganisms in the soil. The chemical characteristics of each specific compound affect the time required for biodegradation. Various parameters have been identified that could be correlated with the degradation period.

For example, the half-life of polycyclic aromatic hydrocarbons, PCBs and dioxins are all related to the Kow. This coefficient is also related to the leaching process and to the persistence of contaminants in the soil.

In fact, compounds characterized by a log Kow > 4.0 rarely mobilize. Therefore the same compounds mentioned above, as well as several organochlorinated pesticides are very persistent and have a very low leaching potential. Monocyclic aromatic

hydrocarbons, some chlorobenzenes, short chain aliphatic compounds and phenols, on the other hand, degrade rapidly and are more easily leached from the soil.

Photolysis, hydrolysis and oxidation (abiotic degradation) also contribute to the disappearance of some organic compounds.

These reactions mostly affect compounds with simple molecular structures, such as phenols and some polycyclic aromatic hydrocarbons (PAHs) with less than four benzene rings. Volatilization also affects volatile substances, which are generally characterized by a reduced molecular complexity.

2.2.3 Soil - animal transfer Depending on the grazing practice, the season and the diet, both organic and inorganic compounds present in soil with a high level of contamination are potentially transferable to animals. In fact, contaminated soil is usually ingested directly during grazing. The average amount of soil ingested by most cattle is around 6% of total removal (d.w, dry weight), i.e. for a typical daily bovine consumption of 15 kg d.w. it can reach 0.9 kg of contaminated soil per day. Assuming that organic compounds in polluted soils are present in concentrations from 0.1 to 10 mg/kg, the amount ingested can vary from 30 to 3000 mg/year Bioconcentration processes are particularly important for persistent and non-polar compounds (low solubility and with high Kow). The BFC bioconcentration factors related to diet, however, are difficult to quantify experimentally. Nonetheless, some models based on the daily intake of organic compounds can provide an estimate of their possible presence in meat or milk.

When ingested these compounds can pass through the gastrointestinal membrane, 11

CNR Environment and Health Inter-departmental Project enter the blood or lymphatic system or into some organs in relation to the lipid content and, depending on the compound (PCBs, dioxins, hexachlorobenzene), may have long half-lives.

Other compounds such as polycyclic aromatic hydrocarbons are not particularly absorbed, but can be partially degraded with the consequent formation of very dangerous intermediate products.

Among foodstuffs, milk is especially sensitive to organic compounds which undergo considerable changes in the concentration of organic compounds even in response to short term changes.

2.2.4. Concluding remarks More research is needed in order to evaluate if the pollutant levels in the environment threaten human health, considering the most susceptible situations (proximity to contamination sources such as landfills or contaminated sites) and the most exposed people, e.g. owing to work activities or diet (6, 23, 54, 57). One very important aspect is an understanding of the transport and transformation of pollutants from the source of origin to the final receptors (environmental ecosystems and humans), through the different soil typologies (64, 103, 106, 129)). On the basis of the duration, frequency and intensity of exposure, it is also important to evaluate the concentrations necessary to produce biological alterations in living organisms, until the onset of a pathology.

Diet represents the principal means through which not only chemicals, but also micro-organisms and mycotoxins, reach humans (52). Research is needed on the bioavailability mechanisms of organic and inorganic pollutants in soil, and the survival and persistence quantification of pathogen agents in the environment.

The detection of microbic interactions 12

is also essential, together with further knowledge of emerging pathologies and molecular toxicology. Finally, studies need to be performed on the long-term effects following chronic exposure to low levels of individual or mixed chemicals, as well as the consequences of exposure to high concentrations of natural elements.

3. PILOT STUDY: INFLUENCE CHARACTERISTICS

ON

THE

OF

MOBILITY

SOIL OF

CONTAMINANTS IN THE INDUSTRIAL AREA OF

GELA (SICILY) 3.1 Soil characteristics of the Gela site Gela is a town in Sicily located in an area of important industrial activity, which has caused over the course of time significant contamination of the environment. In fact, it is so polluted that the Italian government has designated it as an area (in Italian known as “Site of National Interest: SIN�) that is subject to specific regulations in terms of remediation. The Gela site lends itself to a new interpretation of pollution that is no longer confined to a contaminated site, but which has instead spread to a wider environmental area. The Institute of the Ecosystem Studies (CNR-ISE, Pisa) have tried to highlight how important soil characteristics are in defining the contamination pathways of pollutants, and how complicated it is to establish general relations since each type of soil has specific characteristics that differentiates it from other types.

Given the complexity of the matrix, which can contain many organic and inorganic contaminants, the definition of a potential hazard based on soil characteristics, is limited. It is only thorough a characterization of the soil of the industrial area of Gela affected by pollution that it would it be possible to give a more accurate response. However,

The fate of pollutants in soil given the amount of data produced during the characterization phase of the site, we planned to assess the characteristics of the soils derived from analysis certificates, based on the analytes determined. We also wanted to evaluate which soil parameters were missing, which are useful to detect contamination pathways.

As mentioned previously, the potential hazards of a contaminant present in the soil and its risks to human health, particularly through the exposure pathways from the soil through the food chain and finally to man, can be better defined if we know the chemical and physical properties of the soils in which contaminants are present (1, 2, 22).

We examined the documentation collected at the Italian Ministry of the Environment on Gela site to check whether, in addition to the characteristic parameters of contamination (pollutants concentration), there were also parameters describing the characteristics of the soils. This was because these factors would help to predict the environmental mobility and potential bioavailability of the contaminants present.

The parameters that were observed and that are most frequently reported are pH, cation exchange capacity (CEC) and organic carbon (C). These quantities are fundamental to understand what types of soil are in the areas concerned and how they interact with the contaminants in the soil. These data are shown in Figures 2, 3 and 4. However, there are no data on texture, which is of paramount importance.

Such data could possibly be obtained, even if only partially, from the geological description of any probing that might have been done.

Given the significant amounts of data on the characterization of soils within the various industrial areas, some conclusions

can be drawn with regard to heavy metals.

However, based on the data reported on the certificates, it is not possible to make concrete hypotheses regarding organic contaminants. Among the parameters identified, pH is particularly constant and depends on the nature of the mineralogical substrate from which the soil originates.

In fact, it is the most important parameter that governs the concentration of inorganic elements in soil solutions. In the soils from the Gela area there should both be a limited mobility of heavy metals, which move like ions with positive charges (Cd, Zn, Cu, Pb), and a limited bioavailability of such metals. The same should hold true for mercury, however the probable presence of high concentrations of chloride ions can greatly facilitate the mobilization of the element and its diffusion in the environment. Metals that move like ions on the other hand, with a negative charge such as As, may be more easily mobilized, and could become a significant problem in the whole area. The other parameter reported in the characterization analysis of soil is the cation exchange capacity. This quantity expresses the charge density on the surfaces of soil colloids. It varied considerably from one industry to another which meant it was impossible to identify a uniform retention capacity of metals in different parts of the Gela area under investigation.

The importance of pH and CSC is not as significant in organic compounds as it is in inorganic contaminants. These parameters have a limited influence on the mobility of nonionic organic compounds, which are much more influenced by the content of organic matter. Organic matter in the soils of this area appears to be quite low although there are considerable differences between one area and another. Variability in the values of organic matter and the lack of soil texture characteristics are of 13

CNR Environment and Health Inter-departmental Project primary importance for understanding the behavior of organic contaminants. This highlights the need to integrate data from the characterization of the site with the features of the soils, which could be partly drawn from land use maps at a provincial or regional scale. The characterization of environmental matrixes can be a key issue for population exposure estimates, integrated with data on health and epidemiology. As far as soil is concerned, in addition to the commonly used ways of evaluating a contaminated site, such as skin contact and direct ingestion, it is also necessary to take into account the characteristics of this environmental matrix affected by the contamination. Soil properties determine the movement of pollutants and their passage into the food chain (1). This leads to an understanding of low-dosage effects, which are prolonged over time and which are often forgotten in decontamination strategies dictated by the need to solve immediate and acute problems resulting from pollution.

By understanding the specific soil characteristics of the area, if possible along with a related food analysis, uncertainty in the exposure calculations may be reduced and a relationship can be defined between the sources and targets of the contamination.

In order to evaluate the capacity of the soil to interact with different types of pollutants, it is necessary to consider this environmental matrix as a threephase system. In general, the solid phase constitutes 50% of the soil, while the other half is made up of a porous space which, in a good quality soil, contains half water and half air. The solid phase, the degradation of the parent rock, contains organic materials (humic substances) that are concentrated in the upper layers. It also contains inorganic materials which at a certain depth become 14

the exclusive constituents of this phase.

The liquid phase is made up of water that forms a “soil solution�. This solution contains dissolved substances and can dissolve other substances from the solid phase. The soil solution reaches the roots and gets into pores. This is the principle means of transport of all of the substances, including the pollutants. The gaseous phase of the soil is made up of air, which on the surface layer is richer in carbon dioxide because of the high quantities of organic material.

The flow of air into and out of the soil is essential for plant growth and for the decomposition processes of animal and plant residues, as well as all materials of an organic nature.

Mercury is particularly important in the Gela site. Mercury, like other metals in the soil, may be present in a dissolved form as a free ion, or absorbed non-specifically by weak electrostatic bonds, specifically absorbed by covalent bonds, made more complex by organic matter, or precipitated in its solid phase in the form of carbonate, hydroxide or sulfide (18). Depending also on redox conditions, mercury can exist in three valence states, Hg0, Hg and Hg2+. Its bivalent form is generally highly reactive with dissolved ligands, and is highly soluble in water. It very often forms complexes with Cl-, OH-, S2- and with sulfur-containing functional groups of organic compounds and NH3.

Mercury also forms complexes of moderate stability with Br- and I- and some nitrogenous R-NH2-type binding agents.

The factors that control the speciation of the metal in solutions are pH, ionic strength, redox potential, a concentration of dissolved organic matter (DOM), and dissolved ions such as oxygen and sulfides.

The maximum solubility of mercury occurs

The fate of pollutants in soil in an oxygenated environment (Eh 350-400 mV) which is the typical condition found in soil. The principle forms that are found in soil are Hg(OH)2 and HgCl2. With these ions, mercury can form soluble complexes that are environmentally significant because they are very mobile. On the other hand, in anoxic environments these ions form stable and insoluble sulfides.

Methylmercury, CH3Hg+, and dimethylmercury, (CH3)2Hg, are also formed in the soil (49, 90) but they constitute on average less than 2% of the mercury present in the soil. Even at low concentrations, these compounds can cause serious bioaccumulation problems (101).

CH3Hg+ is synthesized by microbe activity (bacteria and fungi) both aerobically and anaerobically. It is soluble in water and forms different compounds such as CH3HgCl, CH3HgOH and CH3HgSH. The anion that binds itself is particularly important because it determines the biological uptake. The speciation of the CH3Hg+ ion is similar to Hg2+ and therefore the parameters that influence it are the same: pH, DOM and ionic strength.

Mercury is mobilized in the soil through the formation of soluble inorganic compounds which include HgCl2 and Hg(OH)2. The degree of mobility of these complexes depends on the type of charge and on the chemical and physical characteristics of the soils in the area. The presence of chloride ions makes the metal highly mobile for the formation of very soluble complexes.

In the presence of high amounts of organic substances, a process that is equally important is the formation of organic complexes of bivalent mercury due to the high affinity of the Hg(II) ion and of its inorganic compounds for the functional groups containing sulfur. A part of bivalent mercury can be complexed by soluble humic substances, such as fulvic acids, and therefore may be present in the liquid phase

of the soil. Mercury loss due to ground runoff is still very small compared to the total percentage, so that in contaminated soils like those in the Gela area the metal can be expected to be released for a very long period, thus affecting human health for many years.

While historically mercury has been the element of greatest concern in the area of Gela, the soil characteristics are more favorable to provide conditions for a great mobility and bioavailability of arsenic. Like other metals arsenic toxicity depends on the chemical form of the element, organic compounds being much less toxic than inorganic ones. The main forms of arsenic in soil are arsenate and arsenite which are highly toxic, because their molecular similitude to phosphate can interfere with the functions of many proteins. USEPA defined arsenic as a human carcinogenic contaminant.

Soil arsenic may influence human health by soil dust respiration, soil ingestion and consumption of contaminated water (3).

Moreover arsenic may enter the food chain via crops and vegetables grown in polluted soil (62).

The total content of arsenic in soil is not a reliable indicator of the potential hazards for health and environment. Its mobility and bioavailability are largely determined by soil characteristics (104).

The retention of arsenic in the solid phase depends on soil pH, mineralogical composition and competing ions in soil solution. In aerobic soils, sorption on metal oxides is the main process that regulates arsenic bioavailability. Arsenate is linked to amorphous Fe and Al oxides, by the formation of inner sphere surface complexes, while arsenite forms inner sphere and outer sphere complexes, the latter specifically with Al oxides.

In the soil from the area of Gela, the trend 15

CNR Environment and Health Inter-departmental Project of arsenic mobility is inverse to that of mercury. Soil pH determines the negative potential of mineral surfaces, which increases with increasing pH. The net effect is a decrease in sorption processes in the solid phase of soil. The transport of arsenic in soil is controlled by sorption/release processes and the alkaline conditions of these soils together with the oxidation – reduction potential, promoting an increase in the mobility of arsenate ions. Movement of the contaminant is determined by a pore space diffusion coupled with a sorption on solid phases which can be described by a Freundlich type equation. The solubility of arsenic can be described by a distribution coefficient Kd of the divalent arsenate ion which is directly dependent on soil pH according to the equation Log10 Kd = log10 (As soluble/H2AsO- 4) = a + b pH Where Kd is the solid solution distribution coefficient, for the arsenate ions Assol the amount released in solution and H2AsO- 4 the free ion activity (127). From this equation potential bioavailable arsenic substantially increases with increasing pH. In the environmental conditions of soils in the area of Gela there is a high probability of the existence of soluble arsenic forms.

The soil characteristics, would seem to indicate that the hazards deriving from this element could be even higher than those deriving from mercury contamination.

Environmental issues regarding metals such as mercury, arsenic (11, 14, 70, 91) are strictly linked to soil characteristics in that immobilization or potential bioavailability is regulated by parameters that are specific to the soil (pH, clay content, organic matter, and cation exchange capacity).

These determine the chemical and physical conditions that may give rise to 16

precipitation or solubilization resulting in an increase in bioavailability and/or leaching, with a danger of polluting the aquifers.

3.2 Contamination pathways of organic compounds In an area with a high degree of pollution such as the Gela site, the main pathways of contamination affecting the soil are, in addition to skin contact and direct ingestion of soil, absorption by roots, transfer to the edible part of plants, and direct soil ingestion by animals during grazing.

The absorption by plants of organic compounds present in soils is influenced by the physical and chemical properties of the compound, by the type of soil and by the characteristics of the plant. It can occur both by radical absorption and by subsequent translocation in the aerial part, both by leaf absorption of volatile compounds and contaminated dust.

These issues vary in importance depending on the compound in question. Hydrophobic substances (PCB) can be absorbed on the root surface and remain bound to the lipid of membranes. This can create serious problems in some species such as carrots, which have an ectoderm rich in lipids. Plant absorption is a complex phenomenon based either on an active process specific to each compound, or a passive process in which organic contaminants are transported by the transpiration water of the plant.

There are several indicators for predicting the transferability of organic compounds from the soil to the plant. For example, compounds with a log Kow between 1 and 2 are those most likely to be moved by the aerial part of the plants. Substances with a half-life of less than 10 days will tend to disappear from the soil before being absorbed by the plants, while the most persistent ones can get into the plant

The fate of pollutants in soil nutrition processes. The most volatile compounds with Henry’s constant > of 10-4 tend to evaporate from the soil and given that they are not absorbed by the roots they may contaminate the plants through the leaves by volatilization. Of course, this is a very schematic approach that can be used for an initial screening of polluted soils in order to understand what the immediate dangers are.

The passage of an organic contaminant from the soil to the food chain can be described as a series of consecutive partition reactions between the solid and liquid phase of the soil, between the soil solution and the roots, and between the roots and the aerial part of the plant. This series of reactions is influenced by the characteristics of organic compounds, in particular by the partition coefficient of octanol/water, so that compounds with a low Kow value can be moved more easily into the aerial part of the plants. On the other hand, substances with a high Kow value (PAHs, PCBs, PCDD/F) are adsorbed by the soil and, if partially uptaken by the plants, they remain in the root system (2). Generally, these compounds are not absorbed, but there can be an accumulation of some compounds in root crops (confined to the outer parts of the roots that are removed before consumption).

Some of the more volatile compounds may enter the leaves, especially through the stomata, by atmospheric deposition or by absorption of the molecule in its vapor state. For semi-volatile compounds with a high Kow, translocation from the root system may be minimal, so the absorption in the vapor state can become an important source of leaf contamination. Compounds with a high lipophilic nature and high volatility may be present with significant concentrations in the leaves.

Inside the plant, some substances may

be metabolized in a short period, others (PAHs, PCBs) much less, though they may be partially degraded at specific sites. For example, some nitrobenzene compounds are degraded in the roots, while some aromatic chlorinated compounds are metabolized only in the leaves.

Metabolism takes place depending on the structure of the chemical contaminant and the type of plant. For example, when degradation increases, it decreases the number of chlorine atoms, and the process is often only partial with the formation of intermediates.

The amount of halogenated organic compounds that can be absorbed by the plant, including hexachlorobenzene, a contaminant of interest in the Gela area, depends on water solubility, the concentration and organic matter content in the soil. The immediate risks stem from whether the plant is able to metabolize or eliminate the compound before being harvested, and whether the compound is transferred to the edible part.

Hexachlorobenzene can result from various industrial processes. It is very stable and not particularly reactive, since it is involved in the adsorption phenomena at the soil surface, which influence the volatilization and leaching processes as well as its preponderance to biological and chemical degradation or uptake by plants.

Since it is a non-ionic compound, it is subject to the adsorption process involving Van der Vaals forces, and it is closely linked to the content of organic matter, particularly in soils with a low clay content. Unlike other chlorobenzenes it has a log Kow > 5.3 and is therefore difficult to assimilate by the plant since it is substantially immobilized by adsorption processes in the soil.

The molecular structure is such that the leaching process should be quite limited.

However, depending on the characteristics 17

CNR Environment and Health Inter-departmental Project of the soil texture, the compound may be found in groundwater, transported through the larger pores in the soils, or in soils that have a tendency to form deep shrinkage structures.

The chemical stability of hexachlorobenzene makes it particularly persistent in soil and resistant to biodegradation, with a half-life of more than 1500 days. This compound has a remarkable permanency in the atmosphere with the possible formation of hydroxyl radicals and a half-life of two years, which could reach the soil as a result of precipitation and atmospheric deposition.

The principal biodegradation mechanism is oxidation, which leads to the formation of hydroxylated aromatic compounds, followed by the breaking of the benzene ring.

Hexachlorobenzene tends to accumulate in the roots of plants and remains bound to lipids of the membranes and the cell walls with less possibility of translocation due to its low solubility.

The potential toxicity of hexachlorobenzene for animals is largely linked to the risk of direct ingestion of soil by animals during grazing or through fodder feeding.

Hexachlorobenzene is characterized by a high volatility that can be an important pollution pathway, through leaf absorption of fodder and consequently by animals.

3.3 Conclusions Soil is a complex system that has allowed life on earth to exist and facilitated the birth of agriculture. In addition to being the most important source of essential nutrients, it is also a source of pollutants that reach humans through the food chain and diet (1). Given that soil quality is vitally important for our health, it is surprising that this issue has been studied so little by the scientific community (86). This probably 18

stems from the fact that identifying and understanding the mechanisms linking soil quality and health, through the intake of agricultural products or processed foods, requires detailed and multidisciplinary expertise which is difficult to coordinate (42, 43, 44, 45, 46).

An innovative solution is to overcome the compartmentalization of environmental aspects and consider a continuum that goes from the presence of a substance in the soil, to its transfer into the food chain with the consequent health effects (94). The main transfer pathways of substances from soil to humans have been studied almost exclusively within contaminated sites. It is assumed in rather simplistic terms, that there is a direct correlation between the concentration in the soil of a given element (or substance) and its absorption by man (39). However, what really needs to be investigated is how, in a broader context the transfer of contaminants from soil to humans follows quite complex pathways (76). These pathways are determined by the chemical and physical nature of soil (115) characterized by physical, chemical and biological equilibriums in a multiphase system that is thermodynamically open.

4. CNR SPECIFIC EXPERTISE: QUALIFIED TEAMS AND EXTERNAL COLLABORATIONS

4.1 CNR Institutes The Institute of the Ecosystem Studies of Pisa (CNR-ISE, Pisa) is involved in the study of the quality of soil and of shallow and ground waters. This is because they play an essential role in life cycles, ecosystems and our quality of life.

The study of soil quality related to human health has not been studied much by the scientific community. The understanding of mechanisms that link soil quality and human health through food ingestion needs

The fate of pollutants in soil

Figure 2. pH variability in soils sampled at the Gela site.

Figure 3. CEC variability in soils sampled at the Gela site. coordinated and multidisciplinary skills. which can increase the risk of pathologies Such mechanisms are not easy to identify is determined on the basis of duration, and to address to projects involving a frequency and intensity of exposure. varied skill set.

The transfer of substances from soil to CNR-ISE’s research is aimed at studying humans follows complex pathways, in the mechanisms of transport and the relation to the physical and chemical transformation of contaminants from properties of the soil and to the sources to soil. The dose of contaminants characteristics of the biological receptor. needed to determine biological alterations, The transport of contaminants needs to be 19

CNR Environment and Health Inter-departmental Project

Figure 4. Organic carbon variability in soils sampled at the Gela site. tackled starting from the highest critical Possible applications of this research • To identify contaminant pathways in state areas. contaminated sites and surrounding The studies at CNR-ISE focus on the areas and the effects of pollutants in effects of chronic exposure to low levels soil on dietary uptake. of contaminants, either individual or in mixtures, and on the consequences of • To evaluate of the transport of contaminants from sources to target via exposure to high doses of contaminants soil-plant system. naturally present in the environment.

In this particular field CNR-ISE is The Institute of Biophysics of Genoa carrying out studies on the bioaccessibility (CNR-IBF, Genoa) has a considerable of contaminants (i.e. heavy metals and experience in electrophysiology and selenium) in soil in relation to their ion channel biophysics in nervous and bioavailability. Bioavailability is the endocrine culture cells, investigated capacity of a contaminant to interact by patch-recording and voltage-clamp with the biological world and involves the techniques, and intracellular calcium remediation of contaminated soil using dynamics, studied by fluorescent probes. In recent years, these skills have been applied green technologies.

Within this field, the CNR-ISE group has to the study of heavy metal accumulation organized national congresses on Soil and toxicity in mammalian cells and the Quality, Food and Health in cooperation modulation of neurotrasmitter-gated ion with the Institute of Clinical Physiology channels by metal ions in primary neuronal (CNR-IFC), University of Bari and the cultures and in recombinant receptors Local Operative Division of Gorizia of expressed in heterologous systems CRA-RPS. The congresses were funded (frog oocytes and/or mammalian cells). by the Italian Ministry of Agriculture and The group has additional expertises in Forestry and the third edition will be held molecular and cellular biology, including PCR and RT-PCR, in vitro transcription in 2010.

20

The fate of pollutants in soil and the functional expression of wild type and mutated protein clones in Xenopus oocytes, cell culture and mammalian cell transfection. They also study the effect of acute and chronic treatment with heavy metals (Pb, Cd, and others) on cell survival and the maturation of neurons in culture by functional and viability tests and apoptosis/necrosis measurements.

Recent work has characterized some Cd and Pb permeation pathways through the neuronal membrane and has identified the location of specific binding sites on the NMDA receptor channel for Pb and Ni. The CNR-IBF group in Genoa also studies the molecular and cellular mechanisms regulating astrocyticneuronal interactions in physiological and pathophysiological conditions. This is done using calcium imaging, immunoblotting and electrophysiological techniques.

They research the features and roles of P2X7 purinoceptors on primary cultures of neonatal and adult astrocytes, in secondary cultures stably transfected with rat P2X7 or expressing truncated P2X7 receptor, cocultures of neuron/glia and on purified nerve terminals (synaptosomes) and astroglial fraction (gliasomes).

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants present in the food chain and in human blood and milk. Exposure to PBDEs during pregnancy and lactation leads to signal pathway modifications, calcium homeostasis alteration and apoptotic neuronal death.

Such events could play different roles depending on the developmental stage of the central nervous system. Scarce reports are available on specific models, allowing dissection of diverse mechanisms involved and temporal sequences of prenatal or neonatal exposure to PBDEs. Relative contributions of neurons and glia, and their bi-directional communication in the

control of glutamate synaptic level and in excitotoxicity triggering and execution, are not clearly defined. Knowledge of the efflux mechanism of the excitatory aminoacids and of how they regulate in the early and late phases of exposure to PBDEs, could lead to the possibility of regulating extracellular excitatory aminoacid levels in different neonatal PBDEs phases. As intracellular Ca2+ accumulation seems to be a prerequisite for neuron damage cascade, the dampening of Ca2+ influx through ionotropic glutamate or purinergic receptors (e.g. P2X7) could significantly reduce neuronal damage. We therefore plan to investigate the glutamate efflux and cellular Ca2+ levels in physiological conditions and during PBDE exposition in vitro models of the neonatal and adult brain.

Parallel studies will be conducted to investigate whether the amino acid release from astroglial cells can be modulated by endogenous signaling molecules through the regulation of swelling-activated Clchannels. The following experimental models (from the cerebral cortex of neonatal and 60 days-old rats) will be used: i) isolated purified nerve terminals (neuron model) and gliasome (astroglial fraction unpolluted by nerve endings, model for astrocytes ex vivo) from the cerebral cortex of neonatal and adult rats after food exposition to PBDEs insult. ii) in vitro cortical neurons and astrocytes.

A functional and pharmacological characterization will be carried on these models out of ionotropic glutamate and purine receptors by studying the “release� of glutamate (or [3H]D-aspartate), and intracellular Ca2+ transients with fluorescence methods (Fura-2).

Possible applications of this research • Identification and validation of cellular models (cultured cells) to establish 21

CNR Environment and Health Inter-departmental Project significant alternatives to in vivo animal tests in toxicology.

• Characterization of metal binding sites on neurotransmitter receptors and other ion channels for designing selective ligands to be used in clinical pharmacology.

• Implementation of biosensors to appraise the bioavailable fraction of toxic metals and to establish the factor of correlated biological risk.

Knowledge of the modes for controlling extracellular glutamate accumulation and cellular Ca2+ overload and their relationship with swelling-activated Cl-channels in neonatal and adult brains exposed to PBDE insult would contribute to a rational therapeutic strategy to neuroprotection with a multipharmacological approach.

In vitro neonatal models, using controlled experimental conditions and the dissection of pollutant mediated neurotransmitter efflux modes are only at a very early stage of development. Furthermore, little is known about the control of the excitatory neurotransmitter efflux from nerve terminals and the role of neuronal and glial counterparts in the control of glutamatergic transmission and of glutamate level at glutamatergic synapses in the developing and adult brain.

The Institute of Biophysics of Pisa (CNR-IBF, Pisa) is involved in the quantification of prospective toxicity in aquatic environments in order to evaluate the relative risk of the introduction of unknown contaminants. Water organisms can be contaminated directly or indirectly.

The former occurs by contact or ingestion of the substance dissolved in water, whereas the latter happens when the contaminant is accumulated in the food chain. Chemicals present in sewage of industrial or agricultural origin are liable to contaminate soil, superficial water as 22

well as groundwater aquifers and thereby represent a risk for all water use: drinking water as well as bathing, irrigation and breeding water.

Contaminants may be of a chemical or biological origin. The former mainly consist of substances with slow degradation rates and which are therefore easily accumulated in the soil and aquifers and thus in the first levels of the trophic chain – photosynthetic organisms, plants and algae. Chemical residua, such as pesticides, herbicides and heavy metals, in animal and human tissues undergo a biological magnification process. Their toxicity, often consolidated by a prolonged presence, represents an important health risk. Among the contaminants of a biological origin, those relevant to health issues are algal toxins: these may be released in the aquatic environment and have a toxic effect on humans.

Possible applications of this research The research group aims to create “early warning” monitoring systems on marine, fluvial and basin waters using microspectroscopy and digital microscopy. In fact, in vivo and in situ microspectroscopic and microfluorimetric measures performed on the photosynthetic compartments of algae present in waters contaminated by either organic compounds and/or heavy metals, reveal the quantitative effects of the pollutants on the chlorophyll:carotenoid ratio and on photosynthetic efficiency. All this information indicates the quality of the water and which of the microalgae analysed may be used either as biosensors or bioremediation, Digital microscopic measures, on the other hand, obtained with optic microscopy techniques and image processing are able to identify and classify algal species (even to identify a single occurrence), to determine water quality

The fate of pollutants in soil and to recognise those species producing toxins dangerous to human health.

The Institute of Agro-environmental and Forest Biology of Rome (CNR-IBAF, Rome) has a mushroom germ plasm bank.

Their research focuses on the: - collection and characterization of the wild germ plasm from different countries; - use of mushrooms for recycling agricultural and agroindustrial waste; - degradation of lignocellulosic materials for animal feeding; - biotechnologies for environmental applications of fungi.

To ascertain the quality and the safety of both the substrates and the fruiting bodies, analyses on the presence of xenobiotic substances, in particular the presence of heavy metals have been performed,.

Moreover, in the field of the alternative use of mushrooms, technologies have been developed aimed at: - obtaining polysaccharides through extraction in fruitbodies and in mycelia; fungal polysaccharides, especially chitin and chitosan, are widely applied in pharmacopeia, cosmetics, diets and environmental applications; - using mycelium biomass for wastewater depuration. Several batch studies have been performed to test the ability of mushrooms to adsorb heavy metals.

Fungal biomass loaded PVA was then used in columns for the depuration of water containing heavy metals.

- using mycelium biomass for mycoremediation, in the degradation of phenols, antimicrobial tetracyclines, polycyclic aromatic hydrocarbons and heterocyclic compounds.

Possible applications of this research Mushrooms can be considered as: a) living organisms or b) food.

In case a) mushrooms can be used for

mycoremediation in water depuration or in organic molecule degradation. It would be interesting to study the role of polysaccharides present in the cell walls in heavy metal adsorption and the degradation of toxic compounds.

In case b) it is important to consider that mushrooms are able to adsorb heavy metals in the soil in which they live or in the growth substrates used for their cultivation. High concentrations of metals are toxic and inhibit growth and fructification, but with different responses for different metals. It would be interesting to evaluate the dose that permits mycelium expansion and carpophore formation but which may still be dangerous if the mushroom is used as human food.

The Institute of Methodologies for Environmental Analysis (CNR-IMAA, Potenza) has considerable expertise in the study of mineralogical and geochemical risks to human health as well as the use of geo-materials for therapeutic treatments (such as pelotherapy and pharmacology).

In recent years CNR-IMAA has applied mineralogical and geochemical information in order to: - investigate the presence of potential toxic elements in waters and rocks outcropping in some areas at risk and also to study the mobility of some chemical elements due to rock-water interaction processes; - identify geo-environmental risk factors (temperature, water quality, trace elements, etc.) affecting the biominerals present in the human body (in particular in kidney stones and bones) and their mineralogical and chemical composition.

Possible applications of this research • The identification of lithologic pollution due to rock-water interaction processes could lead to the production 23

CNR Environment and Health Inter-departmental Project of geochemical maps which could be considered as tools to protect human health.

• The chemical, mineralogical, petrological and textural study, carried out with integrated techniques, can be used to collect useful information on the processes of neo-formation and the transformation of both pathological and non-pathological biominerals present in the human body (stones, osteoporotic bones, teeth etc.).

• The methodological approach involved (epidemiology and geo-environmental features) may enable information to be gathered that would be useful for preventing and treating some diseases.

• The identification of a procedure for characterizing and highlighting the use of mineral sources in paleotherapy and pharmacology.

The research of the Institute for the Dynamics of Environmental Processes, (CNR-IDPA, Venice) is focused on studying the environmental processes, especially the mechanisms of transport and transfer of organic and inorganic pollutants, both at local-regional and global levels.

To understand the accumulation and transfer processes along the trophic web, it is essential to study the behaviour of trace elements and persistent organic pollutants (POPs) at a chemical and biological level.

In addition their chemistry needs to be considered in concentration terms, as well as variations in the chemical species in which the elements can be present.

In order to completely understand the processes and mechanisms that control the involvement of metals and organic compounds at various organisation levels of the ecosystem and the interaction levels between the various trace elements, it is important to closely examine their absorption and transport along the trophic 24

web. The interaction of an element with other parts of a system depends on its chemical form, so it is very important to study the speciation of trace elements and their effects on the interactions between various biotic or abiotic compartments of the environment in depth. As for trace elements, the same can be said for persistent organic pollutants (POPs), which include polychlorobiphenyls (PCBs), polyaromatic hydrocarbons (PAHs), dioxins and hexachloro-cyclohexanes. In addition knowledge of the concentrations of the diverse congeners in the environment plays a key role towards a better understanding of the transport along the trophic web and subsequently the bioconcentration and the biomagnification in biota.

Several studies have been carried out in highly polluted areas and in pristine environments, such as the Venice Lagoon, the Ross Sea in the Southern Ocean, Morocco, Vietnam, Mexico, etc. Different environmental matrices were sampled (seawater, sediments, biota, etc.) and the concentrations of organic micropollutants and of trace elements were assayed; furthermore, the speciation of trace elements were studied. It is known that several organic pollutants, such as PCBs, can bioaccumulate within the trophic web, at a level directly related to environmental levels, and levels within an organism’s diet.

Therefore for an accurate risk assessment, all the information on congener levels in the biota and the environment has been integrated with the WHO Toxic Equivalent Quantities (TEQs).

The Venice Lagoon represents a particular ecosystem, a transition between two very different environments: the Adriatic Sea and a drainage basin. The variety of inputs (fresh waters, including run-off from agricultural soil and contaminated industrial sites, seawater, industrial and urban wastes

The fate of pollutants in soil and the input of pollutants via aerosol) deeply affect the environment, which is also characterised by a high biodiversity and high productivity determined by the input from the drainage basin. Thus, taking into account the particular features of this environment, the Venice Lagoon is among the areas protected by the European Framework Directive (22nd December 2000, aka Water Framework). According to the Water framework, in-depth studies on the environment are required at a morphological and ecological level. These should study the impact of environmental change taking into account any socioeconomic impacts. Of primary importance are methodological studies to develop guidelines to evaluate environmental risks that operate not just at a technical or legal level but also at a scientific level.

Research also focuses on monitoring stress biomarkers in indicator organisms specifically chosen for each environment under study. The environmental monitoring of biomarkers and bioindicators could provide fundamental information. This would contribute towards an improvement of direct measurements, such as chemical measurements by looking at the speciation of potentially toxic elements. Thus for a correct evaluation of environmental risks, analyses need to take a holistic approach.

Biomonitoring and chemical measurements need to be integrated, taking into account the diversity and similarities between organisms and between organisms and their environment. This would contribute towards as complete a vision as possible of all the possible transport routes, and all the possible exposure and assimilation modes, as well as bioaccumulation and toxicity dynamics.

The contamination of waters and sediments in coastal areas and harbours is due to a wide range of organic (POPs, such as

PCBs, PAHs, etc.) and inorganic pollutants (trace elements, such as mercury (Hg), lead (Pb), chromium (Cr), etc.). In these areas sediments may be a significant sink and/ or source of these pollutants. Taking into account the necessity to dredge sediments in order to keep navigation channels open, remediation and environmental recovery are of great consequence in harbour areas. In fact, the management of dredged sediments is crucial for the growth of the port of Venice, due to increasing sea traffic and foreign trade. In view of the dredging of many millions of cubic meters of sediment according to the ‘Piano di Recupero Morfologico’, CNR-IDPA carried out a sediments remediation project (RISED, Azione Biotech III, Regione Veneto), in collaboration with the Venice Port Authority. The main aim of this project was to assess an innovative washing procedure for dredged sediments. The goal was to been environmentally friendly and suitable for the variety of organic and inorganic pollutants, by exploiting the properties of natural organic substances.

Activities, future applications and innovations Analyses of organic micropollutants are carried out in the CNR-IDPA laboratory, by gas chromatography coupled with high resolution mass spectrometry (GCHRMS). In addition to the traditional extraction systems, there is a pressurised solvent extraction system (One-PSE, Applied Separations), an automated sample purification system (Power-Prep, FMS) and an automated system for reducing the sample volume (Turbovap, Zymark) in the laboratory of the institute. Two gas chromatographs (HP 6890 Series), with autosamplers, coupled with EI-MS detectors (one is a high resolution detector coupled with a double focus magnetic 25

CNR Environment and Health Inter-departmental Project sector Thermo Finnigan Mat XP 95, the other is a low resolution quadrupole detector HP 5973) enable several classes of organic pollutants at trace and ultra trace levels (PCBs, PAHs, dioxins, emerging hazardous and/or priority substances, etc.) to be determined in different and complex matrices.

Furthermore, an important future aim is to establish a risk assessment for POPs (persistent organic pollutants), which includes information on toxicity and on the accessibility and availability towards biota in a very efficient and pliant way. For a correct evaluation of environmental risks, an assay for stress biomarkers, specifically for the exposure to organic contaminants is being applied to different species of biota.

A class 100 Clean room minimizes any contamination of samples to be analysed for trace elements. Three differently equipped ICP-MS instruments, namely a Thermo Finnigan Element 2, coupled with an autosampler, an Agilent Technologies 7500i coupled with an autosampler, and an Agilent Technologies 7500cx equipped with a collision cell, enable the analysis of elements at trace and ultra trace concentration levels in different matrices (seawater, sediments, air, food, different species of biota). In order to better understand the bioavailability and the bioaccessibility of trace elements, the study of geo-speciation in sediments is carried out using sequential extraction and analysis

by ICP-MS. Since the bioavailability of trace elements depends on speciation, it is essential that analytical methods are available to determine or predict the bioavailable fraction of a metal. Thus, the speciation of trace elements such as arsenic (As) and mercury (Hg) is studied using various methods. For a correct evaluation of environmental risks, an assay for the stress biomarkers for the exposure to 26

different species of trace elements is being applied to different species of biota.

The use of new technologies for environmental monitoring is fundamental when planning environmental recovery scenarios in order to appropriately manage particular environments. The results obtained in the RISED project were very promising (both organic and inorganic pollutants appreciably decreased after the treatment), due to the holistic approach used for the various classes of pollutants.

Furthermore, dredged sediments are no longer harmfully toxic waste, so they may be a very important resource for recovering the lagoon landscape. This study also underlines the importance of speciation, since according to the most recent frameworks on risk assessment, it is essential to know the bioavailability and bioaccessibility of pollutants in order to plan the most suitable remediation project. Future research includes the possible application of various natural organic substances and the synergy of sediment washing with other remediation techniques, such as bioremediation or phytoremediation.

Throughout the world there is a growing awareness of environmental management, which is associated with careful environmental risk management.

The Biotic Ligand Model (BLM) is an important instrument for assessing the risk posed by trace elements. It can be used to determine the bioavailability of a trace element, and the sensitivity of organisms to it as a function of its aquatic chemistry. Thus this a tool for estimating site specific toxicity factors of the elements under question. Future research needs to apply the best “tool-box� according to the Water Framework, so as to establish the environmental risk assessment from a scientific, technical and legislative point of

The fate of pollutants in soil view.

The Methodological Chemistry Institute (CNR-IMC, Monterotondo, Rome) has carried out studies concerning the performance of chromatographic and electrophoretic methods for the analysis of different classes of organic pollutants and their degradation products in water, soil and plants and milk. The following classes of compounds have been considered: - Pesticides (among which are some EDCs such as hexachlorocyclohexane, fenvalerate, linuron, atrazine) - Phenols - PAH - VOCs ( Benzene, Toluene, Xylene , aliphatic ketones and alcohol in goat milk) In recent bioremediation experiments at IMC, the distribution of hexachlorocyclohexane isomers in soil, plants and rhyzosphere has been studied, together with the degradation of such compounds by bacterial strains inoculated in poplar plants in a greenhouse experiment.

The degradation of PAHs and phenols by ligninolytic fungi and the interaction of PAH metabolites with humic acids have also been studied.

Possible applications of this research Interaction of organic pollutants and their metabolites with humic matter and dissolved organic matter in soil and water.

Identification of biodegradation compounds of organic pollutants in soil by bacteria and fungi and uptake by plants.

Transfer of VOC from the environment to mammalian and milk contamination 4.2 External collaborations The Department of Biology and Chemistry of Agro-Forestry and Environment at the University of Bari (DIBCA, Bari) is mainly involved in various studies related to soil

chemistry and biochemistry, such as: - the monitoring, conservation and improvement of soil fertility in order to maintain soil quality and increase crop productivity; - the role of soil in complex biogeochemical cycles also disturbed by anthropogenic impacts (spillings, amendments, disposal, reclamations, etc.); - the processes and techniques of soil and sediment decontaminations from heavy metals and various xenobiotics; - the physical, chemical and biological indicators for soil health and quality in response to internal and external anthropogenic activities; - the environmental risks deriving from the agricultural use of geneticallymodified organisms (GMO).

These objectives are achieved through a complex theoretical and experimental approach involving simultaneous chemical, physical and biological phenomena and processes occurring in the soil-plant-xenobiotics system. With given environmental conditions, this consequently leads to a correct and rational use of natural soil resources balancing soil productivity and soil protection.

Possible applications of this research Monitoring heavy metal and organic pollutants through the food chain, especially in the soil-plant system: relationships between pollutants and soil organic components and related fractions; degradation and retention phenomena of conventional and organic pesticides in various soil systems.

Evaluation of the physico-chemical transformation of organic components in relation to organic waste amendments.

5. FUTURE PERSPECTIVES AND DEVELOPMENTS Millions of chemicals are released into the 27

CNR Environment and Health Inter-departmental Project environment, and end up in the soil; the impact of most of them on human health is still not fully known. There are also naturally occurring amounts of potentially toxic substances in the soil whose fate in the terrestrial environment is still poorly known. The behavior of contaminants in soil is related to both the contaminants and the characteristics of the soil. The soil properties regulate the distribution of a substance among the soil phases (solid, liquid, gaseous) and thus determine the retention, the release and the migration of each contaminant (26, 62, 102, 107).

Pollution is also regulated by a time factor, which influences the availability from the source emission to the final target. Human exposure to soil pollution is therefore time-dependent both directly and through secondary transfers such as the food chain.

Unlike our knowledge of the exposure of soil to water and air pollutants, our understanding of the effects of soil contamination is still in its early stages, due to the numerous reactions that take place in the soil (sorption, release, degradation, ageing). These reactions modify the bioavailability of contaminants, which is dependent on the specific characteristics of each particular soil. All these reactions are also influenced by natural weathering processes, which contribute to the transport and the erosive migration of contaminants.

As a result, and despite several legislations regarding soil, models of pollutant behaviors are very poorly defined, especially in terms of the terrestrial food chain. The transfer of contaminants from soil to the food chain requires a detailed knowledge of the complex reactions, that influence their bioaccessibility and these reactions are completely different from those defining the source of exposure.

A study of the fate of contaminants in the 28

soil can thus provide a reasonable estimate of exposure. This can then be used as a basis on which to evaluate adverse effects on human health (4, 56, 79, 94). There is still a great uncertainty regarding this issue and the need to tackle the following issues has been recognized at an international level: - the dietary uptake from vegetables grown in polluted soils; - accidental soil ingestion; - bioaccessibility and bioavailability.

Most attention is now focused on the last issue, since the bioavailability of contaminants in soil (65, 71) is quite different from that deriving from the toxicological experiments on which the risk assessment is founded. Therefore only a deeper understanding of the characteristics of the soils that regulate the chemical and biological reactions of contaminants will contribute to decreasing the uncertainty in risk assessments (36, 41, 79, 112), and the consequences of soil pollution on human health.

One of the main objectives of PIAS-CNR project “Environment and Health� was to highlight the close links between environmental matrices and human health. Within this framework, Working Group 1 (WG 1) focused attention on the fate of contaminants in the environment, particularly on the soil ecosystem. The fate of contaminants in the soil depends not only on the original chemical form of the contaminants, but also on the specific characteristics of the soil. The ability to accurately determine the effects of contaminants on individual species, populations, communities and ecosystems is hampered by an uncertainty in the quantification of receptor exposure pathways. Laboratory and field studies have shown that hazards for human health did not derive from the total concentration of a contaminant in the soil, but from the

The fate of pollutants in soil fraction that is biologically available for that population at that specific time and with certain soil conditions. It is now common knowledge that total concentrations are not useful to explain the effects of contaminants.

These effects may differ from soil to soil depending on soil characteristics and environmental conditions. However one of the main shortcomings of the present procedures to evaluate the risks for human health is the inadequacy, or total absence, of incorporating the bioavailability of contaminants.

Half of a century ago, total diet studies were initiated in response to concerns regarding the loss of food quality. Nowadays the list of foods analyzed is continuously updated and through the database of U.S. Food and Drug Administration (FDA) it is possible to know the content of pesticides, heavy metals, dioxins and other contaminants in many foods and beverages. These studies represent a suitable tool for monitoring dietary intake both in industrial and in developing countries. Many studies have also been carried out in Italy and it appears that most of the elements in the Italian total diet derived from plant foods (88).

The WG 1 proposal aims to go beyond total diet studies and to understand mechanisms and processes by which contaminants enter the food chain and influence to various extents nutrition and the health of humans.

The general objective of this proposal concerns the establishment of a knowledge network among all the PIAS Working Groups. This will at the same time enable each WG to promote and conduct research strategies in medical, especially epidemiological circles. However, it is necessary to devote the same attention to soil pollution as has been previously given to air and water pollutions. In this respect, bioavailability processes assume an essential role to efficiently describe

the occurrence of contaminants in food, the effects of soil quality on the quality of food products, and the implications for human health.

The working path of the WG 1 Proposal project starts with the study of contaminant bioavailability in soil. Bioavailability can be defined as the degree to which chemicals present in the soil matrix may be absorbed or metabolised by human or ecological receptors or are available for interaction with biological systems.

In addition, bioavailability depends on time. Due to ageing, contaminants binding to the soil may become stronger, consequently reducing the effects on the environment. On the other hand, due to natural or anthropogenic changes in soil factors (e.g. pH) contaminants may become more available. The task of soil chemistry is to define the available fractions, the potentially available fractions, and the non-available fractions of contaminants in different environmental conditions.

The proposal is to study contaminant bioavailability in three geographical areas characterized by soils of different origins where it is possible to find either areas with a high degree of pollution due to contamination sources (known as “Site of National Interest: SIN�), areas characterized by natural high levels of metals (Cr, As, Hg), and areas characterized by the absence of point source pollution.

The general aim of this proposal is to identify the transfer of contaminants from soil to the food chain and to evaluate the possibility that in highly polluted areas, soil might not be able to exert its essential role as a filter for ground waters.

The outcome of this part of the project is also to produce a model or to examine the possible use of an existing model (such as the Dutch CSOIL model) to evaluate the routes through which individuals 29

CNR Environment and Health Inter-departmental Project are exposed to a given pollutant. The main routes to consider are: ingestion of soil by children, inhalation of soil dust, and the consumption of vegetables. This evaluation must be carried out by inserting bioavailability concepts in the models to obtain reliable data, which would also be useful in deriving the concentration of a contaminant in a specific soil in relation to human health.

All the research centers mentioned in Chapter 4 will provide their own contribution throughout the whole pathway of the project (Figure 5) under the coordination of CNR-ISE (Pisa).

The first step is to detect the pollutant entity (structure, agent) in soil and water. CNR-ISE (Pisa), will provide an identification of contaminant pathways and an evaluation of the transport of contaminants from sources to target via soil-plant systems. DIBCA (Bari) will provide the monitoring of heavy metal and organic pollutants through the food chain, as well as the study of the relationships between pollutants and organic soil components and related fractions. Through the identification of geo-environmental pollution due to rock-water interaction processes, CNR-IMAA (Potenza) will create geochemical maps for use as tools to protect human health.

The second step is to understand the mechanisms which influence the transport of chemicals in the soil-plant system. Using microspectroscopy and digital microscopy, CNR-IBF (Pisa) will examine the transport, diffusion and accumulation processes of pollutants from the accumulating matrix (soil and water) to plants and animals. CNR-IDPA (Venice) will focus on environmental processes, especially the transport and transfer of organic (polychlorobyphenils, polyaromatic hydrocarbons, dioxins and 30

hexachloro-cyclohexanes) and inorganic (trace elements, such as mercury, lead, chromium, etc.) pollutants. CNR-IDPA (Venice) is involved in the analysis of organic micropollutants, using gaschromatography coupled with highresolution mass spectrometry and elements at trace and ultra trace concentration levels in different matrices with three differently equipped ICP-MS instruments. CNR-IMC (Rome) will focus on the analysis of different classes of organic pollutants and on the identification of compounds subjected to biodegradation in soil by bacteria and fungi, and to uptake by plants. CNR-IBAF (Rome) investigates the ability of mushrooms to absorb heavy metals in soil. Mushrooms can be used for mycoremediation in water depuration or in organic molecule degradation.

The third step is the study of pollutant bioavailability in order to plan the most suitable remediation strategy. ISE-Pisa will study the bioaccessibility of contaminants in soil in relation to their bioavailability.

CNR-IDPA (Venice) will use analytical methods to determine or predict the bioavailable fraction of a metal.

The fourth step is an investigation on the transfer of pollutants from soil to humans through the food chain. CNR-IBF (Genoa) will focus on the characterization of metal binding sites in neurotransmitter receptors and other ion channels for the design of selective ligands to be used in clinical pharmacology. CNR-IBF (Genoa) will also study the implementation of biosensors in order to appraise the bioavailable fraction of toxic elements and to establish the correlated biological risk factors. A comprehension of the control mechanisms of neurotransmitter receptors and ion channels in the neonatal and adult brain exposed to a specific class of organic pollutants (polybrominated diphenyl

The fate of pollutants in soil

Figure 5 – Project proposal ethers) would allow a rational therapeutic strategy in a multipharmacological approach to neuroprotection. CNR-IDPA (Venice) will focus on the absorption and transport of metals and organic compounds along the trophic web. Knowledge of the concentration of the different congeners in the environment plays a key role in understanding bioconcentration and biomagnification in biota. The chemical, mineralogical, petrological and textural study, carried out with CNR-IMAA (Potenza) integrated techniques, will enable useful information to be gathered on the processes of neo-formation and transformation of both pathological and non-pathological biominerals present in the human body (stones, osteoporotic bones, teeth etc.). The methodological approach involved (using epidemiological and geo-environmental features) may provide useful information for preventing and treating some diseases.

All the previous steps will be completed by an integration of epidemiological studies carried out by CNR-IFC (Pisa).

With the contribution of WG1 participants: F. Bianchi (IFC), W. R. L. Cairns (IDPA), P. Cescon (IDPA), F. Corami (IDPA), L. Cori (IFC), E. Galli (IBAF), P. Gualtieri (IBF), C. Marchetti (IBF), T. Miano (Univ. Bari DIBCA), M. Nobile (IBF), R. Piazza (IDPA), C. M. Polcaro (IMC), V. Summa (IMAA)

Keywords: contaminated environmental health, heavy organics, soil quality.

site, metals,

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Water and Soil Monitoring for the Protection of Environment and Human Health M. Rusconi, S. Polesello CNR, Water Research Institute (IRSA), Milan, Italy rusconi@irsa.cnr.it

ABSTRACT The first pillar of the protection of the environment and also, as a positive consequence, of the human community living in this environment, is the establishment of protective monitoring programs. Current monitoring programs for water and soil are based on sampling and laboratory analysis of chemical and microbiological variables. Parallel to this traditional approach, methods to measure effects directly on living organisms, at both individual and community level, have been integrated into monitoring plans. The present paper reviews the state of the art of the research activities in the field of water and soil monitoring carried out by the Italian National Research Council (CNR) Institutes: this review is the outcome of a survey conducted by the Working Group 2 established in the framework of the CNR Environment and Health Inter-departmental Project , PIAS CNR. Emerging problems, such as the presence of nanoparticles and perfluorinated compounds in the environment, and future developments of the monitoring techniques are also discussed.

1. INTRODUCTION The protection of the environment and human health from toxic agents is traditionally based on the selection of a list of potentially dangerous substances or agents and the statement of the corresponding emission limit values or quality standards.

Methodologies to establish quality standards are based on physicochemical and toxicological data which are normally collected and organized in a risk assessment document. The prioritization procedure used to establish the list of pollutants is based on the knowledge of the toxicological properties and data on use and production amounts. In order to be validated, all these procedures need a large amount of data which are actually not available for the millions of synthetic molecules prepared and brought into the market.

As a consequence, the approach, based on

emission limit values at the discharge and on environmental quality standards in the receptor compartment, could not be really protective for the environment.

A step forward in the legislation on water quality protection was the publication of the Water Framework Directive (WFD, Directive 2000/60/EC) that introduced the concept of water bodies protection: it moved from a regulation based on emission control to one that is based on the protection of the ecological quality of receiving water bodies. On the assumption that the repression of discharge by imposing emission limit is not sufficient to protect the environment, it is crucial to verify whether the receiving body is able to support activities which are imposed on it, keeping as much intact as possible the ecological community that resides there.

The aim of the WFD is to achieve a good quality status from the ecological point of view, namely to ensure that all bodies

CNR Environment and Health Inter-departmental Project could support a biodiverse ecological community.

In parallel with this innovative approach based on ecological classification, in order to control the chemical pollution, WFD establishes a priority list of compounds which, for production volumes and/or use and hazardous characteristics in terms of toxicity, persistence and bioaccumulation, pose a risk to the aquatic ecosystem or to human health. The pollution control is based on a combined approach which sets limits on emissions (left to Member States) and maximum allowable levels in the receiving water body, expressed as environmental quality standards (EQS) which are fixed in a recently adopted EC Directive (105/2008/EC).

The use of living organisms and their community as monitoring tools has many advantages. Organisms, living in the environment under study, are constantly exposed to the physical, biological and chemical influences of that environment.

Organisms can often accumulate significant quantities of compounds even if exposed to very low concentrations in the environment.

It is nevertheless difficult to correlate the measured adverse effects on the ecological community with the presence of specific classes of chemical compounds because also the hydromorphological and physicochemical alteration of the natural habitat influences the structure of the community. In order to detect sub-lethal effects, single living organisms are the best indicators of environmental alteration: if chemical tests only detect “known” substances, the measuring of effects on organisms by appropriate biomarkers can highlight not only the biological response to unknown substances but can also evidence the synergistic effects that may be caused by a mix of different substances.

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Integrated monitoring systems are the most effective tool to highlight the interactions among substances by pointing out the responses at different levels: responses which can be so lethal that they affect the composition of the community, or sub -lethal responses that act on the bodymetabolic-physiological models or interact with genetic transmission mechanisms.

Low-cost or highly innovative technologies have been developed for the application in field/in situ (spectroscopic or sensing), in order to achieve a rapid characterization of the site with a high spatial/temporal resolution; in alternative to chemical or physicochemical monitoring systems, techniques based on the measurement of biological response have been proposed, as they can evaluate the possible risk, even in the absence of a direct chemical measurement of a particular pollutant. The role of biomarkers, possibly integrated in a biosensor system, is crucial for the development of an early warning system which could prevent adverse effects on the ecological community and human health.

2. WORKING METHODOLOGY IN WORKING GROUP 2 OF THE CNR ENVIRONMENT AND HEALTH INTER-DEPARTMENTAL PROJECT (PIAS) The working group 2 (WG2) on “Monitoring Systems for Soil and Water”, established in the framework of the PIAS-CNR Project, has defined its field of interest in the development and use of monitoring techniques, technologies or innovative methods for soil and water monitoring, where a situation of environmental pollution represents a potential risk for human health.

After a preliminary review of literature focused on the various techniques developed for the monitoring of soils and waters, WG2 researchers were invited

Water and Soil Monitoring for the Protection of Environment and Human Health to put together their scientific expertise and produce a state of the art report of the activities regarding the monitoring of environmental impacts which could be relevant for human health. Assuming the central role of the integrated approach, a review of the different professional profiles present in the CNR Institutes was carried out, to facilitate the creation of a multidisciplinary group of researchers and the sharing of different expertise ranging from instrumental analytical chemistry to ecological assessment.

3. STATE OF THE ART OF CNR ACTIVITIES Ecological risk assessment (ERA) has been defined as ‘‘the practice of determining the nature and likelihood of effects of anthropogenic actions on animals, plants, and the environment’’ (1). A correct analysis of the complex interactions between the pollution caused by humans and the environment requires the application of a multidisciplinary approach and the determination of different parameters, that can describe the exposure levels and convert them into individual warning situations (2).

A clear example of this operating method is described by the Triad approach used in the ecological risk assessment of sediments (2,3) and allows the investigation of the possible negative effects of toxic chemicals at different levels of biological organization, from single organism to population and/or community level (2). The Triad paradigm enables the assessment of potentially hazardous effects on ecosystems by simultaneously considering chemical concentration, bioavailability of pollutants and the ecotoxicological profile of the environmental matrix under observation.

The latter is usually determined by a set of ecotoxicological tests as well as by

monitoring possible ecological alterations, quantified by changes in different structural and functional community attributes. This integrated approach on different levels of monitoring should be adopted to provide full details of the impact on both the water and the soil in the site of interest. Methods that are relevant at different levels of specificity are needed because some parameters (such as biomarkers) describe effects at suborganism levels of biological organization (4) and different phases of stress syndrome evolution in model organisms (5,6). On the contrary, other ecotoxicological endpoints (e.g. survival and reproduction) indicate possible direct effects at population level (7). Chemical analyses reveal the presence of potentially dangerous substances in soils, but cannot be used to quantify bioavailable fractions (8) that play a more relevant role in threatening ecosystem integrity (9-11).

Finally, a direct evaluation of community structure and functionality should clearly detect overall environmental effects on ecosystems (12) .

As everybody knows, biomarkers have been defined as sublethal responses to environmental chemicals at different levels of biological organization (e.g, molecular, cellular, tissutal, physiological, behavioral, of organisms) which evidence an alteration respect to the natural status (13,14).

Toxic effects caused by exposure to environmental pollutants can alter endpoints at different levels of biological organization (4,15) (Fig. 1).

In particular, the classical biological tools applied in ERA (i.e, bioassays and ecological parameters) are able to highlight the impairments from the organism to the population–community level. This analytical system cannot, however, be used to investigate early effects in organisms exposed to pollutants (i.e, from early 41

CNR Environment and Health Inter-departmental Project

Figure 1: Different biological toxic effects From Dagnino et al. (15). sublethal stress syndrome to the onset of reduced survival). The investigation of the initial phases of biological impairment plays a crucial role in determining the vulnerability level reached by the biotic resources in those cases where no evident changes in the traditional, high level endpoints are detected. Therefore, in order to complete the analysis of the spectrum of possible biological effects induced by environmental contamination, the alterations in sublethal endpoints measured on sentinel organisms can be evaluated (6). In spite of the high sensitivity of these types of parameters, it must be stressed that it is possible to clearly infer the stress syndrome degree in organisms exposed to toxic chemicals by the application of ad hoc models. This is done by using a complete battery of biomarkers at different levels of biological organization (i.e, molecule, cell, tissue, organ, organism) on model organisms (5) .

Therefore an extensive monitoring requires 42

induced by environmental contamination. different skills, ranging from purely analytical capacities for the identification of compounds present in the matrix under investigation, up to bio-molecular techniques to assess effects of pollutants on the gene expression.

In the following paragraphs, a survey on the activities of CNR institutes will be presented, covering many of the skills described above and needed for the establishment of an effective monitoring project. The covered field of expertise ranges from advanced chemical analysis, in laboratory as well as field research, to traditional ecotoxicological assays, biomarker assessment in exposed and natural organisms, up to studies about the alterations of structure and function of ecological communities.

3.1 Laboratory chemical analysis The off-site instrumental techniques are characterized by manual sample collection and transfer in centralized

Water and Soil Monitoring for the Protection of Environment and Human Health laboratory units where advanced analytical equipment is available. Many chemical monitoring activities with instrumental methods are currently on-going in CNR Institutes, also in response to the Water Framework Directive (WFD, 2000/60/EC) requirements.

Several groups are involved in monitoring metals in Italian soil, surface and groundwater. Metals present in soil and waters can accumulate in the trophic chain and represent a risk for the final consumers.

The Tuscany region in Central Italy, due to geological and historical reasons (past mining activities present in the territory since the Etruscan era), is particularly impacted by metals and many CNR groups are working on metals contamination in this area.

Trace metal concentrations were monitored in some urban soils of three medium sized towns of coastal Tuscany. (16) Soil samples were collected in roadsides, urban agricultural soils (allotments), playgrounds and public parks. The analysis included total metal content (Pb, Cu, Zn, Ni, Cd), and sequential extraction. Lead reached the highest levels in the soils and was higher near roads. In urban agricultural soils and in allotments Cu was present in noticeable quantities (300 mg/kg). The presence of Cu in urban soils seems to be typical of soils used for a long period as agricultural land, especially vineyards in the area covered by this study. Sequential extractions were performed to evaluate the mobility of the metals and to better understand the impact of the anthropogenic activity on urban sites.

Mercury contamination in the Cecina river basin (Tuscany, Italy) has been studied by Scerbo et al. (17). Mercury was measured in waters, sediments and fish of the river and its most important tributaries. In fish samples the organometallic metabolites of

mercury were also determined. Particularly high concentrations were found in the sediments of the S. Marta channel flowing into the Cecina, where a chloro-alkali plant discharges its wastes, and high levels were still detectable 31 km downstream from the confluence, bioaccumulating also in fish species.

Italy, and particularly Tuscany, is strongly interested by boron contamination because of the presence in its territory of active volcanism, geothermal activity and mineralized areas. The compliance with the EU normative is technically complex and economically very expensive. The limit of 1 mg/l imposed by the European Union for boron in drinkable waters (98/93/EC) is based on the “precautionary principle”, considered that the effect of boron on humans is at present poor and contradictory.

New geochemical and isotopic 18 investigations on waters (δD, δ O, δ11B, δ13C, δ15N, 87Sr/87Sr) and soils (δ11B, 87 Sr/87Sr) were carried out in Southern Tuscany where boron anomalies occur and the assumption of this element through drinking water or agricultural products could have an adverse effect on the health of the local population (18-21).

The determination of abnormal concentrations of trace metals in soil is crucial to highlight the possible presence of contamination. These abnormalities are identified on the basis of the knowledge of natural concentrations, expressed in terms of “background” (natural background levels) or “baseline” (currently found contents). The criteria for the determination of these “natural” concentrations have been the subject of intense international debate for many years. It is therefore necessary to evaluate the potential of other pollution indicators from diffuse sources, complementary to the existing ones (soils 43

CNR Environment and Health Inter-departmental Project and sediments). In order to achieve this, the properly standardized use of higher plants offers a promising tool to establish a precise date for the event and to assess the spatial extension of the contamination (23-25).

In parallel to soil and groundwater studies, presence, distribution and accumulation of metals in sea areas were investigated: samples of Mytilus galloprovincialis were collected monthly during the July 1999-June 2000 period from two mussel culture areas influenced by urban and industrial wastes (26). These stations, subject to different environmental impact conditions, are located in the coastal area of Taranto Gulf (Ionian Sea, Southern Italy). Metals (Cd, Cu, Pb, Zn, Fe and As) were determined by atomic absorption spectrophotometry (AAS) in the whole soft tissue of mussels. Seasonal changes in metal concentrations were observed.

Metals exhibited maximum values in later winter-early spring, followed by a progressive decrease during summer.

Metal concentrations were similar to those detected in other Italian coastal zones, and indicate that the seafood under investigation poses no hazard to human health because metal content is within the permissible range established for safe consumption by humans.

For many years the presence of toxic inorganic fibrous particles such as asbestos in drinking waters has been of great concern for their direct impact on human health.

The assessment of the diffusion of inorganic fibrous particles in the environment is performed through detection, identification and quantification of mineral inorganic particles present in animal and human tissues and biological fluids from impacted areas. In these same areas, a comparison is carried out among the types and amounts 44

of particles detected in the biological samples and the fibers present in aerosol and mineral outcrop. An evaluation of the biological effects of some of the fibrous mineral phases present in the rocks is also performed (27-30).

In the field of the advanced methodologies for the monitoring of organic micropollutants in water, the CNR Water Research Institute (CNR-IRSA) plays a well acknowledged role. Besides the determination on “classical� persistent and priority organic pollutants (chlorinated pesticides, PCB, PAH, alkylphenols), advanced analytical methods based on LC-MS technique are under development for the determination of metabolites and emerging pollutants in various surface, drinking and ground waters in Italy (3133).

Emerging substances are those compounds or groups of compounds produced or used in significant quantities but which are not currently restricted by regulation due to the lack of information about their effective environmental diffusion and toxicity. In this category, many substances are polar substances such as perfluorinated compounds, PPCP (pharmaceutical and personal care products), estrogens. The CNR-IRSA research group is also the national focal point of an international network, NORMAN (Network of Reference Laboratories for Emerging substances) and the one responsible for the substance prioritization procedures at European level under the Water Framework Directive Common Implementation Strategy.

3.2 Biomarker-bioassay Bioassays are typically used to measure the effects of some substance on a living organism.

They can be classified on the basis of the type of response, by discriminating

Water and Soil Monitoring for the Protection of Environment and Human Health the end-point level: a high response level is associated to the survival rate or reproduction inhibition, while a low response level is connected to sublethal effects that are revealed by specific physiological or genomic alterations.

Among the former type, the most used ecotoxicological tests are those based on the measurement of EC50 (Effect Concentration) or LC50 (Lethal Concentration), i.e. the concentrations which exert an effect on 50% of the organisms under test. The latter type of bioassays is based on the measurement of specific biomarkers: experimental tests evaluate effects that are not lethal for the organism, like a change in a metabolic protein or a behavioral modification.

As indicated by Dagnino et al. (15), the separate evaluation of different levels of response helps to avoid a misinterpretation of the ecotoxicological results.

At the CNR Marine Science Institute (CNR-ISMAR), studies are carried out about the health condition assessment of marine invertebrates and vertebrates in relation to environmental stressors. (3436) Moreover, there is a great interest about the deployment of biochemical, histo-cytochemical and histopathological biomarkers as early warning systems in coastal marine environments monitoring that is the object of the research at the CNR Institute of Biomedicine and Molecular Immunology (CNR-IBIM) Cell Stress and Environment Research Unit (37-46).

This research group has contributed to the identification of cellular and molecular stress markers in the marine environment by using the sea urchin as a model system.

This is a common organism in our shores and has a great ecological and commercial importance. Laboratory as well as field experiments showed that, when responding to chemical (heavy/essential

metals) or physical (temperature, acidity, ionizing radiations) stressors, the adult immuno-competent cells and embryos or larvae of the sea urchin express specific markers, better known as stress proteins, including heat shock proteins (hsp70) (39-41), metallothionein, (38) and acetylcholinesterase (37). Other studies have shown that environmental stress causes DNA damage in the form of broken singleand double-strand (44), and variations in the levels of other stress and apoptotic markers in response to exposures to heavy/essential metals (cadmium/manganese) and/or UVB/X radiation (42,43). Results at cellular and molecular (proteins and mRNA) levels from laboratory exposures were compared to those obtained using samples collected in field studies carried out in the Mediterranean and Northern seas, in order to bridge together field ecology and laboratory-oriented molecular toxicology (45,46). Most of the markers tested were sensitive to the stress conditions used.

The results of the research support the suitability of sea urchin cells and embryos as valid tools to bio-monitor the effects of physical and chemical stress on marine aquatic ecosystems.

At the CNR Biophysics Institute (CNR-IBF) studies on the effects of environmental pollutants in eukaryotic microorganisms are ongoing; specific type of physiological, cellular and molecular level responses have been identified in the presence of environmental pollutants (47-52). This kind of studies is a valuable complement to chemical analysis, as it can provide valuable information on the potential toxicity to the organs, in order to detect the first symptoms of exposure. The effects studied include: - the change in the intracellular pool of non-protein thiols (glutathione and phytochelatins) in phytoplankton algae 45

CNR Environment and Health Inter-departmental Project to be used as a biomarker of heavy metals bioavailability.

- the variation of photosynthetic microorganisms in aquatic and motility in response to exposure to environmental pollutants.

- short-term genotoxicity tests on cell cultures of specific strains of yeast used as a model system.

An integrated approach of biological assays performed with different microorganisms can be applied to water and sediment elutriate collected in impacted coastal and inland areas, such as estuaries, ports, urban areas.

In vivo measures by micro-spectroscopy and micro-spectrofluorimetry of the photosynthetic compartment of planktonic species, present in water contaminated by organic compounds and/or heavy metals, can determine the effects of these contaminants on the composition of pigments, the ratio chlorophyll: carotenoids, photosynthetic efficiency (53-56). Toxic algal species identification and quantification could be done using techniques of optical microscopy, and/or fluorescence for taxonomic recognition with image processing techniques.

The Venice Lagoon (Northern Italy) is an attractive area of study due to its historical interest and ecological fragility. A spatial and temporal survey at three sites located in the “canals� of Venice city centre and at a reference site in the Lagoon was undertaken to evaluate stress effects on mussels sampled in Venice urban area, where raw sewage is discharged without treatment (57). A battery of biomarkers (metallothionein, micronuclei, condition index and survival in air) was used to evaluate the stress condition of the animals. At the same time, an alkali-labile phosphate assay (ALP) was performed in mussel hemolymph to find a biomarker 46

of the estrogenic effect for this species.

Biomarker results showed an impairment of the general health condition in the mussels coming from the urban area, in agreement with the chemical analysis.

Another study (58) surveyed the water quality in Venice urban areas in connection with the discharge of untreated sewage directly into canals, in addition to the pollutant load already present in these areas. One way of estimating the impact of these chemicals is the monitoring of the local fauna. In the search for good water quality indicators in Venice urban area, two physiological indices for mussels (Mytilus galloprovincialis) - survival in air and condition index - have been evaluated. Native mussels and also those transplanted into the urban area showed reduced survivability in air and decreased condition index values, indicating a less healthy status in animals collected from the urban canals. Data are discussed in relation with pollutant bioaccumulation.

Coastal environments are highly variable on a daily scale. In these environments, benthic foraminifera, a class of marine Protista, can be used as bioindicator (59).

These organisms can define the extent of similar environmental conditions (biotopes) through the study of the structure of their assemblage (presence – absence - relative dominance). The monitoring results show the capacity of the benthic foraminifera to monitor the changes occurring in unstable environments and to indicate the evolutionary trends of transitional environments.

The use of molecular techniques such as PCR assay (Polimerase Chain Reaction) for the determination of the genotoxic effects induced by the pollutants in the monitoring programs is rather recent.

This tool was employed in an assessment

Water and Soil Monitoring for the Protection of Environment and Human Health study on recharged aquifers (60): this practice presents advantages for integrated water management in the anthropogenic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. In this study, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardo, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of one year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes (ermB, mecA, blaSHV-5, ampC, tetO, and vanA) were adapted or developed for water samples characterized by different impacts.

The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminants and antibiotic resistance genes.

A targeting species-specific PCR assay was combined with a filter system to collect phytoplankton cells and get spatial and temporal series as part of the Mediterranean Sea EU project Strategy (61). The application of PCR allowed a rapid detection of several harmful dinoflagellate species and genera, including Alexandrium spp. Field samples were concentrated on filter membranes, total DNA was extracted from mixed phytoplankton populations and PCR assays were carried out with specific primers. Qualitative

PCR results were compared with light and epifluorescence microscopic examinations.

Results indicated that this molecular assay was able to detect harmful target cells at concentrations undetectable by microscopy. The application of this filter PCR assay to seawater samples showed to be a sensitive and rapid procedure for the routine monitoring of coastal waters.

3.3 Ecological community studies and microbiology The classification of the quality status of a water body through the study of the resident ecological community at different tiers is currently widely diffused in European monitoring programs also thanks to the impulse received by WFD. The effect of stressors on the ecological community is evaluated both at structure and function levels. Macrophytes, diatoms, macrobenthos and fish are the mostly used ecological quality indicators.

Among the other biological components of the ecological community in both water bodies and soils, special attention should be paid to the microbiological community for its functional role, ubiquitous presence and possible direct impact on human health.

In fact, the study on the natural capacity of bacterial communities to remove xenobiotics (pesticides, pharmaceuticals, biocides) in soil and water could have a very important implication for human health protection. The CNR-IRSA is currently studying natural bacterial communities from contaminated sites: specific bacteria strains that, after repeated exposure to xenobiotic, adapted themselves and became able to remove pollutants through metabolic and/or cometabolic processes, were isolated and identified (62-67). This “self-purification” ability can be used for “recovery strategies” (i.e. bioremediation) 47

CNR Environment and Health Inter-departmental Project of contaminated sites.

The knowledge of the microbiological quality of coastal waters and marine organisms (fish, crustaceans and molluscs) (68-70), that is fundamental to assess the sanitary and ecological risk in a coastal zone, is a central research issue for the CNR Institute of Coastal Marine Environment (CNR-IAMC).

A recent work by Caruso et al. (71-75) is focused on the use of bacterial indicators to assess the anthropogenic pressures over coastal aquatic environments. Selected bacterial species (Escherichia coli, Enterococcus spp.) or related parameters allow to track the occurrence and evolution of bacterial pollution, and to prevent human health risks caused by the use of polluted waters.

Up-to-date standard procedures for bacterial pathogens determination and identification are necessary, due to the limitations of the usual culture methods (long response times, low specificity). In recent years, research efforts have been devoted to the improvement of technical equipment (automatic multiple samplers) and methodologies for the assessment of seawater microbiological quality particularly addressing the detection and enumeration of Escherichia coli or Enterococcus spp. in seawater as faecal pollution indicators. Rapid methods such as the fluorescent antibody method and the β-glucuronidase assay have been developed and optimized to monitor bathing waters, allowing the quantitative measurement of target bacterial molecules and accurate quantification of faecal pollution phenomena. Combined fluorescencestaining protocols have also been set up, in order to detect bacteria which may present a pathogenic potential. Data obtained by these new analytical procedures encourage the use of E. coli or related parameters 48

as successful tools for early warning of seawater bacterial pollution and for the screening of polluted coastal areas; therefore they offer interesting perspectives to prevent waterborne diseases.

The microbiological quality of coastal waters is usually estimated by determination of faecal indicator bacteria.

However, bacterial species possibly pathogen for humans could occur also as microorganisms indigenous to coastal marine environments (e.g. Vibrio spp.).

Since their concentration is related to the temperature of coastal waters, and unrelated to classical faecal indicators, monitoring and control of this bacterial group is needed to plan preventive measures for human health protection.

(70,76-78) The Vibrio genus is widespread in coastal waters and includes more than 63 species. The most well-known species is V. cholerae, which causes cholera epidemics worldwide. In addition to V. cholerae, many other Vibrio spp. are recognized as potentially human pathogens causing 3 major syndromes of clinical illness: gastroenteritis, wound infections, and septicemia. Epidemiologic data suggest that the majority of these infections are foodborne disease and associated with raw or undercooked shellfish. In wild and cultivate shellfish the bacterial density may reach high concentration and potential toxic effect for humans. For this bacterial group the standard microbiological criteria used to assess water quality have to be revised.

The analysis of antibiotic resistance patterns (ARPs) of faecal indicator bacteria allows to detect the presence and persistence in the environment of genes linked to antibiotic resistance. Research is in progress to characterize the ARPs of enterococci, as they are emerging pathogenic bacteria, and of E. coli for

Water and Soil Monitoring for the Protection of Environment and Human Health their capacity of acquiring antibiotic resistance and spreading their resistance to other species such as Salmonella, Shigella, Yersinia, Vibrio etc. (79-81).

Studies on E. coli and enterococci have particular epidemiological and ecological relevance because these micro-organisms can occupy multiples niches, including humans, mammals, birds, reptiles and insects.

3.4 Advanced technologies and early warning systems Advanced technologies have been applied to the monitoring of soil and water for the protection of human and environment health.

Among those, interest is growing for the application of innovative microscopy techniques, such as scanning probe microscopy (SPM), atomic force microscopy (AFM) and scanning near-field optical (SNOM) to the environmental field.

These techniques exploit the interaction of a functional tip scanned over the surface of a sample (solid or adhesive on the substrate) to reconstruct the morphology of the sample and, simultaneously, achieve superresolution maps of other properties of the sample (i.e. local optical and fluorescence properties, maps of surface friction and nano-mechanical properties, magnetic domains, etc.). These microscopes are very useful in the study of nanostructured materials, surfaces and interfaces (i.e. sensors, biosensors, biocompatible surfaces), and analysis of nanoscale biosystems that can be investigated at the cellular level, subcellular and macromolecular aggregates. In particular, biosystems can be studied in physiological conditions and can describe temporal, three-dimensional and quantitative evolution (morphometry). (82-88) In addition to the development of innovative tools, expertise has been gained over the

years in the study of intra-cells interactions in culture and exposed to environmental agents (drugs, electromagnetic fields, heavy metals, UV etc.) or nanostructured agents (nanoparticles or nanotubes) and in the study of complex phenomena such as cellular aging or apoptosis.

An important research field is the integration of advanced technologies in measuring systems that can be used for on-line or in-situ monitoring. The final goal is to make available some devices that can act as an early warning system of sudden alteration in the environmental quality status.

In the CNR-IRSA, Liquid Chromatography with Mass Spectrometric detection (LCMS) has been integrated with an automatic sampling and preparation station into an on-line monitoring station for the determination of polar organic substances in drinking water (89). This system can be used in potable water treatment plants for the control of influent and effluent. In this way, a control on the incoming water quality and efficiency of the treatment procedure should be achieved. The operating limitations of this station are linked to the total cost of the equipment, that is still too high for a massive deployment, the reduced frequency of sampling and the need for highly qualified professionals for the frequent maintenance required.

Spectroscopic techniques are suitable to be integrated in small portable or fixed station devices for in-situ monitoring by optical fiber detection.

The concentration of several pollutants, usually present in industrial waste waters, could be predicted by the neural network data processing of absorption and fluorescence measurements in the visible spectral range. Proper network tuning provides quantitative analysis of many pollutants with sub-ppm resolution.

49

CNR Environment and Health Inter-departmental Project Compact optical fiber instrumentation for absorption spectroscopy and an innovative flowcell for fluorescence measurements enable cost-effective, in situ, nonstop monitoring of waste waters (90).

In the framework of the development of new methods for measuring and monitoring soil pollution, the use of magnetic susceptibility as a proxy variable for monitoring heavy metals in soils has been explored (91).

Magnetic measurements are carried out by using a magnetic susceptibility meter with two different probes for in situ field surveys. The relationships between heavy metal levels and magnetic susceptibility values of soil samples were assessed.

Results suggest that a careful check of the experimental procedure plays a crucial role for using magnetic susceptibility measurements in situ monitoring of heavy metals.

Environmental management needs some tools capable of providing, over a relatively short time, integrated responses regarding the levels of contamination and the ecological consequences on different compartments of the concerned ecosystems.

The biosensor “tool�, that responds precisely to this necessity, consists of a biological active element - from an isolated enzyme to a whole organism - immobilized on a transducer system (sensor) for the selective and reversible determination of the presence and/or the concentration of certain chemical molecules in a sample.

In fact selectivity and sensitivity, together with the possibility to have a portable tool are the main advantages of biosensors.

A compact and portable sensing device that combines the production and detection of hydrogen peroxide in a single flow assay has been proposed for herbicide detection in water (92). The principle on which the biosensor is based is that herbicides, under illumination, can inhibit the photosystem 50

II electron transfer. Photosynthetic membranes isolated from higher plants and photosynthetic micro-organisms, immobilized and stabilized, can serve as a biorecognition element for a biosensor.

The inhibition of photosystem II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase.

Systems that use biological responses to detect environmental quality changes in continuous (on-line monitoring) in a simple, quick and economical way can be used as Biological Early Warning System. In this field, the possible use of an electrochemical growth signal transduction of a natural biofilm (microecosystem) on a suitable metallic substrate, was recently investigated with the aim of revealing, in real time, any alteration of the normal development of the microbial community induced by the presence of toxic substances in the aquatic environment (93-95). The prototype of the innovative patented biosensor shows that the biological electrochemical signal is significantly inhibited in the short term (minutes-hours) by known concentrations of a series of reference toxics.

Among the most advanced early warning systems, based on in vivo response of organisms to toxic agents, a Swimming Behavior Recorder System - able to measure the swimming behavior of marine invertebrates larvae exposed to toxic substances and/or environmental matrix under controlled conditions - has been presented (96-99). The methodology for the detection of alteration in swimming uses a prototype system to analyze video graphics, capable of automatically recording the aquatic organisms swimming speed, and providing, on the basis of the alteration of this parameter compared with

Water and Soil Monitoring for the Protection of Environment and Human Health a control, two toxicological endpoints: immobilization (acute) and alteration of swimming speed (sub-lethal).

3.5 Auxiliary techniques for monitoring Geophysical techniques have been used for several years to measure hydraulic parameters in the monitoring and control of groundwater contamination.

The most common techniques are the electromagnetic ones that are more sensitive to any changes in physical parameters (i.e. electrical conductivity) of soils and sub-soils caused by the presence of a particular contaminant in the water tablet or soil porosity. Therefore, the use of geophysics monitoring systems should be able to follow over time and space areas the evolution of a particular case of pollution, such as, for example, a leakage from a dumping site. The study and application of these non-invasive and lowcost technologies, integrated with the more traditional ones (sampling and diagnostic studies), will lead to a knowledge of the land and the environment, in order to provide a better safeguard level and to plan remediation procedures.

In the Hydrogeosite laboratory of the CNR Institute of Methodologies for Environmental Analysis (CNR-IMAA) a simulation plant has been built to perform hydrogeophysical experiments for the integrated study of contaminated soils and subsoils with the aim of creating a standard methodology for practical intervention (100-102).

The activity carried out by the CNR Research Institute for Geo-hydrological Protection (CNR-IRPI) in the field of erosion and hydro-meteorological monitoring is of fundamental importance for the protection of the water bodies and their basin.

4. EMERGING ISSUES The literature review highlights that the monitoring focus is still mainly addressed to some classes of well studied molecules, such as trace metals and persistent organic pollutants, which represent a typology of pollution that emerged several decades ago, but for which there is still concern because of their persistence in soil and water sediments and their capacity to accumulate in the trophic chain.

However there are many relatively new classes of pollutants, which we daily deal with, but are not still regulated by legislation. For these classes, fundamental research and appropriate monitoring plans are needed in order to understand the environmental distribution and fate, which are necessary to assess the effective risks for ecosystem and humans. CNR Institutes are carrying out research on many of these emerging environmental issues and, among them, two classes can be chosen as case studies, i.e. the engineered nanoparticles and the perfluorinated compounds. In the following sections a short critical review of the knowledge gaps will be presented in order to list future research needs for these two emerging classes of compounds.

4.1 Engineered nanoparticles Anthropogenic nanoparticles (engineered nanoparticles; ENPs) are used in nanotechnology to create products used in various fields such as agriculture, electronics, biomedicine, manufacturing, pharmaceuticals cosmetics industry. The use of nanomaterials containing ENPs is expected to continuously increase in the near future. The nanometer size range (0-100 nm) means that nanomaterials exhibit properties and functions other than those owned by the same materials with larger diameter and these properties are 51

CNR Environment and Health Inter-departmental Project

Figure 2: ENPs’ environmental fate (figure adapted from National Institute for Resources and Environment, Japan http://www.nire.go.jp/eco_tec_e/hyouka_e.htm). attributable to the increase of the surface the methods for their determination and area ratio and number per unit mass of study in contaminated environments are ENPs, which lead to increased chemical not yet standardized. reactivity, greater strength and electrical The ENPs’ environmental fate is extremely conductivity and, potentially, a more complex and the processes regulating ENPs’ distribution in the different compartment are pronounced biological activity.

The extensive use of anthropogenic still not exhaustively investigated (Fig. 2).

There are physico-chemical processes that can nanomaterials in large consumption affect their potential environmental toxicity products means that their transport, use (solubility, aggregation, absorption, interaction and discharge is a potential new source of with other toxic substances). The study of pollution in domestic sewage and industrial ENPs physico-chemical behavior influenced discharges, resulting in a diffused pollution by abiotic factors in different matrices is of surface waters and transitional/coastal fundamental to simulate scenarios in laboratory areas by ENPs. ENPs can be transferred to experiments. humans through food and several studies According to Moore, some open issues can be (103-120) demonstrate their potential risk suggested for future research projects(121): • Which is the hydrodynamic behaviour of for human health.

ENPs? Today there are still many uncertainties • Do they behave like larger natural about the environmental fate and toxicity particles? of ENPs in aquatic environments because • How do they associate with larger sediment 52

Water and Soil Monitoring for the Protection of Environment and Human Health

Figure 3: Different levels of biological organization in a laboratory mesocosm (by Dr. Giovanni Pavanello, ISMAR). and natural colloidal particulates? international research institutions.

• Do they bind lipophilic organic and metal For the evaluation model, different classes pollutants, and what are the routes of of anthropogenic ENPs representative nanoparticle uptake into biota? of potential future scenarios of use and • Do ENPs-linked chemical pollutants show impact in the aquatic environment should enhanced toxicity? be chosen. Their interaction with various • Are the particle size and surface properties significant factors in determining toxicity compartments of aquatic ecosystems (water and sediment), in varying chemical in aquatic organisms?

• What is the implication of ENPs exposure and physical conditions (pH, temperature, concentration, natural matter for organisms health and ecosystem ion integrity? concentration) should be determined.

• Can modelled fluxes and predicted impacts For each ENPs’ selected class a of ENPs help to provide an explanatory standardized protocol for measurement framework for their environmental must be developed for each different behaviour and possible impacts? environmental matricx (water, sediment All these gaps could be filled by planning and biota), including all the possible an appropriate laboratory experimental chemical and physical interactions between model which should involve different ENPs and the different matrices. One expertises available in the CNR Institutes, approach could be a careful and critical with the cooperation of other national and 53

CNR Environment and Health Inter-departmental Project analysis/testing of ENPs’ “detection” methods already used in biomedical field (Environmental Scanning Electron Microscope-ESEM, Field-Emission Gun-Environmental Scanning Electron Microscope-FEG-ESEM, Scanning Transmission Electron Microscope-STEM, Transmission Electron Microscope-TEM, Scanning Electron Microscope-SEM, Atomic Force Microscope-AFM, Scanning Probe Microscope-SPM, Confocal Laser Scanning Microscope-CLSM and MultiPhoton Microscope-MPM) in order to adapt them to the specific peculiarities of the environmental matrices.

A crucial issue for a reliable risk assessment on ecosystems is the selection and standardization of ecotoxicological bioassays at different levels of biological organization (molecules, cells, organisms), which must be representative of different aquatic environments and ENPs categories selected as references standards. The selection of model species should be large enough to ensure a good representation of the different compartments of the most representative reference ecosystems.

Although it is reasonable and practical to predict the potential environmental risk on the basis of bioassays with single species models and/or battery of them, it is equally important to investigate how these supramolecular entities interact at ecosystem level. For this purpose, investigation protocols and exposure methods using realistic ENPs concentrations for various environments should be developed using laboratory micro and mesocosms containing sediment, microorganisms, algae, invertebrates and vertebrates, with different levels of ecosystem complexity. (Fig. 3).

The mesocosm study can also supply information on the accumulation and eventual biomagnification along the trophic 54

chain. These data, together with human dietary and environmental exposure studies, are needed to get a reliable risk assessment for human health.

4.2 Perfluorinated compounds in the water environments Perfluorinated alkyl substances (PFASs) are fluorinated man-made chemicals with unique molecular properties, chemical and thermal stability, water- and fat-repellent properties that make them and their derivatives useful in different industrial and household applications. They are widely employed for impregnation of textiles and paper, for cleaning aids, for fire fighting foams, for metal surface treatment and in the production of fluoropolymers.

Among PFASs, perfluorooctanoic (PFOA) and perfluorooctanesulfonic (PFOS) acids are two persistent and bioaccumulative end-stage metabolites of particular concern for human health and the environment due to their potential toxicological effects on animal and also human endocrine system; to date, there is no evidence of biodegradation of these chemicals.

Recent concerns with the toxic effects of PFOS and PFOA began in the early 2000s when the 3M Company, U.S.A, the major perfluorochemical manufacturer, decided to phase out the production of PFOS related products. However, there are still a number of industries, such as the semiconductor etc, that still use PFOS in their production (122) and manufacturers that use and produce PFOA and its related products for consumer usage. Evidence is accumulating regarding their persistence in the environment, potential for long-range transport, tendency to bioaccumulate, and potential toxicological effects (123-125).

Due to their physico-chemical properties, these chemicals leaked into the water, they accumulate in surface waters, and water

Water and Soil Monitoring for the Protection of Environment and Human Health is the major reservoir of perfluorinated compounds (PFCs) in the environment, as well as the most important medium for their transport (126,127). Unlike other classical persistent organic pollutants (POPs), the anphiphilic PFCs have not been shown to accumulate preferentially in adipose tissue. They rather bind to blood proteins and accumulate in the liver of exposed organisms (128). Recent studies have indicated that PFOS can biomagnify in higher trophic organisms through the aquatic food chain (129,130).

PFOA is mainly used as adjuvant in the production of fluoropolymers such as polifluorotetraethylene and similar products used in clothing production, cosmetics and for non-stick cookware coatings. It was estimated that aqueous and gaseous emissions of PFOA or their salts originated in this production represent the majority (about 80%) of environmental release (126). It is also used (but in small amount) in industrial and consumer products (varnishes, inks, paper, floor polishes, cleaning formulation etc.) which represent some direct human exposure routes and diffusive environmental sources not normally considered. PFOA may also form as a degradation product of fluorotelomer alcohols found in a wide range of household and consumer products like hair shampoo, rug cleaners, and food paper products. These are volatile and can be carried for long distances by air currents (131).

In a European study on the major EU rivers carried out by the Perforce consortium (PERFORCE project), river Po watershed was identified as the dominant source of PFOA in Europe. River Po accounted for two thirds of the total PFOA discharge of the rivers studied (127). The PFOA concentration (200 ng/L) in the water of the Po river collected at the basin closure

(Pontelagoscuro near Ferrara) was from 10 to 200 times higher than those measured in the other European rivers. This result suggested is the presence of an industrial source of PFOA in the Po watershed.

Recent survey of perfluorocarboxylic and perfluorosulfonic acids concentrations in the Po watershed (33,132) confirmed the data of the PERFORCE project, and measured a PFOA concentration of 60174 ng/l at Pontelagoscuro (FE). All tributaries, except river Tanaro, showed the PFOA levels typical of a diffuse pollution; on the contrary, the highest PFOA concentrations were determined in river Tanaro (1270 ng/L) and in river Po, downstream the confluence of the Tanaro river (60-337 ng/L), suggesting the presence of a point source for this compound that can be reasonably attributed to an industrial Teflon production site in the Alessandria county.

In the study carried out at the CNR-IRSA (33) an assessment of the contamination by perfluoroalkyl acids (PFAAs) in surface, urban and industrialized waste waters and drinking waters in the river Po basin was carried out. An HPLC method with ion trap mass spectrometry detection was developed for the analysis of perfluorinated carboxylates (from C5 to C10) and perfluorinated sulfonates (C4 and C8) with a LOQ of 2 ng/L. Water sample extraction was performed on weak anion exchange (WAX) cartridges.

In this work three kinds of waste waters, from textile industry discharge, urban wastewater treatment plant (WWTP) and mix urban-industrial WWTP, were analyzed because the industrial activity is considered the main source of PFOA and other homologues. PFOS was found at lower concentrations than PFOA, both in industrial and urban waste waters, This is probably due to the phase out of the 55

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Figure 4. Perfluorinated concentration in tap waters. LOQ = Limit of quantification (2 ng/L); NJDEP = New Jersey Department of Environmental Protection guidance level (40 ng/L) PFOS and PFOS-related compounds in the electrochemical production since 2002.

Three sources of tap waters (Lecco produced from Lake of Lecco water; Milano - produced from ground water; Ferrara - partially produced from the Po river) were also analyzed. The only perfluorinated compound, detected in drinking water above the limit of quantification, was PFOA in the tap water of Ferrara which produces drinking water mixing ground and surface waters from river Po (Fig. 4).

To date, there are no international water guidelines for PFOA in drinking water but some single countries set up their own water guidelines. The strictest standard, (40 ng/L), issued by New Jersey in the USA, was not overcome by Ferrara tap waters. Nevertheless it is necessary to underline that this sampling campaign on drinking waters does not have any statistical representativeness, but it is only a baseline survey which needs further in depth enquiry.

Preliminary results highlighted the presence, in the Po river watershed, of point and diffused sources of perfluorinated compounds. PFAAs were measured in drinking waters produced from 56

contaminated surface waters, revealing the risk for human consumption.

These preliminary campaigns, carried out in different periods and hydrological regimes, gave only a first look at the distribution of perfluorinated compounds in the Po watershed but no data have been collected for the rest of Northern Italy whose waters discharge in the High Adriatic Sea. The available data do not allow to estimate the risk of contamination for the aquatic species present in the transitional and coastal areas.

Furthermore the presence of intensive aquaculture activities in these areas could result in a source of exposure also for humans which, at the present state of knowledge, can not be envisaged.

Therefore, the overall goal of the IRSA research group will be to assess the environmental and health risks posed by the PFCs contamination in the river Po basin and in other river basins and coastal areas of the Northern Adriatic sea. In order to reach these objectives, the following detailed actions should be carried out: • a comprehensive survey of the river Po basin , the coastal areas and lagoons of the High Adriatic Sea in order to identify critical areas and hot spots for PFC impact by: o Monitoring the river Po basin with downstream sampling of the main tributaries; o Evaluation of PFCs distribution in surface and groundwater around fluorochemical industrial sites in Northern Italy; o Monitoring of mollusc aquaculture areas in hot spot areas such as the Po Delta and Northern Adriatic Sea coast o Monitoring of raw and treated drinking water drawn from the Po river or groundwater impacted by industrial activities • intensive monitoring of the critical areas in different hydrological regimes for the assessment of contamination and transport

Water and Soil Monitoring for the Protection of Environment and Human Health processes; • determination of PFCs levels in edible marine and lagoon bivalves, farmed or caught in the critical areas, in order to determine: o PFCs accumulation in different sites, using two edible species with different geographical distribution (site comparison); o PFCs accumulation in two different bivalves, addressing the possible existence of different patterns of exposure (clams in the sediment/mussels in the water column) and of bioaccumulation (species comparison); • Estimation of PFCs risk exposure for humans as a result of the consumption of contaminated mollusks and drinking waters.

5. FUTURE PERSPECTIVES AND DEVELOPMENTS The term “emerging pollutants” means a continuously updated list of the most diverse molecules produced, used and eventually diffused in the environment.

Such list includes synthetic surfactants, endocrine disruptors, pharmaceutical substances, perfluorinated compounds, industrial additives, new generation pesticides, etc, all substances which are not included in the lists of regulated priority pollutants and in ordinary monitoring plans. Considering also that the toxicity and the effects of certain new generation compounds are often not known or fully understood, it is evident how crucial it is to develop monitoring systems suitable to detect the possible responses to unknown toxic agents. In fact it is impossible to measure all the compounds present in a certain environmental compartment and we need to turn over our traditional approach in monitoring: first we measure the effects and then we look for the molecular agents which should be the cause of the effect itself. This revolution in

the monitoring approach will be probably the most outstanding innovation in the next future.

At present, the procedures identified by the words “Toxicity Identification and Evaluation” (TIE) or “Effect Directed Analysis” (EDA) represent the state of the art in this field and for that reason we will present a short review on this topic in this last section.

The first identification process of “guilty” substances and its verification of the observed effects caused by these compounds dates back to 1983 (133).

Subsequently taken over by the United States Environmental Protection Agency (US EPA), this process took the name of Toxicity Identification Evaluation (TIE).

The classic TIE is divided into 3 phases.

During the first phase (Fig. 5a), groups of active compounds are sequentially removed from the water sample with chemical treatment, as long as the toxic effect of the solution on the biological model system does not disappear. In the second phase substances are tentatively identified by the treatment that decreases the toxic response of the sample and the classes of chemicals identified are related to the biological responses. Finally the activity of each identified substance or substances mixture is confirmed using the same biological model.

The Effect Directed Analysis (EDA) procedure, which is the subject of EU research projects in the framework of WFD implementation, is the conceptual and technical evolution of the TIE approach.

Like TIE, EDA is based on the use of the response of a biological reference system in combination with sample fractionation and the chemical analysis of individual fractions for identification of hazardous compounds in complex mixtures present in the environment. Figure 5b schematically 57

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Figure 5: a-An example of TIE’s first phase. b-scheme of EDA process. describes the process which starts from the observation of a “macroscopic” effect on the biotic component in the investigation site. Through the use of a chromatographic separation systems, the mixture is divided into its individual components. Each fraction is tested for activity on in-vitro model systems, the nature of the compound is defined through analytical detection and recognition systems to determine the substance responsible for the effect.

Sample fractionation into individual classes of compounds is achieved using chromatographic columns with different stationary phases based on the principle that each compound has a specific affinity with the stationary phase column at a certain mobile phase composition. Therefore different types of serially connected chromatographic columns allow a sample basic components separation efficiency greater than that obtained using a single column, as in simpler TIE procedures (134,135 ).

The identification and confirmation of compounds in each fraction is achieved by using high-resolution mass spectrometry techniques, hyphenated with chromatographic systems; liquid chromatography (HPLC) is used for the 58

separation and identification of polar compounds and metabolites which are more difficult to analyze but are much more biologically active than classic persistent organic pollutants.

In addition to a more rigorous procedure and a greater degree of complexity and precision in the sample fractionation compared to TIE, EDA introduces the use of in-vitro bioassays that allow a high number of spatial and temporal replicates and provide the capacity to run the tests in batteries, so getting a complete screening of the toxic activities of the sample fraction.

As examples of in vitro tests used in EDA approach we can mention Yeast estrogen screen (YES), Yeast androgen screen (YAS), for the evaluation of endocrine disruption activity (136), bioluminescence tests such as CALUX, Microtox and Mutatox, assays on fish hepatocytes for physiological and accumulation studies: these are all sensitive, specific and rapid tests which allow to provide a full view on the biological activity of the compounds under investigation.

In parallel to those “classic” in-vitro tests, in recent years, the development of the “omics” disciplines helped to expand knowledge about the effects of many

Water and Soil Monitoring for the Protection of Environment and Human Health toxic compounds: toxicogenomics and toxicoproteomics study the relationships between genome (or protein) structure and activity and the biological effects of exogenous agents. This means that if a compound shows, for example, a mutagenic effect, the magnitude of the effect can be determined by the gene expression analysis of an exposed individual.

As an example of the effectiveness of the integrated monitoring and analysis approach, we can mention the study of Keiter et al. (137) who adopted an integrated TRIAD and EDA method to identify the activity of so-called dioxinlike compounds (i.e. molecules whose steric and electronic structure is similar to those structural characteristics of dioxins which exert effects on biological systems) as the cause of a specific adverse effect on river organisms.

The EDA procedure is rather complex and time consuming and, of course, it is not suitable to meet the demand of fast and reliable techniques for early warning systems. Nevertheless, if integrated into routine monitoring programs as a complementary technique for investigative monitoring, it will be absolutely necessary in those situations where the toxic or other biological effect cannot be attributed to a known agent or source. Alternatively it could be used as a screening method to assess the risk of a particular pollution source that is crucial for an effective management of the territory with the aim of human health and environmental safeguarding.

With the contribution of WG2 participants: A. Barra Caracciolo (IRSA), E. Belluso (IGG) G. Caruso (IAMC), L. Da Ros (ISMAR), M. Faimali (ISMAR), M. Girasole (ISM), R. La Ferla (IAMC), M. Mancuso (IAMC), G. Mascolo (IRSA), V. Matranga (IBIM), L.S. Monticelli (IAMC), E. Morelli (IBF), P.

Gualtieri (IBF), M. Pennisi (IGG), M. Polemio (IRPI), E. Rizzo (IMAA), E. Sacchi (IGG), R. Zaccone (IAMC).

With the contribution of: A. Baldi (Fondazione Italiana Endometriosi); E. Fommei (Pisa University and CNR-IFC), G. Iervasi, A. Pierini (CNR-IFC); S. Maffei, C. Vassalle (Fondazione “G. Monasterio” CNR-Regione Toscana, Pisa); C. Roscioli, S.Valsecchi, L. Viganò, D. Vignati (CNR-IRSA, Brugherio).

Keywords: water and soil monitoring, biomarker, bioassay, early warning system, emerging substances.

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Role of Atmospheric Pollution on Harmful Health Effects A. Pietrodangeloa, M. Bencardinoa, A. Cecinatoa, S. Decesarib, C. Perrinoa, F. Sprovieria, N. Pirronea and M.C. Facchinib a. CNR, Institute of Atmospheric Pollution Research (IIA) Monterotondo St. (Roma), Italy b. CNR, Institute of atmospheric sciences and climate (ISAC), Bologna, Italy pietrodangelo@iia.cnr.it

ABSTRACT Gaseous and particulate matter in ambient and indoor air has a key role on the increased morbidity or mortality observed in many clinical studies. Knowledge of the main toxicity patterns of atmospheric pollutants is still at an initial stage, especially as concerns particulate matter. This is mainly due to the varying sizedistributions and chemical composition of PM10 and PM2.5 and to the many-sided toxicity mechanisms of ultrafine particles (UFPs). In this paper, recent findings on toxicity routes attributable to PM matter (i.e. the water-soluble organic fraction (WSOC), studied for the strong oxidative potential to biological tissues), to UFPs and to gases, are reviewed. Toxicity routes are discussed as evidence or hypothetical relationships between sources, diffusion paths, receptor sites and susceptible populations. Finally, strategic points are underlined which will be further developed in the “Pilot study for the assessment of health effects of the chemical composition of ultrafine and fine particles in Italy” project.

1. INTRODUCTION Adverse health effects of atmospheric pollutants have been well documented in Europe and in other parts of the world.

These include many diseases and an estimated reduction of a year or more in life expectancy for people living in European cities. There is also evidence of increased infant mortality in highly polluted areas.

Concerns about these health effects have led to the implementation of regulations to reduce harmful air pollutants emissions and their precursors at international, national, regional and local levels. Further measures – while necessary to further reduce the health effects of air pollution – are becoming increasingly expensive.

There is thus a growing need for accurate information on the health effect of air pollution to plan scientific, effective and well targeted strategies and reduce these effects.

In July 2002 the European Parliament

and the Council adopted the Decision 1600/2002/EC on the Sixth Community Environment Action Programme (Sixth EAP). This Programme sets out the key environmental objectives to be attained in the European Community, one of which (Article 2) is to establish “. a high level of quality of life and social well being for citizens by providing an environment where the level of pollution does not give rise to harmful effects on human health …”(1). The activities of the European Commission to implement the Sixth EAP currently take place within the Clean Air for Europe (CAFE) programme (2). This programme, launched in early 2001, aims at developing long-term, strategic and integrated policy advice to protect against significant negative effects of air pollution on human health and the environment.

The World Health Organization (WHO) in support to the CAFE process, provided updated information on health effects

CNR Environment and Health Inter-departmental Project of air pollutants establishing the project “Systematic Review of Health Aspects of Air Quality in Europe” (3) in the course of which the current state of knowledge concerning health impacts of air pollution has been reviewed. The body of evidence of air pollution effects on health at the pollution levels currently common in Europe has been considerably strengthened by the contribution of both epidemiological and toxicological studies. The latter provide new insights into possible mechanisms to analyse the hazardous effects of air pollutants on human health and complement the large body of epidemiological evidence, showing, for example, consistent associations between daily variations in air pollution and some health outcomes. Exposure to ambient air pollution has been linked to a number of different health outcomes, starting from modest transient changes in the respiratory tract and impaired pulmonary function, to restricted activity/reduced performance, emergency room visits and hospital admissions and mortality. There is also increasing evidence of air pollution adverse effects not only on the respiratory system, but also on the cardiovascular system.

This evidence stems from studies on both acute and chronic exposure. Short-term epidemiological studies suggested that a number of sources are associated with health effects, especially motor vehicle emissions, and also coal combustion.

These sources produce primary as well as secondary particles, both of which have been associated with adverse health effects. If long-term exposure to a specific pollutant is linked to some health effects, cohort studies provide a basis to estimate chronic diseases and lifespan reduction in a given population. This is the case for mortality linked to PM longterm exposure. An expert group led by 70

WHO – the Joint UNECE/WHO-ECEH Task Force on Health Aspects of Long Range Transboundary Air Pollution – recommended the use of risk coefficients from the American Cancer Society (ACS) study (4) to estimate the effects of chronic exposure to particulate matter (PM) on life expectancy in Europe. This study is the largest cohort study published in the scientific literature on the association between mortality and exposure to PM in air, and has involved 550,000 persons between 1982 and 1998. The risk estimates from this study were also used in the WHO Global Burden of Disease project (5). This project estimated that exposure to fine PM in outdoor air leads to about 100,000 deaths and 725,000 years of life lost each year in Europe. It is clear that there is a significant health risk associated with PM. It is also clear that there is a yet not known safe threshold for exposure but that there appears to be a linear relationship between exposure and risk. In addition, it has not yet been possible to identify with confidence which PM chemical constituents are primarily responsible for the different health effects. Therefore, even though the evidence on the relationship between exposure to different air pollutants and health effects has increased considerably over the past few years, there are still large uncertainties and important gaps in knowledge. These gaps can be reduced only by targeted scientific research. Areas in which such research is urgently needed include exposure assessment, dosimetry, toxicity of different components, biological mechanisms of effects, susceptible groups and individual susceptibility (taking into account gene–environment interactions), effects of mixtures versus single substances, and effects of long-term exposure to air pollution. The “Systematic Review” clearly demonstrated the need to set up a more

Role of Atmospheric Pollution on Harmful Health Effects comprehensive air pollution and health monitoring and surveillance programme in different European cities. Air pollutants to be monitored include coarse PM, PM2.5, PM1, ultrafine particles, PM chemical composition, including elemental and organic carbon, and gases such as ozone, nitrogen dioxide and sulphur dioxide. The value of black smoke and ultrafine particles as indicators of traffic-related air pollution should also be evaluated. Furthermore, periodic surveillance of health effects requires better standardization of routinely collected health outcome data.

The “Systematic Review” also showed the need of a system to maintain the literature database and develop meta-analysis to monitor research findings, summarize the literature on health effects and health impact assessment.

2. AIR POLLUTION AND HEALTH Ambient air pollution consists of a highly variable, complex mixture of different substances, which may occur in the gas, liquid or solid phase. Several hundred different components have been found in the troposphere, many of them potentially harmful to human health and the environment. The main sources of air pollution are transport, power generation, industry, agriculture, and heating. All these sectors release a variety of air pollutants – sulphur dioxide, nitrogen oxides, ammonia, volatile organic substances, and particulate matter – many of which interact with others to form new pollutants.

These are eventually deposited and have a whole range of effects on human health, biodiversity, buildings, crops and forests.

Air pollution results in several hundreds of thousands of premature deaths in Europe each year, increased hospital admissions, extra medication, and the loss of millions

of working days. The health costs for the European Union are huge. The pollutants of highest concern for human health are airborne particulates and ozone – indeed no safe levels have yet been identified for either of them. Nevertheless, the “Systematic Review” focused on three pollutants: particulate matter (PM), ozone and nitrogen dioxide, as requested by the CAFE Steering Group. This is not to imply that other substances do not pose a considerable threat to human health and the environment at the current levels present in Europe. It should also be mentioned that PM itself is a complex mixture of solid and liquid constituents, including inorganic salts such as nitrates, sulphates and ammonium and a large number of carbonaceous species (elemental carbon and organic carbon). Thus PM implicitly covers a number of different chemical pollutants emitted by various sources. The term ‘particulate matter’ (PM) is used to describe airborne solid particles and/or droplets. These particles may vary in size, composition and origin. Several different indicators have been used to characterize ambient PM. Classification by size is quite common because size governs the transport and removal of particles from the air and their deposition within the respiratory system, and is at least partly associated with the chemical composition and sources of particles. Based on size, urban PM tends to be divided into three main groups: coarse, fine and ultrafine particles.

The border between the coarse and fine particles usually lies between 1 μm and 2.5 μm, but is usually fixed by convention at 2.5 μm in aerodynamic diameter (PM2.5) for measurement purposes. The border between fine and ultrafine particles lies at about 0.1 μm. PM10 is used to describe particles with an aerodynamic diameter smaller than 10 μm. The particles 71

CNR Environment and Health Inter-departmental Project

Figure 1. Deposition probability of inhaled particles in the respiratory tract according to particle size. contained in the PM10 size fraction may reach the upper part of the airways and lung. Fig. 1 shows schematically where particles are deposited in the respiratory tract, depending on their size.

Smaller particles (in particular PM2.5) penetrate more deeply into the lung and may reach the alveolar region. Ultrafine particles contribute only slightly to PM10 mass but may be important from a health point of view because of their large numbers and high surface area. They are produced in large numbers by combustion (especially internal combustion) engines.

As reported, (3) the most severe effects in terms of the overall health burden include a significant reduction, by a year or more, in average life expectancy linked to the long-term exposure to high levels of air pollution due to fine PM. Many studies have found that fine particles (usually measured as PM2.5) have serious effects on health, such as increased mortality rates and emergency hospital admissions for cardiovascular and respiratory reasons.

Thus there is good reason to reduce exposure to such particles. Coarse particles (usually defined as the difference between PM10 and PM2.5) seem to have effects on, for example, hospital admissions for 72

respiratory illnesses, but their effect on mortality is less clear. Nevertheless, there is sufficient concern to consider reducing exposure to coarse particles as well as to fine particles. Similarly, ultrafine particles are different in composition, and probably to some extent in effect, from fine and coarse particles. Nevertheless, their effect on human health have been insufficiently studied to permit a quantitative evaluation of health risks due to exposure to such particles.

As stated above, PM in ambient air has various sources. In targeting control measures, it would be important to know if PM from some sources or of a specific composition gave rise to special health concern due to their high toxicity. The few epidemiological studies that have addressed this important issue specifically suggest that combustion sources are particularly important for health. Toxicological studies have also pointed to primary combustionderived particles as having a higher toxic potential. These particles are often rich in transition metals and organic compounds, and also have a relatively high surface area. By contrast, several other single components of the PM mixture (e.g. ammonium salts, chlorides, sulphates, nitrates and wind-blown dust such as silicate clays) have been shown to have a lower toxicity in laboratory studies.

Despite these differences found among the constituents studied in laboratory, it is currently not possible to quantify the contributions of the different sources and different PM components on the health effects caused by exposure to ambient PM. 2.1. Modelling approach on health impact Health impact assessment allows to quantify the effects of exposure to an environmental hazard. It plays a central

Role of Atmospheric Pollution on Harmful Health Effects role in assessing the potential health effects of different policies and measures, thereby providing a basis for decision-making. A detailed knowledge of several factors is required for any such assessment. Crucial information on exposure to air pollutants is provided by an integrated approach on ambient air quality monitoring and modelling study.

Air quality modelling, particularly the Integrated Assessment Modelling (IAM), is important in linking pollution levels to emission sources and integrating population data, findings from epidemiological studies, information about the formation and dispersion of fine particles in the atmosphere, assessment of current and future levels of emissions of fine particles and their precursors. In the frame of the UN-ECE Convention on Long-Range Transboundary Air Pollution (CLRTAP), and in the context of the Community Environmental policies of the EU Commission, the RAINS-Europe model provides one of the most relevant examples of successful application of Integrated Assessment Modelling (IAM).

The RAINS model (6), developed at the International Institute for Applied Systems Analysis (IIASA), considers emissions of SO2, NOx, PM10, PM2.5,

VOC and NH3, provides deposition and concentration maps and addresses threats to human health posed by fine particulates (7). The assessment of fine particle health impacts is implemented through the Life Expectancy Reduction indicator (LER), defined as months lost attributable to PM2.5 concentrations. Awaiting further refinements in the scientific disciplines, the quantitative implementation should be considered as preliminary and needs to be revised as soon as more substantiated scientific information becomes available.

The Task Force on Health of the United Nations Economic Commission, when conducting the in-depth review of the RAINS approach for modelling health impacts of fine particles (TFH, 2003), noted “that some data suggested that different components that contributed to PM2.5 mass might not be equally hazardous. In particular, the discussion focused on the role of the secondary inorganic aerosols (including nitrates and sulphates). It concluded that, due to the absence of compelling toxicological data about the active different PM components of a complex mixture, it was not possible to quantify the relative health impact importance of the main PM components at this stage�. Therefore, it was recommended

Figure 2. Changes in EU life expectancy loss in 2000 and in the interim objective in 2020 (Strategy) (9).

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CNR Environment and Health Inter-departmental Project to relate health impacts to total mass of PM2.5 until more specific evidence becomes available (8). The methodology used in the RAINS model, at European and national scale, to estimate losses in life expectancy due to air pollution represents an initial implementation assessing the implications of present and future European policies to control exposure to particulate matter. In the Figure 2, an example of the changes in life expectancy loss in EU in 2000 and in the interim objective in 2020 (9) are reported. The impact assessment of the different policies is based on the analysis of a set of technological measurements with the RAINS model related to various emission reduction scenarios. The ambition level of the Strategy is based on a set of specific measurements which would

need to be undertaken at Community and Member State level.

In the recent years, some European countries such as, among others, Italy, have tackled the issue of implementing the RAINS model at national level, introducing higher spatial resolution in similar models, pursuing the ultimate objective of a more adequate response to the need of evaluating, at national level, cost-effective policy measures to reduce air pollutant emissions, and consequently, the pressure on environment and human health. As a result, the RAINS-Italy model (10) as the national version of the RAINSEurope model was defined considering as emission source areas either the nation as a whole or the 20 administrative Regions. In a recent work (11), the RAINS-Italy model

Figure 3. Losses in average Life Expectancy (months) attributable to PM2.5 concentrations at 2010: a) CLE scenario; b) difference (months) between Air Quality Management Plan scenario and CLE scenario (11).

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Role of Atmospheric Pollution on Harmful Health Effects was used to assess the emission reduction strategies followed in the Regional Air Quality Management Plans (AQMPs) to meet environmental quality targets by means of Technical and Non-Technical Measures. Regarding health impacts (Figure 3a), the most important Italian metropolitan and industrial areas show an average Life Expectancy loss ranging between 12 to 23 months in the 2010-CLE scenario. This higher resolution map shows a better definition of the hot spots present in the urban areas of Turin, Milan, Rome and Naples, as well as in the industrial sites of eastern Sicily and Taranto, in the Apulia region (12). The above mentioned study showed that if compared to the 2010 CLE scenario, the 2010 AQMP scenario reduces PM10, NOx and SO2 emission by 2.8%, 2.4% and 0.5%, respectively. Regions with a more effective AQMP reach higher PM10 yearly average concentrations reductions, with peaks of 7.5% in northern and central Italy, even if this is not sufficient to assure the compliance with air quality standard in 2010. Similarly the improvement in the average Life Expectancy loss indicator (Figure 3b) is of 1 month only in Lombardy (11).

2.2. Indoor vs outdoor air pollution As it is the case for other air pollutants, the total exposure of an individual to suspended particulate matter (of whatever size) is the result of contributions from the two microenvironments, outdoor air and indoor air. The indoor air compartment can be further subdivided into homes, restaurants, car, buses and aircraft, workplaces etc.

Consequently, in studies to detect and quantify the health effects of particles, attention must be paid that exposure is characterised adequately. Generally there are two different ways to obtain such characterization. One is by measuring

total air exposure using personal sampling: the persons under study are provided each with a personal sampler that they have to carry on them or position as close to them as possible for 24 hours consecutively.

Since this is cumbersome for a study participant, the following alternative can be used: total exposure is modelled taking into account the time spent in the various microenvironments (indoors and outdoors) and the concentrations observed in these microenvironments. Personal sampling provides a concentration level that represents the integration of all the concentration levels in all compartments visited by the studied person during the 24-h (or longer) measurement period and, thus, it cannot detect the individual contribution of any compartment. In contrast, the modelling process using the combination of the pollutant concentrations in the different microenvironments and the time spent therein permits to assess the contribution of total exposure to each of these microenvironments. This kind of source apportionment can be of great help to decide what measurements should receive priority in controlling pollutant concentrations. A recent publication on exposure to PM2.5 describing the results of a model approach (13) stated that the “indoor-residential� microenvironment had the greatest influence on total exposure to PM2.5, compared to the other microenvironments considered, namely outdoor and non-residential indoor (office, school, store, restaurant, bar, in-vehicle). It turned out that the outdoor compartment was responsible for a direct contribution of about 5% on average. Another 35% was due to an indirect contribution via infiltration of outdoor air into indoor spaces. Thus, about 60% of the total exposure to PM2.5 could not be influenced by control measurements taken to reduce outdoor air PM2.5 levels.

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CNR Environment and Health Inter-departmental Project 3. INORGANIC AIR POLLUTANTS 3.1 Gaseous matter

Air pollution by inorganic gaseous matter was dealt with since the first major pollution events (i.e. the Great Smog of London, 1952; etc.) put on evidence the strict relationship between levels of chemical species in the ambient (and indoor) air their harmful effects on health and ecosystems. Among inorganic gases, carbon monoxide (CO) is one of the most common air pollutants. It has a low reactivity and a low water solubility and it is mainly released into the atmosphere as a product of incomplete combustion.

CO is not only directly released in the air, but can also originate from the chemical reactions of organic air pollutants, such as methane. Its latency in the atmosphere is about three months. Since at moderate latitudes air masses travel for months and since the CO formation from organic air pollutants takes place everywhere in the atmosphere, a global background level of CO exists, ranging between 0.05 and 0.15 ppmv (0.06 and 0.17 mg/m3) (14). It is estimated that about one-third of CO, including that derived from hydrocarbon oxidation, originates from natural sources.

CO levels in busy city streets are higher than those present near highways, since the amount of CO emissions per kilometre strongly decreases with vehicle speed and also because ventilation in city streets is less. Ambient CO levels are usually highest in winter, because cold engines release much more CO than hot engines and also because the atmosphere tends to be more stable than in summer. It has to be reminded, however, that usually CO ambient levels do not exceed neither WHO guidelines for health protection nor the limits of the EU directives on air quality. Although CO is hardly removed 76

from the air in atmospheric transport at continental level, long range transport does not lead to concentrations of concern at both rural and urban background level.

Also at points of high traffic in large cities, levels exceeding legislation are only occasionally observed. Industrial areas may be affected by large CO emissions; however, when these emissions are released through high chimneys, local ambient concentrations show poor increases and do not pose risks for human health14. CO toxicity patterns are linked to its reaction with haemoglobin in the human blood to form carboxyhaemoglobin (COHb). The affinity of haemoglobin for CO is 200250 times higher than for oxygen, and as a result this binding reduces the oxygencarrying capacity of the blood and impairs the release of oxygen to extra vascular tissues. The most important variables determining the COHb level are CO in inhaled air, duration of exposure and lung ventilation. Physical exercise accelerates the CO uptake process.

The formation of COHb is a reversible process; however, the half-life elimination of COHb is much longer in the foetus than in the pregnant mother. The effects of CO exposure on cardiovascular disease have been studied for a long time (15). However, only limited information is available about the possible cardiac effects of gaseous pollutants at concentrations close to those present in ambient air. Apart from hazards due to high CO concentrations, other health effects seem to originate from the association between CO and other gaseous and particulate matter, especially exposure to exhausts from motor engines. Although attention has recently been focused on the cardiovascular effects of PM, few studies show evidence of the relationship between some cardiovascular diseases and the exposure of different populations to road

Role of Atmospheric Pollution on Harmful Health Effects traffic exhausts. For example, experimental studies have demonstrated mild cardiac effects from both sulphur dioxide (SO2) and ozone (O3)(16). Other studies, where personal exposure to different pollutants has been investigated, have suggested that the estimated cardiac effects attributed to gases, including SO2, are actually effects of other pollutants, specifically PM (17).

At this stage of knowledge, however, it is difficult to differentiate between the effects of PM and those of gases because people are normally exposed to both types of pollutants at the same time. Because of these uncertainties, it seems prudent to further investigate both the effects that low concentrations of gaseous pollutants, alone or in combination with PM, might have on cardiovascular diseases, and the possibility that the associations with gaseous pollutants may actually reflect the effects of PM or some component that is not currently being studied for its health effects (16).

Anthropogenic sulphur dioxide (SO2) results from the combustion of sulphurcontaining fossil fuels (mainly coal and heavy oils) and the smelting of sulphur containing ores. Over the past years, however, there has been a net tendency towards emission reduction in Countries where low-sulphur fuels and emission control measures have been adopted. In addition, the source pattern has changed and moved from small multiple sources (domestic, commercial, industrial) to large single sources releasing SO2 from tall stacks. Volcanoes and oceans are the major natural sources of SO2. After being released in the atmosphere, sulphur dioxide is further oxidized to sulphate (SO4=) and sulphuric acid forming an aerosol often associated with other pollutants in droplets or solid particles having a wide range of sizes. SO2 and its oxidation products are

removed from the atmosphere by wet and dry deposition. Nowadays, it is also recognized that sulphate aerosols play an important cooling role in the radiative climate of the Earth through the phenomena of sunlight scattering in cloud free air and as cloud condensation nuclei. Sulphur dioxide is an irritant and when inhaled at high concentrations may cause breathing difficulties in people exposed to it. People suffering from asthma and chronic lung disease may be especially susceptible to the adverse effects of sulphur dioxide.

Nevertheless adverse effects from high concentrations of SO2 have been observed both on healthy people and asthma patients (18).

Oxidized nitrogen compounds (NO2, NOx, NOy) and ozone (O3) join common patterns in atmospheric formation chemistry, environmental fate and adverse effects on health and the ecosystem. NO is directly released by all combustion processes; once in the atmosphere, it reacts with oxygen and a number of other inorganic (e.g. O3, OH radical, halogens) and organic (VOCs) gases to form NO2, NO3, HONO, HNO3, PAN, nitro – PAH and other organic and halogen nitrates, in the gaseous or particulate phase. Ozone and oxidized nitrogen compounds are strongly oxidant and this aspect mainly characterizes their harmful health action. In particular, the oxidizing potential of these compounds is commonly referred to as “odd oxygen” (Ox) or “odd nitrogen” (NOx), i.e families of chemical compounds that interconvert rapidly among themselves on time scales that are shorter than those necessary to form or destroy the family . Another family is that defined as “NOz”, which refers to the sum of NOx oxidation products (19).

NOx = NO + NO2 [1] 77

CNR Environment and Health Inter-departmental Project Ox = Σ (O(3P)

+ O(1D) + O3 + NO2) [2]

NOz = Σ (HNO3 + HNO4 + NO3 + 2NO2O5 + PAN + other organic nitrate + halogen nitrate + particulate nitrate) [3] Unlike some other compounds whose formation rates vary directly with the emissions of their precursors, O3 differs in that its production changes nonlinearly with the concentrations of precursors.

At the low NOx concentrations found in most environments ranging from remote continental areas to rural and suburban areas, the O3 net production increases with the increasing of NOx. At the high NOx concentrations found in downtown metropolitan areas especially near busy streets and roadways and in power plants, there is a net destruction of O3 by titration reaction with NO. Between these two regimes is a transition stage in which O3 shows only a weak dependence on NOx concentrations. In the high NOx regime, NO2 scavenges OH radicals which would otherwise oxidize VOCs to produce peroxy radicals, which in turn would oxidize NO into NO2. In the low NOx regime, VOC oxidation generates, or at least does not consume, free radicals, and O3 production varies accordingly. Sometimes the terms ‘VOC-limited’ and ‘NOx-limited’ are used to describe these two regimes; also, the terms NOx-limited and NOx-saturated are used. The chemistry of OH radicals, that are responsible of the initiation of hydrocarbons oxidation, shows a behaviour similar to that of O3 with respect to NOx concentrations (19). These considerations introduce a high degree of uncertainty into attempts to relate changes in O3 concentrations to precursors emissions. It should also be noted at the outset that in a NOx-limited (or NOx-sensitive) regime, O3 formation is not insensitive to radical 78

production or the flux of solar UV photons, but O3 formation is more sensitive to NOx. For example, global tropospheric O3 is sensitive to CH4 concentrations even if the troposphere is predominantly NOx-limited.

To get information about the O3-NOx-VOCs relationships and sensitivity, the ratio of summed VOC to NOx concentrations determining whether conditions are NOxsensitive or VOC sensitive is not sufficient to describe O3 formation, since other factors - i.e. the effect of biogenic VOCs (which are not present in urban centres in early morning) - and some important individual differences in VOCs ability to generate free radicals, have to be considered. The difference between NOxlimited and NOx-saturated regimes is also reflected in measurements of hydrogen peroxide (H2O2), another strong oxidant of ambient air. H2O2 formation takes place by self-reaction of photo chemically generated HO2 radicals, so that there is large seasonal variation in H2O2 concentrations, and values in excess of 1 ppb are mainly limited to summer months, when photochemistry is more active (20). Hydrogen peroxide is produced in abundance only when O3 is produced under NOx-limited conditions.

The transition from NOx - limited to NOx - saturated conditions is highly space and time dependent. In the upper troposphere, responses to NOx additions from commercial aircraft have been found that are very similar to those in the lower troposphere.

Moreover, the complex interplay between chemical and meteorological processes gives rise to uncertainties in understanding ozone formation. This is especially true for regions of complex topography. In coastal regions around the Mediterranean Basin, for instance, the combination of mountain and sea breeze re-circulations significantly affects ozone phenomenology. Ozone can also have very specific distributions

Role of Atmospheric Pollution on Harmful Health Effects in mountain areas, and observed concentrations differ significantly between mountain peaks and valleys (20). Nitropolycyclic aromatic hydrocarbons (nitroPAHs) are generated from incomplete combustion processes through PAHs electrophilic reactions in the presence of NO2 (21). Among combustion sources, diesel emissions have been identified as the major source of nitro-PAHs in ambient air.

Direct emissions of nitro-PAHs in PM vary with the type of fuel, vehicle maintenance, and ambient conditions (22). In addition to being directly released, nitro-PAHs can also be formed from both PAHs gaseous and heterogeneous reactions with gaseous nitrogenous pollutants in the atmosphere.

After formation, nitro-PAHs with low vapour pressures (such as 2NF and 2NP) immediately migrate to particles under ambient conditions; therefore harmful effects related to nitro – PAHs are better investigated in the organic fraction of particulate matter. An extended discussion on this topic is reported in par. 3.2.

Also in indoor environments NO2 plays a key role in adverse health effects. It is indeed produced by NO reactions with ozone or peroxy radicals generated by indoor air chemistry involving O3 and unsaturated hydrocarbons such as terpenes found in air fresheners and other household products (23). Nevertheless indoor NO2 is also contributed by indoor – outdoor air exchange. The relationship between personal NO2 exposure and ambient NO2 can be modified by the indoor environment. For example, during the infiltration processes, ambient NO2 can be lost through penetration and decay (chemical and physical processes) in the indoor environment, and the concentration of indoor ambient NO2 is not just the ambient NO2 concentration but the product of the ambient NO2 concentration and the

infiltration factor (Finf, or α if people spend 100% of their time indoor). Indoor NO2 is removed by gas phase reactions with ozone and assorted free radicals and by surface promoted hydrolysis and reduction reactions. The concentration of indoor NO2 also affects PAN decomposition.

These processes are important not only because they influence the indoor NO2 concentrations to which humans are exposed, but also because some products of indoor chemistry may confound attempts to examine associations between NO2 and health. As a matter of fact, NO2 is an oxidant and lipid peroxidation is believed to be a major molecular event responsible for its toxicity. As a result, there has been considerable attention paid to NO2 effect on the antioxidant defence system in the epithelial lining fluid and in pulmonary cells. Repeated exposure to indoor NO2 at concentrations ranging from 0.04 to 33 ppm has been shown to alter low molecular weight antioxidants such as glutathione, vitamin E, and vitamin C, as well as some enzymes involved in cell oxidant homeostasis. NO2 effects on structural proteins of the lungs have raised concern because elastic recoil is lost after exposure. It has been observed that the latter increases collagen synthesis.

This, in turn, shows increases in total lung collagen which, if sufficient, could result in pulmonary fibrosis after longer periods of exposure. Such correlation has yet to be confirmed by in vivo studies involving NO2 exposure; nevertheless some evidence shown in animal studies about asthma, emphysema and other lung diseases. Similarly to O3, NO2 is absorbed throughout the lower respiratory tract, but the major delivery site is the centriacinar region, i.e, the junction between the conducting and respiratory airways in humans and animals (21).

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CNR Environment and Health Inter-departmental Project Ozone is a strong oxidant, and as such can react with a wide range of cellular components and biological materials: damage can occur to all parts of the respiratory tract. The time pattern of these changes in the respiratory system, as determined in laboratory animals as well as in epidemiological investigations, is complex. During the first few days of exposure, inflammation occurs and then persists at an attenuated level. At the same time, epithelial hyperplasia progresses, and reaches a plateau after about one week of exposure. When the exposure ceases, these effects slowly disappear. In contrast to this, interstitial fibrosis increases slowly and can persist even when exposure ceases. In a large number of controlled human studies, significant impairment of pulmonary function has been reported.

Field studies in children, adolescents, and young adults have indicated that pulmonary function decrements, similarly to those observed in controlled studies, can occur as a result of short term exposure to ozone concentrations in the range of 120-240 Îźg/m3 and higher. In comparison with adults, children have a higher intake of ozone and other air pollutants. This is due to a higher basal metabolic rate, resulting in a higher breathing volume per minute and a higher breathing frequency.

Furthermore, their respiratory tract is still under development until the age of six and a half, and it is therefore more susceptible to the inflammatory effects of ozone.

Children’s immune systems are not yet fully developed and are generally under bigger stress. For these and other reasons, children are at higher risk when exposed to ambient ozone concentrations. Hospital admissions for respiratory causes and exacerbation of asthma are observed both in exposures to ambient ozone (and copollutants) and in controlled exposures to O3 80

alone. Other groups at risk are those people exercising outdoors during evening hours or whenever ozone concentrations tend to be highest (e.g. in photochemical smog events). Due to the irritant nature of ozone, capable of inducing airway inflammation and bronchoconstriction, asthma patients are deemed to be at enhanced risk from exposure to ozone and photochemical smog, because inflamed airways contribute to the pathogenesis and exacerbation of the disease and to morbidity and mortality for asthma. Results from recent epidemiologic studies have suggested that ozone might have serious cardiovascular effect (24 and references therein). Although a large number of toxicity animal studies have been performed on respiratory and other effects of NO2, O3 and other gaseous pollutants on metabolic and physiological functions (body weight, hepatic, renal, brain, etc.), results are often affected by serious limitations, due to both the necessary animal-to-human extrapolation of concentration-response data and the fact that controlled exposures to a single pollutant alone provide incomplete information.

Human clinical studies attempt to recreate in laboratory the atmospheric conditions of ambient pollutant atmospheres, paying great attention to concentrations, duration, timing, and other conditions which may impact responses. These studies allow the measurement of health symptoms and physiological markers resulting from air breathing. This carefully controlled environment allows researchers to identify responses to individual pollutants, to characterize exposure-response relationships, to examine interactions among pollutants, and to study the effects of other variables such as exercise, humidity, or temperature. Susceptible populations, including individuals with acute and chronic respiratory and cardiovascular

Role of Atmospheric Pollution on Harmful Health Effects diseases, can participate with appropriate limitations based on subject comfort and protection from risk. Endpoint assessment has traditionally included symptoms and pulmonary function, but more recently a variety of markers of pulmonary, systemic, and cardiovascular function have been used to assess pollutant effects.

It is reasonable to consider, however, that human clinical studies have limitations.

For practical and ethical reasons, studies must be limited to relatively small groups, to short durations of exposure, and to pollutant concentrations that are expected to produce only mild and transient responses. Findings from the short-term exposures in clinical studies may provide limited insight about the health effects of chronic or repeated exposures. Moreover, the choice of previous- and after-exposure time lags for the observation of health effects is critical in assessing the role of a pollutant in toxicity events. Many studies have shown that NO2 has a fairly consistent, immediate effect on health outcomes, including respiratory hospitalizations and mortality. Several studies also observed significant NO2 effects over longer cumulative lag periods, suggesting that in addition to single-day lags, multiday lags should be investigated to fully capture a delayed NO2 effect on health outcomes.

Finally it should be kept in mind that, although many biochemical changes are not necessarily toxic manifestations of the pollutant per se, such changes may anyway impact the metabolism and toxicity of other chemicals in humans and animal species (21).

The EU regulates the main harmful inorganic gaseous pollutants by the EC legislation of the Air Quality Framework (25). Other legally binding Protocols have been established since the 1979 Geneva Convention on Long Range Transboundary

Air Pollution (LRTAP) (26). Guideline levels aimed at health and environment protection have been set by WHO and other institutions, too, to be used for impact assessment. The first edition of the WHO “Air quality guidelines for Europe (AQG)” was published in 1987 (27). To determine critical or guideline levels, quantitative relationships between the pollutant exposure and its studied effect are needed. However, any such relationships have a certain degree of uncertainty, and the data necessary to produce them are often scarce. Therefore, the establishment of guideline values, such as levels at which acute (or chronic) effects on public health or ecosystems are likely to be not relevantly harmful, impose the support of biological, clinical and epidemiological evidence, often not available or inadequate.

Different legal tools aimed at protecting and improving the health and quality of ecosystems from air pollution have been used in recent years. The 1992 fifth action programme of the European Commission (EAP) on the environment recommended “the establishment of long-term air quality objectives” for many inorganic gaseous pollutants (CO, NO2 and NOx, SO2, O3).

The list of key requirements, also includes the need for “studies to analyze the effects [on health ant ecosystems] of the combined action of various pollutants or sources of pollution and the effect of climate on the activity of the various pollutants examined”. Under the 5th EAP the Air Quality Framework was established, within which the 96/62/EC Directive and the following four Daughter Directives have been adopted. This law establishes limits and threshold values for SO2, NO2 and NOx under the 99/30/EC, CO under the 2000/69/EC and O3 under the 2002/03/ EC for EU Member States. In the 6th EAP, further steps have been taken toward health 81

CNR Environment and Health Inter-departmental Project / environment protection by the Clean Air for Europe (CAFE) programme. The CAFE is conceived as a process based on technical analysis and policy development to achieve the adoption of a Thematic Strategy on Air Pollution. The major elements of the CAFE programme are outlined in Communication COM(2001)245 (2).

The programme, launched in early 2001, aims at the development of a long-term, strategic and integrated policy advice to protect against the significant negative effects of air pollution on human health and the ecosystem. Within this process, the 2008/50/EC Directive on ambient air quality and cleaner air for Europe has been adopted and will enter into force as from 11 June 2010, when the Directives 96/62/ EC, 1999/30/EC, 2000/69/EC and 2002/3/ EC shall be repealed.

3.2 Composition and size distribution of the inorganic fraction of suspended PM 3.2.1 Inorganic fraction of suspended PM The history of air pollution is very long, and since its very first occurrence - smoke from heating and cooking activities in prehistoric dwellings - particles have been addressed as one of the most important issues. Pollution from combustion sources and specifically suspended particles have been responsible for the most relevant pollution disasters (e.g. Mause Valley, Belgium, 1930, the Big Smoke, London, 1952), which led to increasing efforts towards pollution monitoring, the understanding of main pollution processes, political awareness and, finally, regulations.

At European level, pollution from particulate matter (PM) has been first addressed by the First Daugther Directive (1999/30/EC) to the Air Quality Framework Directive (1996/62/EC); recently, a new Directive 82

(2008/50/EC, published in June 2008) summarised most of the existing legislation on ambient air and introduced some new requirements. As far as PM is concerned, the First Daugther Directive addressed only PM10, setting limits for its annual average concentration (40 μg/m3) and the number of exceedances (35 per year) of the 50 μg/ m3 daily concentration limit. Air quality limits also for PM2.5 were introduced only by the recent Directive 2008/50/EC (25 μg/m3, with a 20% margin of tolerance that will be reduced to zero on 1st January 2015). In addition to the measurement of PM mass concentration, Directive 2008/50/ EC also includes the measurement of PM2.5 chemical composition in background sites, listing a number of components that must necessarily be determined in each PM2.5 sample (sulphate, nitrate, chloride, sodium, ammonium, potassium, calcium, magnesium). This new issue is related to the increasing awareness of the complexity of this “pollutant”, which is a mixture of thousands of different chemical species, each one with its own properties and possibly its own environmental and health effects.

Unlike gaseous pollutants, where the concentration is generally sufficient to define the system, for the atmospheric aerosol many parameters have to be defined. Physical parameters include the geometric and aerodynamic diameter, shape (spheres, fibres, etc.), phase (solid, liquid, mixture of both), density, electrical charge, hygroscopicity etc. and are necessary to understand particles behaviour in the atmosphere as well as inside the respiratory system. The most complex issue in aerosol characterisation, however, is its chemical composition, which includes a variety of components, whose determination requires a variety of analytical techniques.

Role of Atmospheric Pollution on Harmful Health Effects The knowledge of health effects caused by inhalation of atmospheric particles has been improved a lot during the last decade and there is no doubt that particles can be harmful to human health. PM is associated with a wide variety of both acute and chronic cardiovascular and respiratory effects. Acute effects include increased hospital admittance for respiratory disease or premature mortality for cardiovascular disease, while chronic effects include a number of diseases leading to longevity reduction. The increase in respiratory and cardiovascular morbidity and mortality is in the order of a few percent for a PM increase of 10 μg/m3 (28-34).

The study of the link between particulate matter and health is extremely complex and poses many problems, including the difficulties in assessing the role of particle size and particle composition, in quantifying the real exposure and understanding the biological mechanisms that are responsible for the effects, in evaluating the impact of the different sources and, last but not least, in detecting the real concentration and composition of the atmospheric aerosol.

The size of atmospheric particles varies among five orders of magnitude, from a few nanometres to hundreds of micrometers.

The size of the aerosol influences its lifetime in the atmosphere (and thus the spatial range of influence of any single source) as well as its pathway inside the human body. Basically, the atmospheric aerosol consists of three modes, which are closely linked to their formation mechanism: the coarse mode, predominantly mechanically generated (e.g. by erosion and by resuspension), the accumulation mode, produced by condensation from vapours and coagulation from smaller particles, and the nucleation mode, which includes particles smaller than 0.1 μm originating

from combustion processes (e.g. vehicle exhausts, biomass burning). Natural aerosol, originating from the sea and the soil, is mostly in the coarse mode and is generally considered as less harmful than anthropogenic aerosol, generated by combustion sources, found mainly in the fine mode and able to penetrate deeply in the respiratory tree. The harmful role of nanoparticles, able to reach the alveoli and to be directly transported inside the body cells, is still a matter of debate. The chemical composition of an atmospheric particle depends on its source as well as on its “story” from the time of its emission or formation to the time when it reaches the receptor (e.g. the human body). Some particles are directly released from their source into the atmosphere (primary PM), but the characterisation of any emission source is quite complex, as it generally changes with time and operative conditions.

In addition, once formed, particles often undergo chemical and physical transformations and for this reason what is measured at the receptor may be also very different from what is released at the source. Even more difficult to trace are the other particles formed in the atmosphere as a result of chemical reactions between gaseous compounds or gas-to-particle conversion; of particular relevance, in this framework, is the oxidation of biogenically released VOCs. Information about PM sources can be obtained by analysing their chemical composition. Only a limited number of compounds constitute more than 1% of the overall PM mass: a few metals (Al, Si, Fe), the main anions and cations (chloride, nitrate, sulphate, carbonate, sodium, ammonium, potassium, magnesium and calcium), elemental carbon and organic material.

This last category is the most important, as it generally represents 20 – 60% of PM, 83

CNR Environment and Health Inter-departmental Project but, unlike the other main components, it is not a single chemical compound but it is constituted by many hundreds of compounds, none of which constitute more than 1% of the total PM mass. Although the determination of the listed compounds is in most cases sufficient to obtain the mass closure (i.e the sum of the single components equals the gravimetric mass), the determination of micro-components is generally necessary to obtain a picture of PM sources and effects (35-45). Although quite complex, the determination of most inorganic PM components has been one of the targets of field research during the last 10-20 years. By determining inorganic PM macro-components it is possible to trace natural sources (sea-spray, desert dust, local crustal components) and to measure the contribution of secondary compounds (ammonium sulphate and ammonium nitrate); inorganic micro-components, on the contrary, may be of help in determining the contribution of anthropogenic sources, e.g. dust re-suspension and industrial sources (46-48). Much less understood and quantified is the organic fraction, as the chemical analysis is generally able to identify no more than 15-20% of total organic mass.

Once we are able to measure PM concentration and to determine its chemical composition, we need to clarify the link between concentration and exposure. This is a critical point in the scientific studies about health effects, as the reference PM values are generally those measured by local Protection Agencies, i.e. outdoor values sometimes taken at traffic hotspots, while people generally spend most of their time in indoor environments, including homes, working places and vehicles, and only a small part of their time outdoors.

Considerable work is still needed to develop models able to simulate the behaviour of 84

individuals in indoor microenvironments.

Also, we need information about the composition of indoor PM, that may greatly differ from the composition of outdoor particles (49-56). For example, in indoor environments we may be exposed to much more particles produced by peculiar sources such as domestic wood burning or cooking than to particles emitted by traffic sources.

As a consequence of the many difficulties arising when relating PM concentration to the results of epidemiological studies, the scientific community is now trying to find a relationship between health effects and individual chemical components.

This attempt requires the availability of long time series of PM composition study and is still in its infancy. The other pathway to elucidate the link between PM and health effects is the study of the PM toxicological effects, that is the specific mechanisms that lead PM to cause the observed health impacts. These studies include animal models, human exposure during occupational activities and experimental exposures. The mechanisms of PM effects on human health are still quite uncertain, but given the variety and degree of observed health associations, it is likely that more than one of them are involved. Basically, particles entering the tissue cells may cause inflammation; researchers increasingly find that reactive oxygen compounds (in the PM or produced by stimulated cells) play a role. Because of the presence of particles in the respiratory tract, changes in the respiratory function may occur. Particles in the blood may increase viscosity, causing thrombosis or myocardial infarct. Of course, individuals with pre-existing deficiencies of the cardiovascular or respiratory system may suffer more severe effects (56-61).

It is clear that a more integrated approach

Role of Atmospheric Pollution on Harmful Health Effects is needed to get insights into PM health effects. In particular, the link between health effect and PM component and size and the biological specific mechanism of its action requires further combined interdisciplinary studies.

3.2.2 Transition (heavy) metals In contrast to gaseous specific compounds such as benzene or carbon monoxide, the assessment of metal and metalloid compounds in ambient air is complicated by the fact that different species with considerably differing toxicity and/or carcinogenic potency may be encountered.

Therefore, to fully evaluate the health effects, it is important to know which compounds do occur in the environment or at least which compounds form the main constituents. In ambient air, metals, metalloids and their compounds are mainly encountered as part of particulate matter. They may be present in the non soluble, non stoichiometric mixture phase (for example as spinels) or as soluble ionic compounds (salts). To a lesser extent and under certain environmental conditions, gaseous forms (i.e, organometallic compounds) may or may not be adsorbed by particles. In respect to their effects on the environment and on human health, these compounds can be characterized by other parameters, such as water solubility (extended to solubility in biological fluids), particle size distribution, morphology and specific surface area, and chemical heterogeneity of their particles (for example, a metal compound encapsulated in another aerosol or surface enrichment of volatile compounds), or the concentration of metals and metalloids in the particles ultimately contacting target tissues in the human body. All parameters mentioned will influence bioavailability and possible effects. In addition, metal and metalloid

containing substances can undergo various chemical and physical transformations in the atmosphere on their way from the source to a possible receptor. For example, As (III) compounds may be oxidized to As (V). Unfortunately, analytical methods normally identify only the elements present in atmospheric particles, since a specific analysis is extremely difficult in the concentration range occurring in ambient air (typically several ng/m続). In addition, the state of oxidation may change during sampling. Consequently, information on the concentration of different compounds in ambient air is very limited at present.

Another possibility to gain some insight into them is to analyze which compounds are emitted by the most important natural (i.e, weathering processes) and anthropogenic sources.

Some metals are naturally found in the body and are essential to human health.

Iron, for example, prevents anaemia, and zinc is a cofactor in over 100 enzyme reactions. They normally occur at low concentrations and are known as trace metals. In high doses, they may be toxic to the body or produce deficiencies in other trace metals; for example, high levels of zinc can result in copper deficiency, another metal required by the body. Heavy metals (HMs) (or toxic metals) are trace metals with a density at least five times that of water. As such, they are stable elements (meaning they cannot be metabolized by the body) and bio-accumulative (passed up the food chain to humans). These include: mercury (Hg), nickel (Ni), lead (Pb), arsenic (As), cadmium (Cd), aluminium (Al), platinum (Pt), and copper (Cu) (the metallic form versus the ionic form required by the body). Heavy metals have no function in the body and can be highly toxic. Once liberated into the environment through air, drinking water, 85

CNR Environment and Health Inter-departmental Project food, or countless human-made chemicals and products, heavy metals are taken into the body via inhalation, ingestion, and skin absorption. If heavy metals enter and accumulate in body tissues faster than the body’s detoxification pathways can dispose of them, a gradual build-up of these toxins will occur. High concentration exposure is not necessary to produce a state of toxicity in the body, as heavy metals accumulate in body tissues and, over time, can reach toxic concentration levels. Human exposure to heavy metals has risen dramatically in the last 50 years as a result of an exponential increase in the use of heavy metals in industrial processes and products. Today, chronic exposure comes from mercuryamalgam dental fillings, lead in paint and tap water, chemical residues in processed foods, and “personal care” products (cosmetics, shampoo and other hair products, mouthwash, toothpaste, soap).

The effects of Heavy Metal toxicity studies confirm that heavy metals can directly influence behaviour by impairing mental and neurological functions, influencing neurotransmitter production and utilization, and altering numerous metabolic body processes. Systems in which toxic metal elements can induce impairment and dysfunction include the blood and cardiovascular system, detoxification pathways (colon, liver, kidneys, skin), endocrine (hormonal)system, energy production pathways, enzymatic, gastrointestinal, immune, nervous (central and peripheral), reproductive, and urinary systems. Breathing heavy metal particles, even at levels well below those considered nontoxic, can have serious health effects.

Virtually all aspects of animal and human immune system functions are compromised by the inhalation of heavy metal particulates. In addition, toxic metals can increase allergic reactions, cause 86

genetic mutation, compete with “good” trace metals for biochemical bond sites, and act as antibiotics, killing both harmful and beneficial bacteria. For the most toxic HMs, atmospheric concentrations for Pb, As, Cd, Ni and Hg in ambient air have been regulated by European Commission directives (Directive 1999/30/EC from 22 April 1999 for Pb; Directive 2004/107/EC from 15 December 2004 for As, Cd, Ni and Hg). For Pb, an annual limit value of 0.5 g m-3, entered into force 1.1.2005, has been set. The pertaining As, Cd and Ni Target Values are, otherwise, reported in Table 3.1.

Table 3.1 Target values for As, Cd and Ni. 2004/107/EC Pollutant

Target Value (1)

Arsenic

6 ng m-3

Cadmium

5 ng m-3

Nickel

20 ng m-3

(1) For the total content in the PM10 fraction averaged over a calendar year

Specifically, from chronic arsenic exposure, the greatest dangers are lung and skin cancers and gradual poisoning, most frequently derived from living near metal smelting plants or arsenic factories.

Arsenic toxicity has been recognized for centuries, and hair shows significant correlation with its intake. As can be released to the atmosphere from metal transformation, fuel combustion and the use of pesticides.

In the air, As exists predominantly absorbed on particles, and is usually present as a mixture of arsenate (As(+V)) and arsenite (As(+III)), except in areas of arsenic pesticide application or biotic activities, where organic species are predominant (27,62). Recent data display a wide range of As concentrations in atmospheric particulate matter, for samples collected

Role of Atmospheric Pollution on Harmful Health Effects at various sites in Spain (ranging from 13 to 144 mg kg-1 for a rural area and an industrialised area, respectively) (63). All studies performed in the Mediterranean basin (64-65) agree with an enrichment of As in the atmosphere as shown by (66).

Cadmium is an element that is naturally found in the earth’s crust. Cadmium is often found as part of small particles present in air. Cadmium has many uses in industry and consumer products, mainly batteries, pigments, metal coatings, and plastics.

Cadmium can enter the environment in several ways. It can enter the air from the burning of coal and household waste, and metal mining and refining processes.

Cadmium attached to small particles may get into the air and travel a long way before coming down to earth as dust or in rain or snow. Cadmium does not break down in the environment but can change into different forms. Most cadmium stays where it enters the environment for a long time. Cadmium has no known good effects on health.

Breathing air with very high levels of cadmium severely damages the lungs and can cause death. Breathing lower levels for years leads to a build-up of cadmium in the kidneys that can cause kidney disease. Other effects that may occur after breathing cadmium for a long time are lung damage and fragile bones. Workers who inhale cadmium for a long time may have an increased chance of getting lung cancer. The greatest danger from chronic nickel exposure is lung, nasal, or larynx cancers, and gradual poisoning from accidental or chronic low-level exposure, the risk of which is greatest for those living near metal smelting plants, solid waste incinerators, or old nickel refineries. Nickel combined with other elements is naturally found in the earth’s crust, in all soils, and it is also released from volcanoes. Nickel is the 24th most abundant element, and in

the environment it is found primarily in the form of oxides or sulphides. Nickel is also found in meteorites and in lumps of minerals on the bottom of the ocean, and it is known as sea floor nodules. The earth’s core is believed to contain large amounts of nickel. Nickel is released into the atmosphere during nickel mining and by industries that convert scrap or new nickel into alloys or nickel compounds or by industries that use nickel and its compounds. It is also released into the atmosphere by oil-burning power plants, coal-burning power plants, and trash incinerators. The nickel that comes out power plants’ stacks is attached to small particles of dust that settle to the ground or are transported in the air by rain. It will usually take many days for nickel to be removed from the air. If the nickel is attached to very small particles, removal can take longer than a month. Given nickel’s ability to cause contact dermatitis, and its observed perturbation of immunoglobulin levels, elevated hair levels may serve as an indicator of possible immune dysfunction, as well as a potentially useful marker of cardiovascular problems.

Pb is one of the most released HMs in the Mediterranean atmosphere. Traffic remains the main source at global scale (67), but its relative importance varies from region to region. In Europe, the phasing out of alkylleads in gasoline resulted in a decrease in atmospheric lead concentrations. As a result, industrial emissions (lead smelting and steelworks) became predominant in Europe and discernable from traffic emissions at continental scale (68). The annual emission of Pb in the Mediterranean region has been estimated to be about 1.1 104 Mg yr-1 in 2005 by (69). Lead is a known neurotoxin (it kills brain cells), and excessive blood lead levels in children have been linked to learning disabilities, 87

CNR Environment and Health Inter-departmental Project attention deficit disorder, hyperactivity syndromes, and reduced intelligence and school achievement scores.

3.2.3. Mercury (Hg) and impact on human health Although mercury is an element found in nature and as such it will always be present in the environment, human activities have significantly increased global atmospheric mercury deposition since pre-industrial times. A significant increase in mercury emissions in the atmosphere occurred during the industrial revolution due to fossil fuel combustion and other human activities.

Mercury is today a severe and chronic pollution problem in the environment.

It is released into the atmosphere from a variety of anthropogenic (i.e, cement production, waste incineration, power generation facilities, smelters) (70-71) and natural sources (i.e, volcanoes, crustal and vegetation degassing, oceans) (7274) in different chemical and physical forms.(75). In the troposphere, the most important forms are gaseous elemental mercury (Hg0), divalent reactive gaseous mercury, Hg(II), which consists of various oxidised compounds, and particle-bound Hg (Hg-p), which consists of various Hg compounds. It should be noted that information on the speciation/fractionation of these different chemical and physical forms is largely operationally defined.

Conversions between these different forms provide the basis of Hg’s complex distribution pattern on local, regional and global scale. Hg cycles in different environmental compartments depends on the rate of different chemical and physical mechanisms (i.e, dry deposition, wet scavenging) and meteorological conditions as well as on the anthropogenic variables that affect its fate in the global environment. Experimental field data and 88

model estimates indicate that anthropogenic mercury emissions are at least as great as those from natural sources, and contribute to the global atmospheric pool. A threefold increase of mercury deposition since preindustrial times was in fact observed from the analysis of lake sediments, ice cores and peat deposits in both hemispheres (7479). Recent studies have highlighted that in fast developing countries (i.e, China, India) mercury emissions are rapidly and dramatically increasing due mainly to a sharp increase in energy production from coal combustion. Recent emission estimates highlighted that the Asian emissions are considered to have a global impact.

Evidence shows that an increase in ambient air levels of mercury is linked to an increased load of toxic mercury in ecosystems (80). The atmospheric input of this element in aquatic and terrestrial ecosystems is driven by particle dry deposition and wet scavenging by precipitation mechanisms (81-83). The most important from a toxicological point of view are the metallic forms. In fact, the impact of mercury on human health and the environment depends on several mechanisms, which, in turn, depend on the toxicokinetic of its major chemical forms present in different environmental media including elemental mercury (Hg0), inorganic mercury (i.e, HgCl2) and organic mercury (i.e, methylmercury).

This toxicokinetic mechanisms include absorption, distribution, metabolism and excretion. Therefore, according to the relevant chemical form of mercury, the combination of these mechanisms will determine the risk associated to the human exposure. For instance, the absorption of Hg0 vapour occurs rapidly through the lungs, but it is poorly absorbed from the gastrointestinal tract.

Role of Atmospheric Pollution on Harmful Health Effects Once absorbed, elemental mercury is readily distributed throughout the body, it crosses both placental and blood-brain barriers (84-86) Elemental mercury is oxidised to inorganic divalent mercury by the hydrogen peroxidase-catalase pathway, present in most tissues. The distribution of absorbed elemental mercury is limited by the oxidation of elemental mercury into mercuric ion as the mercuric ion has a limited ability to cross the placental and blood-brain barriers. Once elemental mercury crosses these barriers and is oxidised to mercuric ion, its return to the general circulation is impeded, and mercury can be retained in brain tissue.

The elimination of elemental mercury occurs via urine, faeces, exhaled air, sweat and saliva. The excretion pattern depends on the extent to which elemental mercury has been oxidised to mercuric mercury (87-90). Absorption of inorganic mercury through the gastrointestinal tract varies with the particular mercuric salt involved and decreases with its increasing solubility and can reach even 20% (91). Available data indicate that absorption of mercuric chloride from the gastrointestinal tract results from an electrostatic interaction with the brush border membrane and limited passive diffusion. Increases in intestinal pH, high doses of mercuric chloride causing a corrosive action, a milk diet and increases in pinocytotic activity in the gastrointestinal tract have all been associated with increased absorption of inorganic mercury. Inorganic mercury has a limited capacity of penetrating bloodbrain and placental barriers. There is some evidence indicating that mercuric mercury in the body following oral exposure can be reduced to elemental mercury and excreted via exhaled air. Because of the relatively poor absorption of orally administered inorganic mercury, most ingested doses in

humans are excreted through the faeces.

Methylmercury is rapidly and extensively absorbed through the gastrointestinal tract (92). Absorption information following inhalation exposure is limited. Epidemic of mercury poisoning following high-dose exposures to methylmercury in Japan and Iraq demonstrated that neurotoxicity is the most worrisome health effect when methylmercury exposure occurs to the developing foetus. Dietary methylmercury is almost completely absorbed into the blood and distributed to all tissues including the brain. It also readily passes through the placenta to the foetus and foetal brain. Methylmercury in the body is considered to be stable and it is only slowly demethylated to form mercuric mercury in rats. It has a relatively long biological half-life in humans (44-80 days) and it is excreted through faeces, breast milk and urine.

4. ORGANIC AIR POLLUTANTS 4.1 Volatile organic compounds The atmosphere is formed by a restricted number of macro-components, namely gaseous nitrogen, oxygen, argon, carbon dioxide, solid particulate matter and water; the latter exists as vapour, or in liquid and solid forms. Thousands of microcomponents are dispersed in the gas phase and/or participate to aerosol composition 93 . Although occurring often at very low levels (even below one part-per-trillion), they nevertheless intervene in the physics and chemistry of the atmosphere, heavily influencing our life. Among the micro-components, a key role is played by hydrocarbons and their derivatives, cumulatively called “volatile organic compounds� (VOC), which are in gas form.

Congeners of VOC occur in particulates as adsorbed on soot or dissolved in water 89

CNR Environment and Health Inter-departmental Project drops and crystals.

In its chemical structure, VOC include: i) aliphatic hydrocarbons (linear, branched and cyclic); ii) arenes having at least one aromatic group (Ar), namely benzene, alkylbenzenes and some polyaromatics (naphthalene); iii) alcohols (ROH) and ethers (ROR1); iv) carbonyls, comprising aldehydes (RCHO) and ketones (RCR1O), v) carboxy-acids (e.g. formic, HCOOH) and phenols (ArOH); vi) organic halogenides; vii) nitrogen, sulphur and phosphorus compounds; ix) heterocyclic and x) mixed functionality types (94-96).

Widely varying in concentration, structure and properties, different classifications have been proposed for VOC. One worth of mention distinguishes four groups of substances according to major aftermaths induced onto the environment, and their chemical formulas. They are: i) gases promoting the Earth warming (greenhouse effect VOC); ii) compounds responsible for the stratospheric ozone hole; iii) hydrocarbons promoting (or involved in) the tropospheric ozone and secondary particulate generation (photochemical smog); and iv) toxic compounds. At this regard, it seems worth to remark that: - The global Earth warming has been overall associated with carbon dioxide and water vapour. Nevertheless, it is well known that other compounds promote this phenomenon, e.g. methane, nitrogen protoxide, sulphur hexafluoride, and chlorofluorocarbons (freons or CFC) (97-98). The worldwide use of these substances and/or their release into the environment as by-products of human activities caused a strong increase in their respective atmospheric loads, and long, expensive and concerted actions must be launched to control and remediate global warming.

- The “ozone hole” first observed on 90

stratosphere Antarctica has been associated with CFC, which capture the solar light and start a reaction chain with those transforming molecular oxygen (O2) into ozone (O3). The variety and intensity of CFC use, combined with their long lifetime, caused their accumulation in the air and transport by winds in remote regions. This phenomenon leads to an increased ultraviolet radiation that reaches the ground, causing in particular an increase in skin tumours. This is the reason why CFC have been banned and replaced by other chemicals characterized by shorter lifetimes; nevertheless, they have lower heat capacities and higher prices, so their true use gains place with difficulty (99-100).

- At ground level, ozone represents a sanitary risk for humans and causes damages to crops and materials, thanks to its strong oxidant potency. Ozone is primarily generated in reactions involving oxygen, nitrogen oxide and dioxide, and “active” sunlight (l <430 nm) (93). The natural ozone background regulated and limited by them. Nevertheless, this equilibrium is modified by VOC that trigger processes leading to the formation of ozone without NO2 consumption.

Thus, the ozone concentration can largely increase. A lot of other oxygencontaining compounds are generated in the form of molecules (carbonyls, carboxy-acids, phenols, epoxides) and free radicals (OH, RO, RO2, HO2); when oxidized, hydrocarbons easily condensate giving raise to nuclei around which nano-particles are formed. Not all hydrocarbons participate equally to the photochemical smog formation.

Methane, short-chain alkanes and benzene are quite non-reactive,

Role of Atmospheric Pollution on Harmful Health Effects similarly to acids and ketones. By contrast, alkenes and alkylbenzene have high photochemical ozone formation potentials (101). A special role is played by isoprene and terpenes (biogenic hydrocarbons), which are very reactive vs. OH and NO3 radicals as well as vs. O3; they can induce high airborne concentrations of O3 in rural areas (forests, crops). Through nanoparticle and oxidant formation, reactive VOC indirectly affect the air quality.

Some VOC have been recognized as toxicants for their acute and/or chronic effects and have been included in the priority list of pollutants (102). For instance, benzene is known as tumour promoter, similarly to many halogensubstituted hydrocarbons (e.g. bromoform, methyl tetrachloride). Numerous VOC are carcinogenic (butadiene, diazomethane), mutagenic (chloroform) or induce cough, skin, eye and noose sensitization, throat irritation (aldehydes, organic halogenides), faint, loss of knowledge (tetrachloroethylene, methanol, xilene), diarrhoea, liver and kidney damages (aniline); some of them are poisonous (ethylene oxide, camphor, methanol, monomethyl mercury), or psycho-active inducing euphoria, depression, headache (methyl bromide, ethanol). Acids, bases and strong solvents are also caustic (trichoroacetic acid; dimethylamine; methanol, acetone, chlorobenzene).

As far as the above mentioned features of VOC have been clarified, dedicated legislations have been issued to preserve the environment and health (103-112).

Regulations refer to industrial and car emissions, power and heat production, agriculture, waste management, materials, food, open air, indoor and work places.

4.2. Organic particulate matter The organic fraction of particulates is well known, yet much remains not understood.

Thousands of chemical substances have been identified in airborne and emission particles. They include n-alkanes and non-polar aliphatic or alkylbenzenes, polynuclear aromatic hydrocarbons (PAH) and the corresponding alkyl-, nitro-, amino-, carbonyl-, oxy-, sulphur- and azaderivatives, halogen- and phosphoruscontaining pesticides, phthalate esters, acids and phenols, alcohols and nitriles (113-115). Most of these substances show scarce hydro-affinity, whilst shortchain, mixed functionality acids are water soluble. Finally, polymers and macromolecules, often of biogenic origin, contribute to the bulk (116). Several studies have been carried out to elucidate chemical composition, with two main objectives in mind: to draw an indirect toxicity parameterization of emissions or environment, to solve the biogenic/ anthropogenic origin of particulates and evaluate the relative contribution of their sources (117-121). Finally, the detection of reactive compounds and their corresponding by-products has allowed to put in evidence the oxidation capacity of the atmosphere, deriving from both the presence of oxidants (ozone, free radicals, nitrogen oxides) and light (122). This seems particularly important in the case of reactions involving gaseous substances that are converted into particles (secondary pollution), and whenever the degradation products are much more toxic than their parent compounds (116). Despite extensive and prolonged efforts made in this field, the complete characterization of organic fraction of particulates is far from being reached. It has been demonstrated that the organic fraction accounts for 10 to over 80% of total airborne particulates.

91

CNR Environment and Health Inter-departmental Project This variability widely depends not only on the environmental contour investigated (locality, emission sources impact, orography, meteorology, indoor or outdoor), but also on the approach adopted to measure it. In particular, very different results are found if the sole â&#x20AC;&#x153;organic solvent extractable fractionâ&#x20AC;? is considered, the water soluble fraction is taken in account, or optical automatic methods are adopted.

Otherwise, the WSOC of aerosol contains many different compounds that, to date, are poorly characterized. Experimental evidence suggest that these compounds are at least partly responsible for the main oxidizing and toxicant properties of urban and non-urban aerosols (56,116). Unlike elemental carbon, which is generally associated with the presence of humans (the sole exceptions consist of forest spontaneous fire and volcano emissions), the organic fraction of particulates has a twin origin, i.e. biogenic and anthropogenic.

That can be easily explained through an example. Linear alkanes globally account for a few percents of organic particulate matter. Their group presents two well distinct composition behaviours.

The saw-tooth distribution dominated by long-chain odd homologues (C29H60, C31H64 or C33H68) is typical of biogenic sources (e.g. high vegetation), whilst the mono-modal (or bell shaped) distribution characterizes anthropogenic emission like motor vehicles; in this case, the maximum centred at C19H40áC25H52. With the exception perhaps of macromolecules and acid compounds having many hydrophilic groups (carboxyl-, hydroxyl-, carbonyl-, epoxy-), organic substances are, individually, micro-components of soot accounting for parts per million down to parts per trillion of the particulate mass.

Together, they form a mixture often adhering to the surface, while the particle 92

core is made of elemental carbon and inorganic elements. The polarity and size of molecules influence the hydro-affinity of particles, their growth capacity and then their time life in the atmosphere.

Most organic compounds are neutral, although exhibiting different polarities; basic species are a few (they include aromatic amines and aza-PAH), whilst a lot are acidic (phenols, carboxy-acids), even when lypophilic. This variety is of environmental concern, since its mediumand long-term toxicity is strictly dependent on the polarity features of these elements.

Studies conducted in Italy and abroad have demonstrated that organic fraction is a major contributor to particulate toxicity. In Table 5.2. UFPs/NPs natural and anthropogenic sources.

Natural

Anthropogenic Unintentional

Intentional (NPs)

Gas-toparticle conversion

Internal combustion engines

Controlled size and shape, designed for functionality

Forest fires

Power plants

Metals, semiconductors, metal oxides, carbon, polymers

Volcanoes (hot lava)

Incinerators

Nanospheres, -wires, -needles, -tubes, -shells, -rings, -platelets

Jet engines

Untreated, coated (nanotechnology applied to many products: cosmetics, medical, fabrics, electronics, optics, displays, etc.)

Viruses

Metal fumes (smelting, welding, etc.)

Polymer fumes Other fumes Heated surfaces Frying, broiling, grilling Electric motors

Role of Atmospheric Pollution on Harmful Health Effects fact, up to 90% of total carcinogenic and/or mutagenic potency of particulates results to be associated to the corresponding organic extracts, although the substrate can act as promoter of synergic effects.

Two main aftermaths can be detected, the former acute (cyto-toxicity, causing cell death) and the latter chronic (cell damage, inducing carcinogenicity, mutagenicity, teratogenicity). Both of them generally appear as associated to neutral polar and acidic compounds taken as pools, and a number of very strong toxicants have been identified among them, including nitro-lactones and dioxins (123-124). By contrast, non-polar components are not toxic, although can exalt the potencies of active species. Despite that, the true contribution provided by each primary toxicant to environmental toxicity cannot be evaluated, since the interaction with the matrix is neglected in terms of synergic/ antagonistic actions, and with respect to net exposition of humans. At this regard, two points must be taken in account.

First, due to combination of its nature and airborne concentration, to ambient conditions (temperature and humidity), to soot concentration and characteristics, each organic species partitions between gas and particulate (125). Thus, their interaction with human body is variable.

Secondly, the total load of organic compounds is distributed among the particle size fractions, with the general tendency to accumulate into the ultra-fine and fine particles (126-127). It is worth remarking that the strongest toxicants (e.g.

PAH, chlorinated hydrocarbons, nitrocompounds) accumulate into the ultrafine and fine particles, while components with less pronounced potencies are more equally distributed. Very polar components, deriving from the oxidation of gaseous emissions, act as condensation

nuclei, triggering the formation of nanoparticles. These latter are considered, as a consequence of their number and size, the main cause of air toxicity and an emerging “hot issue” for environmental safety preservation. Thus, the association of organic species to them has heavy environmental consequences.

The importance of the organic component of particulates is well depicted by legislation issued to preserve health, and especially the health of workers (103106, 128-130). In fact, a set of organics is listed by WHO (UNEP) among the most important toxicants; these include a dozen of persistent pesticides, polychlorinated dioxins, furans and biphenyls. On the other hand, PAH are quoted in European Union Directives concerning air quality or integrated pollution prevention and control.

The target value of 1 nanogram per cubic metre of air, calculated as yearly average, has been established for benzo(a)pyrene, kept as an index of aerosol carcinogenicity.

European and Italian legislations require to measure also other seven relevant carcinogenic PAH. Toxicants like dioxins and furans are also quoted somewhere, however non target or limit values are provided.

In terms of future actions, certainly the role played by organics in particle generation and accumulation, in troposphere properties (e.g. radiance, heat absorption, albedo, water condensation, global warming), human health and environmental preservation has a key importance. Concern would be ascribed also to “new” pollutants like polyfluorinated acids, pesticides and plastic surrogates, psychotropic substances and poly-functionalized species (oxyacids, polycarboxylic acids, which are suspected to affect our world.

93

CNR Environment and Health Inter-departmental Project 4.3. Research in organic air pollutants: the past, the present and the future Organic substances are at the core of a series of studies conducted by CNR with the aim of clarifying the features and dynamics of the environment, in the frame of international and national programmes.

Special attention has been paid to the composition of the atmosphere, the effects induced by anthropogenic activities and by natural events and sources, the kinetics of pollutants in the presence of oxidants and light, the assessment of mobile and stationary emissions and pollution sources, particulate generation and properties, and sanitary relevance of pollution. According to recent developments, some items seem to deserve better analysis: the clean (green) energy generation, the chemistry of radicals, the air/sea, air/ice and air/soil interactions, the influence on meteorology and climate, the strict relationships between chemical composition and toxicity. As concerns this latter, interesting issues have been identified in nanoparticles, water-soluble organics, organo-metallic compounds and psychotropic substances. Finally, the economic, social and legislative aspects cannot be neglected in view of life quality promotion.

5. PARTICLES

IN THE ULTRAFINE

(UFP)

AND

NANO-SIZE FRACTIONS

5.1 Sources and formation of ultrafine particles Ultrafine particles are the dominant contributors of particle numbers in the PM2.5 fraction. Particles with dimensions < 100 nm are defined as nano-sized and ultrafine particles (NSPs, UFPs), according to their manufactured or environmental / biological origin. In particular, NSPs are called ‘ultrafine’ particles (UFPs) also 94

by toxicologists, ‘Aitken and nucleation mode’ particles by atmospheric scientists and ‘engineered nanostructured materials’ by materials scientists(132). The simplest examples of naturally occurring ultrafine particles are those found in the biological tissues of organisms. For example, biogenic magnetite is a natural NSP found in many animal species. Other nanosized materials, including fullerenes, are naturally originated from combustion processes such as forest fires and volcanoes. Sources of ambient UFPs are either primary or secondary. In urban environments, the dominant contributors of primary UFPs are combustion products found in motor vehicle exhausts, which are usually black carbonaceous soots with particle dimensions 200–300 nm. Atmospheric oxidation of gas-phase primary exhaust species can produce lower vapor pressure compounds that readily condense onto existing particles and produce secondary mass. Particle composition and size can also evolve due to interaction/reaction of gas- and liquid- or solid-phase species at the particle surface or in the bulk solution, as well as through coagulation of existing particles. In some conditions, photochemical oxidation of gas-phase species can directly produce new UFP (133). Most atmospheric UFPs are usually <50 nm and evidence has been found that the particle count distribution peaks at 20–30 nm at roadsides with heavy traffic134. Diesel-exhaust particles (DEP) are major components of PM2.5. Although installing a diesel particle filter (DPF) can reduce the number of larger particles in the exhaust, nanoparticles are produced during DFP regeneration. Environmental or atmospheric UFPs contain semivolatile alkanes that originate from fuels and lubricants (135). In particular, primary exhaust emissions of particles consist

Role of Atmospheric Pollution on Harmful Health Effects on the type of product. In table 5.1 (136) main differences between environmental and manufactured UFPs are reported.

Environmental nanoparticles

Manufactured nanoparticles

Size

≤50 nm (≤100 nm: ultrafine)

1–100 nm (biomedical nanoparticles can be larger than 100 nm)

Dimensions

Two or three dimensions are on the nanoscale

Three dimensions are on the nanoscale (nanoobjects with one and two nanoscale dimensions are called nanoplates and nanofibers, respectively)

Name

Table 5.1. Differences between environmental and manufactured nanoparticles

Nanoparticles, fibrous nanoparticles

Nano- : particles, spheres, tubes, rods, fibers, wires, ropes, sheets, eggs, liposomes, dendrimers, etc.

Components

mainly in a mixture of elemental carbon and organic compounds, and traces of heavy metals and sulphur. Tire wear also contains carbonaceous materials, while brake wear is rich in heavy metals136.

Moreover, particle hygroscopic growth measurement techniques have been used to show that ultrafine particles may show two separate modes of ‘‘less’’ and ‘‘more’’ hygroscopic particles (137). The less hygroscopic particles are those that exhibit little or no growth when exposed to a high relative humidity (RH) (typically 80–90%) and are thought to be mainly composed of hydrophobic chemicals such as waterinsoluble organic compounds and soot, and must be attributed to primary emissions from traffic and other combustion sources.

More hygroscopic particles grow by a larger factor (e.g, 1.38–1.69 for 35–265 nm particles at 90% RH) and have been shown to contain inorganic chemicals such as nitrate, sulphate, sodium and potassium and sometimes organic carbon as well. The more hygroscopic particles can result from the conversion of gaseous compounds into particles, or from the modification and oxidation of pre-existing particles. Such chemical and physico-chemical processes, named “secondary”, may occur even far from the emission sources, and may influence the concentrations of ultrafine and fine particles outside urban areas. It has been shown that the nucleation of ultrafine particles from condensation of reactive gases is responsible for the increase of ultrafine particle number concentrations in many rural areas, including polluted and natural sites. The hygroscopic behaviour of the aerosol is an important quality, as this will determine how they interact with clouds (138), which, in turn, affects the lifetime of the particles in the atmosphere.

Conversely, components of manufactured or engineered nanoparticles vary, depending

Carbon soot, hydrocarbons (alkanes), heavy metals, sulfur

Carbon (fullerene, nanotube), metal oxide (TiO2, ZnO), CdSe, metalloids, transition metals, polymers

More recently, even smaller particles in the nucleation mode with peak diameters around 4 nm have been observed. Humans have been exposed to airborne NSPs throughout all their evolutionary stages. Nevertheless, such exposure has increased dramatically over the last century due to anthropogenic sources. These can be classified as unintentionally or intentionally produced, depending on weather UFPs represent a sub-product or the major product coming from an anthropogenic source. Although the mass of UFPs in ambient air is very low, approaching only 0.5–2.0 μg/m3 at background levels, it can increase severalfold during high pollution episodes or on highway. In urban areas motor vehicle particle emissions are a dominant pollution source, where more than 80% of particle number concentrations are found in the 95

CNR Environment and Health Inter-departmental Project ultrafine size range. However, very little information can be obtained about particle number from particle mass measurements, and as current air quality standards are mass and not particle number-based, this means that the greater proportion of motor vehicle particle number emissions are not controlled or regulated (141). The latter case is represented by nanotechnologies.

Main natural, unintentional and intentional sources of NSPs are reported in table 5.2 (141).

5.2 Role of UFPs and nano-particles as atmospheric toxicants Inhalation of particulate matter leads to pulmonary inflammation and reduction in lung function (142) with secondary systemic effects or, after translocation from the lung into the circulation, to direct toxic effects on cardiovascular function (143) and on the coagulation pathway thus contributing to the onset of coronary events (144). Through the induction of cellular oxidative stress and proinflammatory pathways (144), particulate matter augments the development and progression of atherosclerosis (145).

The main factor of these adverse health effects seems to be combustion-derived nanoparticles that incorporate reactive organic and transition metal components.

An important source of these particles is new diesel cars with oxidizing converters, such as modern taxis in North Europe.

Many epidemiological, human clinical, and animal studies showed that ultrafine particles (UFPs) penetrate deeply into the lungs initiating an inflammatory response leading to respiratory diseases and may be absorbed directly into the circulating blood, causing cardiovascular diseases146.

Recent studies highlighted the importance of identification of susceptible subpopulations and mechanisms of involved effects. Several chronic clinical conditions 96

are good candidates to define the susceptible population to the acute effects of UFP, while elevated levels of oxidatively altered biomolecules are important intermediate endpoints that may be useful markers in hazard characterization of particulates.

Overall, despite the increasing amount of data provided by both laboratory and field studies, the nature of the fraction of aerosol particles responsible for health effects is still a matter of debate.

The issue is of importance, because the different constituents of the aerosol exhibit distinct sources and emission/ formation processes (147-148). Therefore, linking toxicological and epidemiological impacts of atmospheric particulate matter to chemical composition is a key for the evaluation of effective pollution abatement strategies (149-150).

The potential role of UFPs as strong toxicant species of ambient air derives also from the following considerations (151): 1. smaller particles have a greater total surface area per unit of mass than larger particles; thus, for a given mass, smaller particles may present a larger surface area for interacting with airway tissue or for transporting toxic material associated with the particle surface into the airways.

2. In vitro studies suggest that ultrafine particles may not be as effectively phagocytosed (ie, ingested for removal) as larger particles by cells of the innate immune response.

3. On the basis of size, models predict that a higher proportion of ultrafine particles of ~ 20 nm than of larger particles reach the air-exchanging alveolar region of the lung. On the basis of mass, however, more larger particles than smaller particles reach this lung region.

4. When particles have been instilled intra-

Role of Atmospheric Pollution on Harmful Health Effects tracheally into animals, on the basis of mass, ultrafine particles were more effective than fine particles in inducing airway inflammatory responses.

5. In some recent studies ultrafine particles appeared to move rapidly out of the airways and into the circulation.

Similarly to gases, when inhaled, specific sizes of UFPs are efficiently carried by diffusional mechanisms in all regions of the respiratory tract. The greater surface area per mass compared with larger-sized particles of the same chemistry renders NSPs more active biologically. This activity includes a potential for inflammatory and pro-oxidant, but also antioxidant, activity, which can explain early findings showing mixed results in terms of toxicity of NSPs to environmentally relevant species (150). It has not been well investigated whether nanoparticles are responsible for pulmonary and extrapulmonary health effects of PM2.5, although the fine particles are reportedly associated with mortality from cardiovascular diseases (149).

Nanoparticles can permeate through tissue walls, translocate to other tissues from the deposition sites, and cause cardiovascular dysfunction. However, we do not have a clear answer as to how far nanoparticles have a more distinctive toxicological aspect and are more toxic than larger particles (151).

The extraordinarily high number concentrations of NSPs per given mass will likely be of toxicant significance when these particles interact with cells and subcell components. Likewise, their increased surface area per unit mass can be of key importance. The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph circulation, to reach potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and heart. Access to

the central nervous system and ganglia via translocation along axons and dendrites of neurons has also been observed. NSPs penetrating the skin distribute via uptake into lymphatic channels. Endocytosis and biokinetics are largely dependent on NSP surface chemistry (coating) and in vivo surface modifications (151).

The importance of surface area becomes evident when considering that surface atoms or molecules play a dominant role in determining bulk properties; the ratio of surface to total atoms or molecules increases exponentially with decreasing particle size, as reported in table 4.3 (141).

Table 5.3. Particle number and particle surface area per 10 μg/m3 airborne particles.

Particle diameter (μm)

Particle no. (cm–3)

Particle surface area (μm2/cm3)

5

153,000,000

12,000

20

2,400,000

3,016

250

1,200

240

5,000

0.15

12

There are many debates about the dosemetric which best describes the toxicity of manufactured nanoparticles. The most commonly accepted dose metric is probably the surface area. Particle shape (e.g. fibrous or spherical), chemical composition, and the chemistry of the particle surface, including the zeta-potential, are also important factors that determine the toxicity of nanoparticles. It has been reported that the carcinogenic potency and toxicity of asbestos (151) largely depend on fiber length. Fibrous titanium dioxide particles have been shown to be much more cytotoxic than spherical nanosize titanium dioxide particles to alveolar macrophages (149). Special attention should be paid to 97

CNR Environment and Health Inter-departmental Project fibrous nanoparticles, because fiber length may be predominant metric determining the toxicity of biopersistent fibrous nanoparticles 5.3 Innovative techniques of chemical– physical characterization of the UFP fraction A major limitation of traditional impaction and membrane technologies is that the detection limits of the laboratory analysis instrumentation necessitate the collection of a large enough mass of sample for analysis, so temporal resolution is limited to greater than several hours for ambient samples. Also, the sample may be affected by evaporation or condensation of semivolatile components during or after sample collection and chemical reactions may take place within the sample itself or between oxidants in the sample gas and the collected particles, affecting the results (148). The size resolution of these instruments is generally poor to moderate because of the limitations of aerodynamic particle separation and the need to increase sampling times and analyze a larger number of samples with increasing size resolution. Nevertheless, examples exist of nano-size impactors (e.g. NanoMoudi by MSP Corporation, USA) and thermophoretic precipitators (152) which allow single particle collection aimed at analysis of UFPs chemical composition, i.e. by scanning transmission electron microscopy (STEM).

As a matter of fact, anyway, the size distribution of ultrafine particles is expected to evolve rapidly in urban air and such knowledge is essential to the evaluation of human exposure. Differential mobility analyzers (DMA) and other particle evolution / growth measurement techniques have been used since recent years to evaluate the behaviour of airborne UFPs in the ambient air, especially at 98

urban sites. In example (153), combined the use of the scanning mobility particle spectrometer (SMPS), electrical low pressure impactor (ELPI) and TEM techniques to characterize the evolution of the particle size, morphology and composition distribution during dispersion of traffic – related emissions at Birmingham (UK). The ELPI (Dekati, Finland) measures real-time particle concentration and size distribution and the SMPS (TSI, USA) measures particle size distributions with a high resolution. Combined use of these two instruments enhances the quality of particle size distribution measurements.

Measurement size range of the ELPI is 3010000 nm, 50% cut size and the SMPS is 9.6-352 nm. The analytical problem posed by the wide range of atmospheric PM sizes is dynamic range. A 1 nm diameter particle might have a mass of 10-21 g (zeptograms), whereas a 10 mm particle a mass of 10 ng.

Reliable sampling and accurate chemical composition determination of a single nanogram particle is a tough analytical challenge; for a zeptogram particle, it is nearly impossible. In addition to PM size, composition and mass loading, many other physical and chemical properties are of interest, and should be preferably measured simultaneously. Research on climate changes, i.e, needs to correlate size and chemical composition with a particle’s ability to scatter and absorb radiation from the infrared to the near ultraviolet; health scientists hypothesize that a particle’s surface area and surface composition may be a key to understanding how it interacts with lung tissue to affect pulmonary functions and transfer chemicals into the blood stream (152); finally, the physical phase (liquid or solid), surface area, and surface composition can strongly affect the interaction of atmospheric trace gases with airborne particles, impacting the chemical composition of both the gaseous

Role of Atmospheric Pollution on Harmful Health Effects and condensed phase components of the atmosphere.

Real-time instruments that measure physical properties such as particle number densities, mass loadings, and particle mobility or aerodynamic size distributions have been available since recent years.

However, real-time instruments that characterize the chemical composition of atmospheric PM, ideally as a function of particle size, are a more recent development (153). Some near real-time PM chemical composition instruments, operating with measurement cycles of 10–60 min, can characterize the average PM content of one or more key PM constituents for an ensemble of particles in a size range defined by the sample collection system.

Examples include the particle into-liquid sampler (PILS) that utilizes automated ion chromatography to quantify average major anion or cation PM content or an automated carbon analyzer to determine the water-soluble organic carbon (OC), instruments based on particle collection followed by thermal decomposition and gas phase chemiluminescence or absorption spectroscopy allow for semi-continuous measurements of sulfate and nitrate (153 and references therein). However, the universal nature of mass spectrometric detection for atomic and molecular species makes this technique eligible as most comprehensive and sensitive to characterize the chemical content of atmospheric PM. Over the past decade, several research groups have made major strides in adapting mass spectrometric techniques to meet this challenge and three major directions of evolution of mass detection techniques can be currently identified. One major theme involves the use of lasers to both vaporize and ionize individual atmospheric particles sampled into a mass spectrometer’s source region.

This class of instruments focuses on single

particle measurements. A second class of aerosol mass instruments uses thermal vaporization of individual or collected particles followed by various ionization techniques. Separation of the vaporization and ionization steps enables quantitative detection of PM chemical composition and mass loading. In addition, the simplicity of thermal vaporization allows the use of a variety of ionization techniques that will produce less sample fragmentation than traditional electron impact (EI) methods, such as chemical ionization techniques(154). However, the most widely used technique is thermal vaporization aerosol mass spectrometer (AMS), which was designed and developed at Aerodyne Research, Inc. (ARI). The initial version of the ARI AMS was designed to measure the real-time non-refractory (NR) chemical speciation and mass loading of fine aerosol particles with aerodynamic diameters between <50 and 1000 nm as a function of particle size155. The original ARI AMS utilizes a quadrupole mass spectrometer (Q) with EI ionization and produces ensemble average data of particle properties. Later versions employ time-offlight (ToF) mass spectrometers and can produce complete mass spectral data for single particles (153).

6. FUTURE

PERSPECTIVES AND DEVELOPMENTS

IN THE FRAMEWORK OF THE

“PILOT STUDY FOR

THE ASSESMENT OF HEALTH EFFECTS OF THE CHEMICAL COMPOSITION OF ULTRAFINE AND FINE PARTICLES IN ITALY” PROJECT

Despite the increasing amount of data provided by both laboratory and field studies, the nature and role of aerosol particles responsible for health effects, as well as of gaseous mixtures especially in urban areas, is still a matter of debate. In the former case the issue is of importance also because the different constituents of 99

CNR Environment and Health Inter-departmental Project the aerosol exhibit distinct sources and emission/formation processes. Therefore, linking toxicological and epidemiological impacts of atmospheric particulate matter to chemical composition is a key for the evaluation of effective pollution abatement strategies.

Results of size-segregated aerosol chemical analyses for Italian stations have already been published during the last ten years and are available in the peerreviewed literature and in project reports (40). These data generally refer to sparse measurements employing multi-stage impactors in both urban (e.g, Bologna, Catania, Rome) and rural/background sites (e.g, Monte Cimone); information on the inorganic and organic composition of ultrafine to coarse particles have been retrieved by chemical determinations of size-segregated fractions.

Nevertheless, an increasing series of data on the aerosol chemical composition and size-distribution has been provided by short-term intensive field studies performed in the frame of national and European research projects(46). During these experiments, state-of-the-art instrumentation has been deployed for aerosol characterization.

In this direction, the â&#x20AC;&#x153;Pilot study for the assessment of health effects of the chemical composition of ultrafine and fine particles in Italyâ&#x20AC;? project, recently approved by CNR, will combine the results of two advanced activities in the field of atmospheric ultrafine particles composition and their toxicological properties, carried out by CNR-ISAC and CNR-IIA, with two new advanced health studies carried out by CNR-IFC and CNR-IBIM, aimed at exploring short-term effects due to air pollutants exposure in subjects with preexistent arrhythmia and lung diseases.

As far as CNR-ISAC and CNR-IIA are 100

concerned, in the project a collection of all these data will be performed and a data-base of size-resolved chemical compositions of the aerosol classified according to site characterization and sampling period will be provided as outcome. The databases emerging from the above activities will be further integrated and the results evaluated to derive conclusions on the available knowledge on the size-segregated chemical composition of the aerosol in the different environments explored. In this view, a further step forward will be also to identify systematic behaviours in the contributions of inorganic and organic chemical constituents as a function of particles size, and depending on site classification (urban, sub-urban, rural, marine, high-altitude, etc.), as well as to provide a summary of the constituents of ultrafine particles. This will help interpreting clinical and epidemiological observations under an enhanced awareness of the behaviour of particulate pollutants in different environments. Finally, a comparison with published results of analogous measurements performed in other European countries will be carried out, to identify singularities to be further investigated in the future. New directions of research in the field of understanding impact routes of ambient and indoor air on health relate to selected species showing a precise toxicant action and to their size-segregated behaviour in aerosols. In this view, research activities will be run concerning either the characterization or the toxicant and reactive behaviour of the water-soluble organic fraction of PM. Besides transition metals and PAHs, on which some peer-reviewed literature already exist, the chemical analysis of fine particulate samples has shown that even in urban areas the water-soluble fraction of the aerosol contains large amounts of

Role of Atmospheric Pollution on Harmful Health Effects poorly-characterized organic compounds (WSOC, “water-soluble organic carbon”), in contrast to the paradigm of many toxicological studies which attributes the organic-soluble and water-soluble fractions of the aerosol to organic and inorganic compounds, respectively.

On the contrary, recent findings point to WSOC as a major agent for aerosol toxicity and oxidizing properties (56,116).

Although a number of bioassays have been adopted in previous studies to provide fast and sensitive measurement of the aerosol chemical reactivity, mechanistic pathways for toxicity were not established.

Relevant bioassays will be thus tested, for the scopes of evaluating the oxidative potential of airborne aerosol in humans and animals. Among possible bioassays, those will be selected which are sensitive to reactive oxygen species, like superoxide and hydrogen peroxide. The latter species, indeed, can be produced in biological liquids and tissues by organic compounds via redox reactions. Such tests include for instance those based on dithiothreitol (DTT) consumption rate, or employing dichlorofluorescin (DCFH) (56,156). The key importance of testing these methods to provide, i.e, an optimal application to the analysis of the water-soluble organic extracts of ambient aerosol samples is a matter of evidence. Finally, a stateof-the-art analytical technique like the Aerosol Mass Spectrometry (AMS) will be employed for the quantitative mass determination of UFPs. This is at date an obligate step towards enhancing the knowledge about responsibilities of the atmospheric pollution on health impacts.

Indeed, the AMS is currently the only technique providing unique information on the short-term processes controlling the concentration and composition of ultrafine particles and their interaction with larger

particles. Moreover, the data analysis of the emerging results from AMS will allow comparison with the more consolidated outcomes from available measurements by multi-stage impaction methods.

In summary, by examining the priorities for the evaluation of upcoming research activities of CNR for linking atmospheric aerosols composition and properties to their health effects, at least two specific key issues can already be addressed and dedicated to a) ultrafine particles and b) WSOC. Keywords: PM, UFP, WSOC, AMS, oxidizing potential.

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110

Human Biomonitoring E. Leonia,b, A. I. Scovassia

a. CNR, Institute of Molecular Genetics (IGM), Pavia, Italy b. Salvatore Maugeri Foundation, Pavia emanuelaleoni@gmail.com

ABSTRACT Human biomonitoring is the discipline devoted to the identification of biomarkers useful to measure environmental exposure, to monitor its biological effects and causal relationship with pathological conditions, and to possibly define the genetic susceptibility of the general population. The search for reliable biomarkers, i.e. characteristics that are objectively measured and validated as health or disease requires the expertise of scientists with diversified specializations, who are able to tackle problems of ever increasing complexity using complementary approaches. Within the frame of the PIAS-CNR project “Environment and Health”, we have constituted a Biomonitoring network, composed by highly qualified scientists from CNR, and external teams. This action aims at promoting a scientific strategy to develop and validate biomarkers of effect, exposure and susceptibility.

1. INTRODUCTION 1.1 Background Chemicals are present in the air, ingested

food and water, at the workplace as well as at home; the exposure to them can occur through inhalation, cutaneous contact, and ingestion. The evaluation and measurement of the impact of chemicals on human health is achieved by “Human Biomonitoring” (HBM), defined as the “systematic standardized measurement of substance or its metabolites in body fluids (blood and urine) of exposed persons” (1-4). The different steps of HBM ranging from the exposure step to the health impairment (and eventually disease occurrence) are schematized in Figure 1.

The estimation of the dose really taken up after the exposure (internal dose) can be addressed by qualitative and quantitative assays able to detect chemicals and/or metabolites in biological fluids; this action provides basic information to identify

exposure biomarkers.

The HBM survey is further extended to the analysis of biochemical and biological effects: as illustrated in Figure 1, biochemical effects could be monitored through the reaction of reactive substances (or their metabolites) with biological macromolecules, such as DNA and proteins. The study of biological effects induced by chemicals implies cellular, cytogenetic, genetic, biochemical, metabolic and immunological approaches.

This strategy aims at defining a panel of effect biomarkers. Furthermore, it is widely recognized that biomarkers of susceptibility may influence each element in the exposure-to-disease paradigm (Figure 1). This class of biomarkers refers to individual factors (e.g. genetic or metabolic) that influence the sensitivity to hazardous compounds. Markers that are measurable at low exposure or dose, have the greatest potential utility to prevent disease, while later markers are

CNR Environment and Health Inter-departmental Project

Figure 1: The exposure-to-disease paradigm. most closely related to the endpoint of risk about the link between blood mercury and dioxin and fish diet (6). However, to better exposure, that is disease development (5).

As discussed in the special issue of the exploit these results, a harmonised strategy Bulletin Epidémiologique Hebdomadaire allowing comparisons between countries (6), HBM can help set priorities for public is required. health and regulatory follow-up. In fact, The selection of chemicals allowed to periodic measurement of biomarkers in enter a biomonitoring program is very the population reveals how body burdens complex. The Centre for Disease Control of chemicals vary from season to season, and Prevention (CDC) has identified a year to year and decade to decade; by number of important variables able to comparing the results over the time it is influence the so-called process, including possible to evaluate the trend of people’s the evidence of exposure, the presence exposure to environmental chemicals. and significance of health effects after a Large biomarker studies can highlight given level of exposure, the development exposure differences among racial, of assays for accurately measuring geographic or socioeconomic groups. As biological concentrations of the chemical specified in the above document (6), it is agent, available specimens, in particular, urgent to define the association between the blood and/or urine samples, and costlevel distribution of chemicals in humans and effectiveness. geographic/social/demographic parameters, As reported in the literature, many in order to generate exposure maps and to chemicals released into the environment can promote better-targeted risk assessment disturb the development of the endocrine and risk management actions. A number of system and of the organs that respond to examples demonstrated that a policy change endocrine signals and can interfere with can reduce people’s exposure to pollutants, the correct functioning of endocrine e.g. the decision of way out leaded petrol organs. Such chemicals are generally because of its established association with named “Endocrine Disruptors” (EDs) (7). blood lead, the information campaigns EDs causing abnormalities and impaired 112

Human Biomonitoring reproductive performance in some species, are associated with changes in immunity behaviour and skeletal deformities and are responsible for apparent changes observed in human health patterns over recent decades, including an increased incidence of certain types of hormone-sensitive cancers (7,8).

1.2 State of the art The exposure of the general population to xenobiotics through different routes is a matter of growing concern. To face this problem, the European Commission adopted in 2003 a Strategy on Environment and Health (9). Then, the EU launched the “2004-2010 Environment and Health Action Plan”, designed to give the EU the scientifically grounded information to reduce the adverse health impacts of certain environmental factors and to endorse better cooperation among actors in the environment, health and research fields (10,11). The final goal of the Action Plan is to promote and integrate environment and health information, and to identify emerging issues, reviewing and adjusting risk reduction policies and improving communication. Of course, this action implies the development of a HMB policy aiming at monitoring activities in human beings, using biomarkers, which focus on environmental exposures, diseases and/or disorders and genetic susceptibility, and their potential relationships (12).

The mid-term review of the action plan pointed out the difficulty of collecting and comparing HBM data from different countries, given that implemented methodologies and sample collection protocols differ (13). In 2007, the EU Council invited the Commission to ensure adequate funding for the EU pilot project on HBM, to pay attention to the existing regulatory frameworks and, most important thing, to

demonstrate the added value of HBM as policy tool and support to public health interventions (14). Despite the big effort of the EU action, experiences of HBM programmes at country level reveal the need for harmonisation. In this respect, last December, in Brussels, 35 partners coming from 27 European countries and including governments, research institutes, the Health and Environment Alliance (HEAL) and the European Chemical Industry Council (CEFIC) established a consortium to perform human biomonitoring at European scale (COPHES) (15). This consortium aims at developing a functional framework that contributes to the definition, organization, and management of a coherent approach towards HBM in Europe, including strategies for data interpretation and integration with environmental and health data. A further priority of COPHES is to regulate data communication and dissemination and to provide key information to all stakeholders from the public to the policy markers. The WHO 5th Environment and Health Ministerial Conference (Parma, March 10-12, 2010) is the next milestone in the European environment and health process, now in its twentieth year. Focused on protecting children’s health in a changing environment, the Conference will drive Europe’s agenda on emerging environmental health challenges for the years to come (16).

HBM aims at estimating people’s exposure to pollutants, thus providing information about possible health effects and options of policy measures to reduce exposure.

Persistent organic pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food, and pose a risk of causing adverse effects to human health and the environment.

Priority POPs include pesticides (such 113

CNR Environment and Health Inter-departmental Project as DDT), industrial chemicals (such as polychlorinated biphenyls, PCBs) and unintentional by-products of industrial processes such as dioxins (PCDDs) and furans (PCDFs). Pesticides, PCBs, PCDDs, PCDFs, and other emerging substances have been detected in foodstuffs and are potentially toxic to human health. In addition to the diet, indoor and outdoor air pollutants are possible sources of health risk; however, available data are scarce and inconclusive. POPs are transported across international boundaries far from their sources, even to regions where they have never been used or produced. For most of these chemicals, we simply do not know how they pass through the environment, whether they are accumulated, dispersed or transformed, and how they affect living organisms at different concentrations (17).

Consequently, POPs pose a threat to the environment and to human health (18).

Among POPs, some trace elements (TEs) e.g. cadmium, arsenic, lead, mercury and nickel, are very dangerous for health because they tend to bioaccumulate, thus enhancing their concentration in a biological organism over the time. TEs may enter the human body through food, water, air, or absorption through the skin when they come in contact with humans in agriculture and in manufacturing, pharmaceutical, industrial, or residential settings. Human exposure to TEs has risen dramatically in the last 50 years as a result of an exponential increase in their use in industrial processes and products.

In general, TEs are systemic toxins with specific neurotoxic, nephrotoxic, and teratogenic effects; they can induce impairment and dysfunction in excretive organs (colon, liver, kidneys, skin), endocrine and energy production pathways, enzymatic, gastrointestinal, immune, nervous, reproductive, and 114

urinary apparatus (19).

A special class of toxicants is represented by Endocrine Disruptors (EDs). In 1991, at the Wingspread Conference, expert scientists at a work session on endocrine-disruptors concluded that â&#x20AC;&#x153;Many compounds introduced into the environment by human activity are capable of disrupting the endocrine system of animals, including fish, wildlife, and humans. Endocrine disruption can be profound because of the crucial role hormones play in controlling developmentâ&#x20AC;? (20). EDs interfere with the normal function of endocrine system and can exert adverse effects on the reproductive and other, indirectly related, physiological systems.

EDs are natural or synthetic compounds, derived primarily from anthropogenic activities, ubiquitous in the environment.

Many of them are resistant to biodegradation, due to their structural stability, and persist in the environment.

EDs present in the environment include a variety of potent human and veterinary pharmaceutical products, personal care products, nutraceuticals and phytosterols.

Substantial research has been carried out on the mechanisms and effects of EDs. Nevertheless it is still unclear to what extent and/or in what situations and population subgroups EDs may represent a significant, long-term health risk.

To monitor the potential environmental and health impacts of EDs, the EU adopted a Communication to the Council and European Parliament on a Community Strategy for EDs in December 1999 (21). In the proposal for a new policy for chemicals (Registration, Evaluation, Authorisation and Restriction of Chemicals, REACH), EDs are covered by the authorisation procedure for substances of very high concern. Despite the joint efforts of many organizations, there is still a need for agreed

Human Biomonitoring test methods that can confirm whether or not â&#x20AC;&#x153;identified candidatesâ&#x20AC;? (more than 500 compounds) are real EDs. As stated also by the American EPA (Environmental Protection Agency), validated methods able to evaluate specific effects of EDs are still being developed (22). Remarkably, studies on environmental/occupational exposure to EDs and reproductive risks have been recently carried out, aiming at dissecting the impact of these chemicals on fertility. Exposure in utero at critical developmental periods may modify the normal path of reproductive and genitourinary development (23,24), and induce hypospadia that is the most frequent genital malformations in the male newborn and results from an abnormal penile and urethral development (25).

2. FROM BASIC TO APPLIED RESEARCH HBM fits with the scope of translational research given that the expected results include the validation of conventional/ new effect and exposure biomarkers, the elucidation of the biomolecular mechanisms of action of selected toxicants, the identification of genetic susceptibility markers in the Italian population, and the definition of the criteria leading to the evaluation of real exposure to toxicants.

In the early 1960s, powerful analytical techniques allowed to measure very low concentrations of chemical substances in biological tissues caused by environmental exposure. Due to the improvement of these techniques essentially through the effort of basic research, it is now possible to detect very low concentrations of agents (parts per trillion and parts per quadrillion) with a high degree of accuracy and precision. This general consideration implies that measurement procedures need a continuous validation

and that up-to-dating of basic knowledge of biological effects of chemicals could help in the development of new procedures of risk assessment evaluation. The analysis of benzene exposure, for instance, has taken advantage from the old evidence that trans,trans-muconic acid represents the urinary metabolite, thus prompting scientists to develop an ad hoc assay (26).

Many disciplines acquired a growing and growing relevance in biomonitoring, e.g. biophysics, whose contributions have gone well beyond the mere application of physical techniques to the study of living systems. Biophysics plays a crucial role in the development of new methodologies and establishes closer links with other frontier areas of the biological and medical sciences (structure-function relations in biological molecules, molecular biology, bioenergetics, bioinformatics).

This evolution has widened the range of skills required by the individual researcher and has increased the need for teams with diversified specializations.

Given that biomonitoring is not confined to the exposure to toxicants but covers also the identification of effect and susceptibility markers, the final goal of HBM is to define a toxicogenomic approach, considered as an integration of genomics (transcriptomics, proteomics and metabolomics) and toxicology. This scientific field investigates how the genome is involved in responses to environmental stressors and toxicants.

It combines studies of mRNA expression, cell and tissue-wide protein expression and metabolomics, to understand the role of gene-environment interactions in disease. One of the important aspects of toxicogenomic research is the development and application of bioinformatics tools and databases in order to facilitate the analysis, mining, visualizing and sharing of the vast amount of biological information 115

CNR Environment and Health Inter-departmental Project being generated in this field. This rapidly growing area promises to have a large impact on many other scientific and medical disciplines as scientists could now generate complete descriptions of how components of biological systems work together in response to various stresses, drugs, or toxicants. Of course, this approach requires the joint effort of a panel of experts, who accumulated a solid experience through basic research, and can transfer it to health applications. This is the case of the teams involved in the PIASHBM project, which are characterized by complementary expertise and capability to cope basic and translational research.

3. THE CNR

SPECIFIC EXPERTISE: QUALIFIED

TEAMS AND EXTERNAL COLLABORATIONS

3.1 CNR Institutes The coordination of the HBM network has been entrusted to the Institute of Molecular Genetics (CNR-IGM, Pavia).

CNR-IGM research activity covers a wide range of biological, biochemical and genetic topics, and ensures the participation of most CNR-IGM scientists to the HBM project. This feature, i.e. the potential commitment of a whole CNR Institute to the Environment and Health Inter-departmental project PIAS, has rendered CNR-IGM especially suitable for the coordination of the HBM group. The HBM coordinator (Giuseppe Biamonti, the present Director of CNR-IGM, then replaced by A. Ivana Scovassi) was in charge to identify CNR Institutes with the expertise required to join CNR-IGM in order to establish a PIAS-HBM network based mainly on the analysis of the effects of EDs (see below).

CNR-IGM is mainly devoted to basic research on the control of cell proliferation, DNA replication and apoptosis in 116

human cells; viral replication; analysis of hereditary genetic disorders with defects in DNA damage repair pathways, chromosome X-linked diseases and muscular dystrophies; post-transcriptional regulation of gene expression during cell response to stress treatments and tumor progression, and analysis of the genetic structure of human populations. Longlasting interactions and collaborations within the CNR-IGM team members are attested by joint peer-reviewed publications. The scientific excellence is completed by a qualified translational research in collaboration with SMEs, which originated a number of patents.

CNR-IGM researchers are active in the development of new techniques, protocols and instruments as well as in the identification and characterization of new compounds with therapeutic properties.

Considerable effort is put in the training of undergraduate, graduate and post-doctoral students. A group at IGM was recently investigating the biological effects of toxic metals and demonstrated the activation of stress response mechanisms after cadmium administration (27-29).

The Institute of Biophysics (CNR-IBF, Genoa) covers a wide range of research fields, sharing as a common feature the application of typical methodologies and techniques of the physical sciences to develop interdisciplinary approaches to the study of the structure and functions of biological systems. A relevant interest concerns physico-chemical investigations of the impact of anthropic and nonanthropic environmental factors on ecosystems. CNR-IBF devotes much effort to the training of young people for research in the fields of Biology and Biophysics, in close collaboration with local universities.

The team involved in PIAS-HBM has a solid

Human Biomonitoring research experience in electrophysiology and ion channel biophysics in nervous and endocrine culture cells investigated by patch-recording and voltage-clamp techniques, and intracellular calcium dynamics, monitored by fluorescent probes. The group studies heavy metal accumulation and toxicity in mammalian cells and modulation of neurotrasmittergated ion channels. The effect of acute and chronic treatment with toxic metals (lead, cadmium and nickel) on cell survival and maturation of neurons in culture is currently analyzed by functional and viability tests and apoptosis/necrosis measurements (3036).

The Institute of Biomedicine and Molecular Immunology (CNR-IBIM, Palermo) is involved in research activity, technological transfer and training in the following areas: molecular, cellular and morphological study of early embryo development and mechanisms involved in the differentiation and in the degenerative mechanisms of eukaryotic cells; molecular study of proteins involved in the allergic reaction; synthesis and characterization of bioactive molecules; pathophysiology of the cardio-respiratory system, lung diseases, systemic hypertension and renal failure insufficiency; organ transplantation; bioeffects of magnetic fields; epidemiology.

An CNR-IBIM team developed the concept that sea urchin embryos as a new friendly model for ecotoxicological studies (37-41). Indeed, marine organisms are highly sensitive to many environmental pressures, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms and for application in environmental studies. Sea urchin

represents a simple though significant model system where to test: 1) the impact on the biology of development in association with gene expression on embryos, and 2) the effects on gene expression and DNA damage on adult immuno-competent cells, which are contained in the coelomic cavity of the adult sea urchin, generically called coelomocytes, studied since many decades, but only recently used as bio-indicators of stress. Due to the capability to respond to injuries, host invasion and cytotoxic agents, coelomocytes have been regarded as the immune effectors of the sea urchin.

Another CNR-IBIM group investigates the impact of environmental substances both on hormone metabolism and nervous system, focusing on the identification of molecular mechanisms at the basis of neurodegenerative diseases, included some retinopathies. The group carries out in vitro studies to elucidate the effect of target chemicals on neurosteroidogenic pathways and the interactions of these chemicals with ER and AR receptors. On the other hand, in vivo analyses using animal models (mice) could be helpful in elucidating the effects in age-related neurodegenerative disorders. RT-PCR, receptor binding assay, recombinant mammalian and yeast cell based transcription assay, western blot, immunocitochemical and histochemical, proteomic and functional genomics are currently used.

The Institute of Clinical Physiology (CNR-IFC, Pisa), the largest biomedical institute of the CNRâ&#x20AC;&#x2122;s Department of Medicine, whose mission is â&#x20AC;&#x153;Innovation for better patient careâ&#x20AC;?, is involved in the study of systemic, neuroendocrine and metabolic factors implicated in many diseases.

Molecular medicine, clinical biology, and clinical biochemistry are devoted to the study of experimental physiology, and 117

CNR Environment and Health Inter-departmental Project to the relevant diagnosis and treatment.

CNR-IFC is a leader institute in the field of clinical and environmental epidemiology, population registers, and research on health services. CNR-IFC researchers established a network of scientific collaborations with many Italian and international Institutions.

CNR-IFC was the first public body in Italy to achieve the status of pharmaceutical developer (“Officina farmaceutica”) with its own production site for injectable sterile radiodrugs with Good Medical Practice certification. This achievement was made possible through close collaboration with a leader Company in the field of biomedical technology and diagnostic imaging, which signed a contract for the production and distribution of radiodrugs for diagnosis in Positron Emission Tomography.

The Epidemiology unit works on epidemiological surveillance, air pollution and health, waste and health and is involved in the national strategic programme “Environment and Health”, funded by the Ministry of Health and coordinated by ISS. Human biomonitoring represents the priority research area of this unit (42,43).

IFC has a long-standing experience in the field of surveillance and research on congenital malformations, as coordinator of the EUROCAT and ICBDSR “Tuscany Registry of Birth Defects”. To investigate the correlation between EDs exposure and reproductive dysfunctions, malformations such as hypospadia or cryptorchidism, reduced sperm counts, testicular cancer and endometriosis, are studied (44).

3.2 External collaborations Each CNR team collaborates with a number of Italian and international Institutions, including Universities, the Scientific Institute for Research, Hospitaization and Health Care (IRCCS), the National Institute of Health (ISS), the World Health 118

Organization (WHO) and SMEs operating in the biomedical/biotechnology field.

To increase the competences of the CNR Biomonitoring team, external collaborators, i.e. Salvatore Maugeri Foundation (FSM, Pavia), Perrino Hospital (Brindisi) and National Institute for Occupational Safety and Prevention (ISPESL, Rome) have been added to the CNR network on the basis of their pre-existing cooperations with CNR Institutes and taking into account their complementary expertise.

FSM is a leading institution in the field of the occupational health and prevention, including biological and environmental monitoring of exposure, reference values setting and prevention of occupational risks. The prevention of occupational risks activity is supported by the Laboratory for Environmental and Toxicological Testing, which has been working for decades on environmental monitoring, with respect to occupational exposure to both organic and inorganic substances (risk assessing and sampling techniques), and on the development of new biomarkers and analytical techniques aiming at the evaluation of occupational and environmental exposure to xenobiotics.

This laboratory is fully equipped to develop and validate analytical methods for the determination of trace elements (ICP-MS), organochlorinated compounds such as PCB and DDTs (HRGC-MS), phthalate metabolites (HPLC-MS/MS), and other emerging substances in biological and environmental matrices, including foodstuffs. The Unit has recently validated reliable methods for the determination of EDs in biological fluids and in foodstuffs (45-48).

ISPESL is a technical-scientific body in the National Health Service and reports to the Ministry of Health as regards all aspects of

Human Biomonitoring Table 1: PIAS Human biomonitoring network.

CNR INSTITUTE (DEPARTMENT)

Group leader

Expertise

Patrizia Guarneri

Impact of EDs on steroid hormone metabolism and neurodegenerative conditions

Valeria Matranga

IBF-CNR, Genova (Materiali e Dispositivi) http://www.ibf.cnr.it

Validation of biosensor methods for the analysis of the effects of stress conditions on marine invertebrates

Carla Marchetti Gianfranco Prestipino

Identification of the mechanisms of action of toxic metals in mammalian nervous cells

IFC-CNR, Pisa (Medicina) http://www.ifc.cnr.it

Anna Pierini Fabrizio Bianchi

Epidemiological studies on the impact of chemicals on human health

IGM-CNR, Pavia (Medicina Scienze della Vita) http://www.igm.cnr.it

Ivana Scovassi

Identification of effect biomarkers (in vitro and in vivo approaches)

Fondazione Salvatore Maugeri, Pavia http://www.fsm.it/

Claudio Minoia

Biomonitoring of toxicants and their metabolites in biological fluids from exposed people; total diet studies

Ospedale Perrino, Brindisi http://www.asl.brindisi.it/

Giuseppe Latini

Biomonitoring of phthaltes: impact on infants and human fertility

ISPESL, Roma http://www.ispesl.it/

Elena Sturchio

Model systems: C. elegans; DNA biosensors; miRNA platform

IBIM-CNR, Palermo (Medicina Scienze della Vita) http://www.ibim.cnr.it

OTHER INSTITUTIONS

occupational safety, health and prevention.

Among the various activities carried out by ISPESL, of interest are research, analysis, experimentation and drafting of criteria and methodologies for the prevention of accidents and professional diseases, identification of safety criteria, prevention against chemical, physical and biological exposure risks at work, standardization of test to evaluate occupational safety and health risk assessment.

The ISPESL Department of Production Plants and Anthropic Settlements (DIPIA), carries out research, experimentation, consultation, assistance to the enterprises, proposal of rules, laboratory controls, standardization of methods and procedures of evaluation, analysis of the systems for purposes of safety and environmental compatibility connected to the interaction between the production premises and

the external environment. DIPIA is also concerned with the complex problems arising from biotechnologies, particularly with the safety and risk assessment of contained use of genetically modified microorganisms and the evaluation of genetically modified organisms safety and their traceability in food and feed products. DIPIA staff carries out many projects supported by Ministry of Health on eco-genotoxicity studies, biomonitoring of polluted sites with toxicity testing, and molecular analysis; it focuses on indicators to detect the state of environmental health after a release of pollutants from â&#x20AC;&#x153;falloutâ&#x20AC;?. To develop potential biomarkers of susceptibility, toxicity tests and plant and animal models are employed, including the nematode C. elegans, where toxicant effects on phenotype, reproduction, apoptosis and micro RNA profiles are tested. About this 119

CNR Environment and Health Inter-departmental Project latter point, the analysis of miRNAs for the detection of early indicators in various diseases is a distinctive feature of the PIAS-HBM team.

The Director of the Neonatology unit at the Perrino Hospital (Brindisi) has recently joined the EU pool of experts of risk assessment. His main scientific interest is the study of the effect of phthalates, to whom general population is exposed through consumer products, as well as diet and medical treatments. In fact, animal studies showing the existence of an association between some phthalates and testicular toxicity have generated public and scientific concern about the potential adverse effects of environmental changes on male reproductive health. In addition, prenatal exposure to phthalates seems to play a relevant role in determining these adverse effects given that human exposure has been demonstrated to begin during the intrauterine life. A link between antenatal phthalate exposure and abnormal fetal development exists, thus justifying the need of therapeutic tools to fight the adverse effect of this exposure.

Numerous maternal lipophilic compounds are eliminated into milk during lactation, their concentrations reflecting fetal in utero exposure. The reported effects of the exposure to phthalates through breast milk in infants confirm that human milk may represent an additional potential source of phthalate exposure in a population at increased risk (49-51). This research is made in collaboration with CNR Institutes.

The members of the â&#x20AC;&#x153;PIAS-HBM networkâ&#x20AC;? are listed in Table 1, which reports also the specific (and unique) expertise of each team.

120

4. RELEVANT FINDINGS The CNR-IBF team has depicted the molecular mechanisms of nickel and lead toxicity in neural cells. Focusing on the inhibition of N-methyl-D-aspartate receptor (NR) channel in a voltage-dependent manner, the group recently identified specific heavy metal interaction with NR channels (30) and defined the relevance of NR composition in modulating the effect of toxic metals (31). The survey of lead effects represented the first structural work addressing the location of lead interaction site on NMDA receptor channel, thus providing original electrophysiological data (30); the new results obtained for nickel allowed the characterization of its involvement in synaptic currents and transmission (31).

At CNR-IBIM, it has been established the suitability of sea urchin as a sentinel organism for the assessment of the macro-zoobenthos health state in biomonitoring programmes. A recent survey of sea metal contamination around Pianosa and Caprara Islands revealed that sea urchin coelomocytes might be used as biosensors of environmental stress (52,53).

This observation further supports the evidence that sea urchin as well as marine invertebrates are useful as bioindicators of environmental stress.

The SEBIOMAG project represents a good example of an integrated approach to HBM. SEBIOMAG (Studio Epidemiologico Biomonitoraggio Area di Gela) was promoted by World Health Organization, coordinated by Fabrizio Bianchi (CNR-IFC), and carried out in collaboration with the Laboratory for Environmental and Toxicological Testing at FSM (Pavia). The project was conceived as the biomonitoring

Human Biomonitoring Table 2: Published monographs of relevant EDs. ED

Authors

HBM teams

Reference

Bisphenol A

Minoia C, Leoni E, Turci R, Signorini S, Moccaldi A, Imbriani M

FSM

[55] G Ital Med Lav Erg 2008; 30:214-24.

PFOS, PFOA

Minoia C, Leoni E, Sottani C, Biamonti G, Signorini S, Imbriani M

FSM IGM

[56] G Ital Med Lav Erg 2008; 30:309-23.

Sturchio E, Minoia, Zanellato M, Masotti A, Leoni E, Sottani C, Biamonti G, Ronchi A, Casorri L, Signorini S, Imbriani M

FSM IGM ISPESL

[57] G Ital Med Lav Erg 2009; 31:5-32.

FSM IFC ISPESL

[58] G Ital Med Lav Erg 2009.; 31:325-70.

Arsenic

PCDDs

Turci R, Minoia C, Leoni E, Sturchio E, Boccia P, Meconi C, Zanellato M, Signorini S, Benzoni I, Mantovani A, La Rocca C, Bianchi F, Imbriani M

of the exposure of target population living in Gela (Sicily). The study consisted of the evaluation of toxicant levels in biological fluids, focusing on trace elements (antimony, arsenic, beryllium, cadmium, mercury, lead, copper, selenium, thallium, vanadium) and organochlorine compounds (PCB, aldrin, dieldrin, DDT, chloroesane, chlorobenzene). Trace elements and organochlorine compounds were measured by inductively coupled plasmadynamic reaction cell-mass spectrometry (ICPDRC-MS) and gas chromatography coupled to mass spectrometry (GCMS), respectively. The target population consisted in 184 subjects aged 20-44 years living in Gela (116), Niscemi (39) and Butera (29). The analysis revealed that the levels for organochlorine compounds, antimony, selenium, thallium, beryllium and vanadium were comparable to control population. The most relevant finding was the peculiar profile of widespread exposure to arsenic recorded in blood, plasma and urine samples, where the values were higher than reference values of nonexposed subjects. In conclusion, the results underlined a widespread exposure to

arsenic, and recommended the evaluation of different forms of arsenic (inorganic arsenic, arsenobetaine, arsenocholine) to better understand the potential sources of exposure (environmental, seafood intake) in this crucial area. The results of this study have been presented in Gela, on July 16, 2009, at the SEBIOMAG meeting.

Remarkably, this analysis represents an added value to a previous survey conducted on the same geographical area and providing environmental data about contaminants present in water, earth and air. These data are reported in the special issue of the journal Epidemiology & Prevention devoted to â&#x20AC;&#x153;Environment and health in Gela (Sicily): present knowledge and prospects for future studiesâ&#x20AC;? (54). The joint effort to evaluate the environmental contamination of an industrial area, and the consequent exposure of the general population, is an example of an efficient strategy to combine clinical and research competences for improving the experimental approaches useful to depict the effects of environmental risk on human health.

121

CNR Environment and Health Inter-departmental Project Of note, some members of the network cooperate for preparing toxicological information profiles of specific chemicals.

Based on a huge amount of literature data, these reviews provide relevant scientific information actually on EDs, including bisphenol A, perfluoroalkyl agents (i.e.

PFOS/PFOA), trace elements (i.e. arsenic) and dioxins (i.e. PCDDs) (55-58). The papers, listed in Table 2, are published on the “Giornale Italiano di Medicina del Lavoro e Ergonomia”, that is the official Journal of FSM. Each paper includes general aspects of exposure source, chemical and physical properties, metabolism, toxicological and carcinogenic potential, food intake and diet exposure, mechanism of action, genetic susceptibility, analytical procedures and general population levels.

This body of information represents a very useful tool not only for clinicians but also for researchers in the environmental and occupational context. In fact, even if extensive toxicity data for a chemical are available, they are almost always in a form that is difficult to combine with biomonitoring-generated exposure values to assess risk (15).

An example of scientific literature that aims at making attractive and understandable to the general public a so complex topic as Environment&Health is represented by the recent book “Ambiente e salute: una relazione a rischio. Riflessioni tra etica, epidemiologia e comunicazione” by the scientists from the CNR-IFC Epidemiology unit (59). The book uses a number of case studies to define the concept of epidemiology, population study, genetic susceptibility, risk complexity, scientific communication, and future perspectives in risk assessment.

The hot topic “Environment&Health” is discussed at different levels, that is ethics, 122

epidemiology and communication also in a recent publication (60).

5. FUTURE PERSPECTIVES AND DEVELOPMENTS In the last years there has been a call for increased epidemiological and experimental research to substantiate the disruptive effects of environmental chemicals in humans. This is currently recognized as an important issue of concern in the protection of human environmental health.

Human biomonitoring is an important tool to evaluate the internal exposure through the environment and to provide early warning indicators for possible long-term adverse effects. Biomonitoring has two main goals: i) determination of the levels of toxicants in biological fluids from a general population; ii) search for new exposure, effect and susceptibility biomarkers.

The here described PIAS-HBM network combines solid clinical expertise with advanced research work. With an adequate funding, it could act in a coordinated way to address the following crucial points: 1. To handle the Environment&Health problem with a multidisciplinary approach, combining medical tools with a biological approach based on biochemistry, biophysics, cell and molecular biology, bioinformatics, molecular genetics and genomics. This goal could be achieved through a strict cooperation between the different members of the network, sharing the respective competences and working in tight association.

2. To validate conventional/new exposure biomarkers. The growing number of chemicals potentially toxic for human health implies a continuous effort to develop new specific assays and to

Human Biomonitoring validate them; in parallel, conventional procedures have to be periodically checked for their efficacy by different laboratories. As for exposure markers, it could be of interest to share biological samples collected at Hospitals and IRCCS in order to increase the number of selected biomarkers. As a new perspective, it could be useful to evaluate the real exposure of the general population through the diet, which is considered to be the main source of body burden of several contaminants. This approach, based on a validated protocol that estimates the dietary intake of compounds, could contribute to face the lack of data on the real exposure to several contaminants and to develop ad hoc protocols to measure their levels in food. Quantification of the risk by the ingestion of pollutants in food is complex and depends on many factors (species, diet composition, duration of exposure, efficiency of pollutant absorption, subsequent metabolism, sensitivity of target organs and stage of development). While the effects of high doses of single chemicals are proven, dietary exposure generally involves prolonged, low-level exposure to a large number of compounds, each of which has different chemical characteristics, different biological effects and is present at varying concentrations.

3. To validate conventional/new effect biomarkers. The search for effect biomarkers implies a panel of approaches, spanning from cellular to molecular biology, focusing on the effects of chemicals in terms of cell proliferation, cell death, signal transduction, and neurodegeneration.

The PIAS-HBM team possesses the

ability to focus on the dissection of the biochemical steps of basic biological processes, such as DNA replication and repair, transcription, translation and post-translation. In vivo assays on cell lines of different origin (e.g. neuroendocrines, neurals, fibroblasts, keratinocytes, transformed) may allow the identification of the effect of chemicals on different cellular (adhesion, cell cycle, proliferation and death, motility) and biochemical (DNA damage, electrophysiology, inflammation, mitochondrial metabolism, neurotoxicity, ionic transport, replication and repair, oxidative stress) parameters. Also the analysis of the impact of chemicals on different classes of receptors (e.g.

ER alpha/beta, AR, PR, GR, ThRs, retinoic acid, aryl-AhR, pregnaneX-R) is appealing. The measurement of the activity of a number of reference enzymes (e.g. acetylase, 5-alfa reductase, aromatase, kinase, DNA polymerase, phosphorylase, ligase, nuclease, PARP, protease, telomerase, topoisomerase, reverse transcriptase, sulfatase, sulfotransferase) represents an original tool to elucidate the biomolecular mechanisms of action of selected toxicants and to identify new effect biomarkers to be tested in the population through validated protocols.

4. To identify genetic susceptibility markers in the Italian population. The search for susceptibility markers in the Italian population is a priority issue.

In addition to cytogenetic and genetic assays, new molecular biology tools such as microRNA and epigenetic regulation of gene expression are useful to obtain predictive markers of noxious effects of pollutants 123

CNR Environment and Health Inter-departmental Project correlated to the genetic background of individuals. The final goal is to identify toxicogenomic markers in the Italian population.

In addition to the above action plan, which is strictly based on the specific know-how of the single teams, other hints emphasize the power of our network: a) Human biomonitoring is directed to human beings. However, basic research on organisms other than humans could speed up the process of biomarker identification and limit the use of human material. The investigations at ISPESL aiming at validating C. elegans as model organism for the analysis of the impact of toxicants on living organisms could be very useful. In fact, the fast growth and reproduction time of this animal as well as the exhaustive knowledge of this genome renders C. elegans an extremely easy model. As underlined in the 2002 Nobel prize award (61), the identification of key genes regulating organ development and programmed cell death in C. elegans was instrumental to the knowledge that corresponding genes exist in higher species, including man. Similarly, the body of evidence accumulated at CNR-IBIM on the use of sea urchin as biosensor could have more applications than the actual ones.

Since the publication of the sequence of this marine invertebrate organism (62), new experimental procedures have been developed to test the effect of chemicals both on whole marine organisms and on coelomocytes. This is a new and original tool. b) It is widely agreed that exposure in utero to toxicants may modify the normal path of reproductive development 124

and cause genital malformations (hypospadia, cryptorchidism). In the same scientific area, it has been shown that prenatal exposure to pollutants and/or the exposure through the maternal milk have adverse effect of infant health. The incidence of congenital malformations may represent an early biological indicator for environmental and/or occupational exposure to contaminants. The search for factors affecting the maternal-fetal environment is currently addressed at CNR-IFC and Perrino Hospital. c) A correlation between neurological disorders and environmental risk has been found. The expertise of CNR-IBF and IBIM in the field of the study of the effects of pollutants on the nervous system strongly supports an in-depth examination of the problem of genetic and environmental factors that modulate the occurrence of such diseases. d) There is an increasing need for epidemiology studies, to develop a strategy of protection of human environmental health. Within our PIAS-HBM network, the expertise of CNR-IFC guarantees a correct approach to the problem. e) The compilation of reviews on the toxicological profile of pollutants is extremely useful to the scientific community. Further work is required to increase the number of considered chemicals; to this purpose, the involvement of all the members of the PIAS-HBM team is desirable. This activity will facilitate clinicians and researchers.

In conclusion, it is clear that in order to investigate the relationship between environment and health, the most

Human Biomonitoring important prerequisite is the availability of an appropriate panel of biomarkers as indicators of individual exposure to environmental chemicals. This attempt requires a cross-interaction among toxicology, cellular and molecular biology in order to identify a potent body of biomarkers of exposure, effect and susceptibility. In this respect, our PIASHBM network fits with the purposes of modern HBM, which has expanded beyond its origin in occupational medicine to cover a wide variety of diagnostic procedures and assessments of environmental pollution, leading to the identification of potentially hazardous exposure before adverse health effects appear and to establish exposure limits for minimizing the likelihood of significant health risk methods. The network we established can be enlarged to other Italian teams providing additional competences, thus ensuring a multifaceted research approach, possibly under the aegis of CNR. The research of the groups belonging to the PIAS-HBM network is funded by different granting agencies. CNR-IGM activity is globally supported by several international grants from the European Framework Programmes, as well as from private and public national agencies (Italian Ministry of Health, MIUR, ISS, AIRC, Telethon, Cariplo, Regione Lombardia). Special funds from CNR have been allowed to CNR-IGM to coordinate the HBM group within the frame of the PIAS project.

The granted sum has covered a one-year research fellow, and the organization of and attendance to the PIAS-CNR Seminar “Endocrine disruptors for EnvironmentHealth evaluation” (Rome, March 16, 2009), to the kick-off HBM meeting (Rome, March 17, 2009), and to the PIAS-CNR Workshop “Advancement and perspectives” held in

Rome on June 18, 2009. FSM is mainly granted by Italian Ministry of Health.

On the behalf of Research Agreements, CNR-IBF carries out several projects in collaboration with the University of Science and Technology of China, a group in Poland, and the Institute de Biotecnologia/ UNAM Cuernavaca, Mexico. CNR-IBIM activity in the biomonitoring domain is supported by various grants, including the Bilateral Italy (CNR)-Japan (JSPS) Seminar on “Physical and Chemical Impacts on Marine Organisms”. CNR-IFC has institutional liaisons for exploiting innovation, with Regione Toscana, local industry, ISS and WHO. Among the financed Environment&Health projects, of interest are the following: “Epidemiological study of biomonitoring” in Campania Region (SEBIOREC) funded by ISS; “Epidemiological study of biomonitoring in Gela Area” (SEBIOMAG) funded

by WHO. ISPESL received a financial support from Ministry of Health to carry out projects in the field of “Prediction, prevention and protection of human health”, and “Development of advanced biosensors for environmental monitoring”.

That each member of the PIAS-HBM team has been/is granted to work in the risk assessment field is an evidence of the qualification of the scientists belonging to the network. However, to establish a coordinated and multidisciplinary work strategy, a global grant from a public agency, covering the expenses of an integrated project, is absolutely required.

We would like to thank the colleagues who shared with us their expertise in the Environment&Health field and accepted to participate in the PIAS-HBM team.

In particular, we kindly acknowledge the active collaboration of Patrizia Guarneri, Giuseppe Latini, Carla Marchetti, Valeria 125

CNR Environment and Health Inter-departmental Project Matranga, Claudio Minoia, Anna Pierini, Gianfranco Prestipino and Elena Sturchio.

We are indebted to the PIAS coordinator, Fabrizio Bianchi, for his continuous and invaluable support.

13.

14.

Keywords biomarkers, biomonitoring, EDs, epidemiology, pollutants.

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Linzalone N, Bianchi F. Human biomonitoring to define occupational exposure and health risks in waste incinerator plants. Int J Environment and Health 2009; 3: 87-105.

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Towards the Effective Surveillance of Hypospadias. Environ Health Perspect 2004; 112:398-402.

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Turconi G, Minoia C, Ronchi A, Roggi C. Dietary exposure estimates of twenty-one trace elements from a Total Diet Study carried out in Pavia, Northern Italy. Br J Nutr 2009; 101:1200-8.

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Latini G, Del Vecchio A, Massaro M, Verrotti A, De Felice C. In utero exposure to phthalates and fetal development. Curr Med Chem 2006; 13:2527-34.

Latini G, Del Vecchio A, Massaro M, Verrotti A, De Felice C. Phthalate exposure and male infertility. Toxicology 2006; 226:90-8.

Latini G, Wittassek M, Del Vecchio A, Presta G, De Felice C, Angerer J. Lactational exposure to phthalates in Southern Italy. Environ Int 2009; 35:236-9.

Pinsino A, Della Torre C, Sammarini V, Bonaventura R, Amato E, Matranga V. Sea urchin coelomocytes as a novel cellular biosensor of environmental stress: a field study in the Tremiti Island Marine Protected Area Southern Adriatic Sea, Italy. Cell Biol Toxicol 2008; 24:54152.

Matranga V, Yokota Y. Responses of marine organisms to physical and chemical impacts. Cell Biol Toxicol 2008; 24:471-4.

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Environmental Health Surveillance Systems E.A.L. Gianicoloa, A. Brunia, M. Serinellib

a. CNR, Institute of Clinical Physiology (IFC), Lecce, Italy b. Regional Environmental Protection Agency, Puglia, Bari emilio.gianicolo@ifc.cnr.it

ABSTRACT An integrated environmental health surveillance system is the systematic, ongoing collection and analysis of information related to disease and environment, and its dissemination to individuals and institutions.

This type of system provides scientific evidence and tools for implementing and evaluating policies aimed at preventing, controlling and protecting health and the environment. An integrated environmental health surveillance system can be realized by setting up environmental and health indicators. Indicators are useful for understanding the spatial and temporal trends of environmental parameters and related health effects, both acute and chronic.

In recent years, much attention has been focused on surveillance systems, and, in particular, on developing methods for combining and integrating information in order to better understand these phenomena. Several analytical approaches have been proposed for classifying environmental and health indicators: Thackerâ&#x20AC;&#x2122;s model; the DPSEEA framework (WHO) which represents an evolution of the PSR model (OECD) and the DPSIR framework (European Environmental Agency).

As part of the Environment and Health Inter-departmental Project (PIAS-CNR), the Working Group on Environmental Health Surveillance Systems has been tasked with the development of a protocol to be tested in areas with different environmental risks, in order to monitor environment and health indicators and to provide useful tools for primary prevention programs and communication. The goal is to select a set of environmental and health indicators to be assessed against their utility and availability in time and space.

1. INTRODUCTION Environmental effects on health are associated with many different factors: environmental degradation, such as air, water and soil pollution, and food contamination; global environmental problems, such as the reduction of biodiversity, the degradation of the ecosystem through deforestation, global warming, ozone layer depletion and contamination by persistent organic chemicals, waste cycle mismanagement and industrial disasters.

As part of the Environment and Health Inter-departmental Project (PIAS-CNR), the Working Group on Environmental

Health Surveillance Systems has been charged with developing a protocol to be tested in areas with different environmental risks, in order to monitor environment and health indicators and to provide useful tools for primary prevention programs and communication. The analysis of the scientific literature on this subject, and the study of the most advanced international experiences are the basis on which a protocol can be defined and tested in areas with different degrees of environmental degradation. In Italy, the Apulia region, a territory with three areas at high risk of environmental crisis (Brindisi, Taranto and Manfredonia), is an interesting experimental site.

CNR Environment and Health Inter-departmental Project The areas chosen to test the surveillance system require epidemiological and environmental characterization, that can be performed using available statistical information or conducting specific surveys.

These elements, based on a conceptual model (Thacker, DPSEEA or DPSIR) integrated with established international experience and shared with local and national stakeholders, are the platform on which to build an information system that can provide information on environmenthealth interaction and lead to preventive and communication actions.

This chapter aims at providing a summary of our knowledge on this subject, with particular regard to several international experiences that are generally considered as more advanced.

Major conceptual models in the field of environment and health indicators will be discussed.

2.

ENVIRONMENTAL

AND

HEALTH

SURVEILLANCE: STATE OF THE ART

2.1 Definitions and purpose Public health surveillance is “the ongoing systematic collection, analysis, and interpretation of outcome-specific data, closely integrated with the timely dissemination of these data to those responsible for preventing and controlling disease or injury“ (1). The modern definition of surveillance, which at the beginning included only transmittable diseases (2), currently refers to chronic and acute diseases, reproductive health, work, domestic and road accidents, environmental and occupational risk, and behavior (3).

In 1988, the Centers for Disease Control and Prevention (CDC) of the United States defined environmental health 130

surveillance as ”the ongoing, systematic collection, analysis, and interpretation of health data essential to the planning, implementation, and evaluation of public health practice, closely integrated with the timely dissemination of these data to those who need to know. The final link of the surveillance chain is the application of these data to prevention and control. A surveillance system includes a functional capacity for data collection, analysis, and dissemination linked to public health programs.” (4).

Most surveillance systems are usually implemented in order to: - Provide estimates on the size of a health problem; - Investigate emerging health problems and epidemics; - Document the distribution and diffusion of health events on a given territory and in specific populations; - Provide the basis for epidemiological research and clinical trials; - Describe the natural history of a disease; - Monitor trends in risk factors related to specific diseases; - Identify changes in health practices; - Monitor the spatial and temporal variation of the occurrence of diseases and risk groups; - Evaluate programs for prevention and disease control (1).

In recent years, attention has been increasingly focused on the need to improve environment and health monitoring systems by developing methods designed to combine information from different information-systems and to support an integrated knowledge of phenomena.

An environmental health surveillance system must be able to assess, analyze and disseminate the information necessary to properly plan policy-makers‘ actions in

Environmental Health Surveillance Systems

Figure 1. the process by which an environmental agent produces an adverse effect and the corresponding types of public health surveillance. health care.

Environmental indicators (quality of the environment, environmental contamination and results of specific monitoring) and health indicators (e.g, indicators of morbidity, mortality and reproductive health) which can be obtained by current health information flows, from a pathology registry or from specific surveys, are the bases of an integrated environmental health surveillance system.

Environmental and health indicators provide a quantitative summary of the phenomenon under study and are useful to understand the spatial and temporal patterns of healthimpacting environmental parameters , acute and chronic health effects and social and demographic factors.

2.2 Conceptual models Several models have been proposed to provide a conceptual synthesis of the monitoring of environmental and health problems. These include - The Thacker model (5);

- DPSIR and DPSEEA model (6, 7).

They emphasize the role of social and environmental macro-determinants and consider exposure to be a central event in environmental causes and in the occurrence of disease. Therefore, exposure is the key element in environmental and health surveillance. In fact, epidemiological surveillance systems have developed, from disease surveillance to surveillance of collective risk factors (8).

2.3 The Thacker model The Thacker model (5) proposes three different kinds of surveillance (Fig. 1): - Hazard surveillance; - Exposure surveillance; - Outcome surveillance.

Thacker defines hazard surveillance as the â&#x20AC;&#x153;assessment of the occurrence of, distribution of, and the secular trends in levels of hazards (toxic chemical agents, physical agents, biomechanical stressors, as well as biological agents) responsible for disease and injuryâ&#x20AC;? (9).

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CNR Environment and Health Inter-departmental Project

Figure 2. The DPSIR model Data on risk factors can be derived from the amount of hazardous agents produced, sold, used or released, or from the concentrations of these agents in various environmental matrices (air, food, soil and dust, water) (10).

Exposure surveillance is the monitoring of individual members of the population to assess the presence of an environmental agent or its clinically unapparent (e.g, subclinical or preclinical) effects. (5, 11).

The definition of target groups for surveillance in areas with documented or presumed environmental pressure is one of the most critical points in the researcherâ&#x20AC;&#x2122;s decision-making chain, since it depends on many factors and considerations.

Actually, exposure is defined as the relationship between the environment (external factors) and the individual (internal factors) as a result of inhalation, ingestion, dermal contact, or via fetus or placenta.

The need to establish a relationship between environmental monitoring and health-related policies and actions led to the addition of a fourth monitoring category regarding the assessment of policy options (10).

2.4 The DPSIR and DPSEEA The methodology of the (Determinant, Pressures, 132

DPSIR State,

Impacts, Responses) allows us to organize environment-health indicators implemented in an environmental health surveillance system (12) (Fig. 2).

The DPSIR framework is a system to analyze the Driving forces responsible for change, the resulting environmental Pressures on the State of the environment, the Impact of changes on environmental quality, and Societyâ&#x20AC;&#x2122;s Response to these changes.

In this model the determinants (or sources, e.g. Agriculture, industry, transport, settlements, animal husbandry, mining) identify the factors influencing the environmental conditions as sources on which to act. They are useful to identify relationships between the factors responsible for pressure and the pressure itself.

Pressures (e.g. emissions of pollutants, waste, noise emission , vibration and radiation) identify the direct effects of increased human activities (i.e. the variables responsible for the degradation) and are useful to quantify the causes of environmental change.

States (e.g. quality of air, water, soil, vegetation, fauna, ecosystems, landscape, physical agents, public health) represent environmental quality and the environmental resources that should be protected. They are useful to evaluate environmental conditions in terms of degree of impairment.

Impact refers to the effects of a pressure: they are major changes in the environment compared to a state-based condition, taken as a reference.

Responses (laws, plans, rules) are actions taken to address the impact, and take different forms depending on the level of the model on which to act (e.g. demands of structural determinants, interventions prescriptive or technology, etc.) (13).

Environmental Health Surveillance Systems Each of the areas identified above can be summarised by using specific indicators, as resulting from current environmental and health data, environmental bio-monitoring and biomarkers of human exposure.

The model comes from the general concept when applied to specific environmental areas such as environmental matrices that define the real component within which chemical, physical and biological agents act.

Matrices are generally identified as air, water, soil, waste, physical agents, and foods. To better address the effect of the human exposure to environmental factors, the World Health Organization (WHO) extended DPSIR to the DPSEEA model (Determinant, Pressures, State, Exposure, Effect, Action).

The introduction of health effects evaluation involves a refinement of the DPSIR conceptual model, translating the concept of impact into ”exposure“ and ”effect“ and the Responses into ”actions”.

In the DPSEEA model, according to the classical epidemiological model, Determinants and Pressures are recognized as determinants of disease, distinguished in individual and contextual determinants.

State, Exposure and Effect represent the extent of the problem (respectively, in terms of emissions, exposure and health effects) (13).

The number of indicators relating to the areas outlined above is very high. There is abundant literature on the selection of indicators useful to describe the state of the environment and health (7, 14).

The indicators include both environmental and health indicators for which the relationship between exposure to environmental hazards and health effects is already established.

Wills and Briggs (15) define two categories of indicators: - Health-related environmental indicators

(HREIs); - Environment-related health indicators (ERHIs).

The first relates to environmental conditions that suggest potential harmful health effects; the latter relates to health outcomes that suggest an environmental cause or a contribution from environmental factors.

In environmental impact assessment studies, an indirect measure of the level of exposure (e.g. concentrations of pollutants or emissions) is used as an environmental indicator (16).

In studies of health impact assessment of environmental pollution, indicators that describe health outcomes caused by exposure to polluted matrices are used.

To determine which diseases are related to the environment (e.g. infant mortality, mortality from respiratory causes), it is necessary to carry out studies on risk assessment from exposure (16).

In general, indicators should have specific requirements: - Validity, reliability and representativeness of data; - Availability of data, their systematic measurement in time and space (not separated from their representation); - Usefulness, i.e. the indicator has to be oriented to the action.

3. ENVIRONMENTAL AND HEALTH SURVEILLANCE EXPERIENCES

Integrated environment and health surveillance systems have recently been developed at international level.

The CDCs, the Californian Policy Research Centre (CPRC), WHO-Europe, and the Institut National de Santé Publique du Québec have produced comprehensive reports on the strategy to implement an for environment and health monitoring 133

CNR Environment and Health Inter-departmental Project system.

The strengths and weaknesses of these systems are summarized in Table 1 (10).

These three systems differ as for their state of the art and completeness, but may represent a good basis to initiate a program of environmental and health surveillance in Italy or in specific areas of the country.

3.1 USA In the United States, the CDC ”Pew Environmental Health Commission“ report first defined the purpose of a surveillance system (17). This document underlined the gaps in our knowledge of environmental medicine and recommended implementing a national environmental health surveillance system.

In 2002, CDC in collaboration with the Environmental Protection Agency (US-EPA) and the National Aeronautics and Space Administration and Member Partners the development of this system was started (18).

The goal of the system is to ”monitor and distribute information about environmental hazards and disease trends, advance research on possible linkages between environmental hazards and disease, develop, implement, and evaluate regulatory and public health actions to prevent or control environment-related diseases“ (19).

In 2002, the State of California initiated the first environmental health surveillance system (20). The ”Strategies for Establishing an Environmental Health Surveillance System“ report led the early development of this system.

It defined the objectives and usefulness of developing and planning an environmental health surveillance system, estimating its costs, defining diseases, environmental hazards and exposures to be monitored and describing the related political, ethical 134

and legal issues (10).

In summary, the program of the CDC has three objectives: - to develop the technological infrastructure, such as the use of GIS for mapping the use of pesticides and the concentrations of pollutants in urban areas; - to improve data availability and use; - to promote the translation of knowledge into policy actions.

In this surveillance system, environmental and health indicators (Environmental Public Health Indicator, EPHI) are divided into the following four categories (21): - Hazard indicators: Conditions or activities that identify the potential for exposure to a contaminant or hazardous condition.

- Exposure indicators: Biological markers in tissue or fluid that identify the presence of a substance or combination of substances that could harm an individual.

- Health effect indicators: diseases or conditions that identify an adverse effect from exposure to a known or suspected environmental hazard.

- Intervention indicator: Programs or official policies that minimize or prevent an environmental hazard, exposure, or health effect.

Indicators are also divided into three topics: a) pathways or sources (e.g, air, water); b) agents (e.g. lead, pesticides); c) events (e.g. sentinel events, environmental disasters).

Topics may also overlap due to the complexity of environmental and public health laws and programs. However, an indicator is generally included under only one topic, although it may be relevant to several.

Environmental Health Surveillance Systems Table 1. CDC, EU and Quebec environmental health tracking systems: strengths and weaknesses.

Quebec (Institut national de santé publique du Québec 2006) 2004)

European Union (WHO Europe

Centers for Disease Prevention and Control (CDC 2006b)

Initiative

Strengths

1. Partnership with federal, state and local government agencies, academic and community groups, healthcare organizations 2. Strong stakeholder input 3. Pilot projects well coordinated

1. Includes upstream driving forces 2. Includes home, work and ambient exposures 3. Includes population exposure and health impact assessment (air quality, noise) 4. Linked to health-based policy action programs (NEHAPs) 5. Developing a children’s environment and health indicator set

1. Common surveillance with occupational and infectious diseases within Ministry of Health and Social Services 2. Annual reporting 3. Research in environmental health surveillance since 1997 with Geomatics for Informed Decisions National Centre of Excellence (GEOIDE NCE) 4. Strong public health surveillance mandate in 2001 Public Health Law 5. Stable funding 6. Strong Quebec Public Health Institute [Institut national de santé publique du Québec (INSPQ)]

Weaknesses

1. Varying levels of state readiness 2. Early in development: • First national report, 2008 • Network launch 2008

1. Diverse data systems across EU 2. Gaps in survey and biomonitoring data 3. Still to define outputs (printed reports and Webbased data)

1. Not all indicators completed 2. Gaps in data for some proposed indicators

Indicators Topics Air, ambient (outdoor) Air, indoor Disasters Lead (Pb) Noise Pesticides Sentinel events Sun and ultraviolet Toxics and waste Water, ambient Water, drinking Indicator Types Hazard Exposure Health effect Intervention

160 indicators proposed in: Air quality Housing Noise Traffic accidents Water and sanitation Food safety Chemical emergencies Radiation Workplace

Twenty-six of 41 indicators reported.

Environmental Indicators: Recreational water quality (beaches) Drinking water quality Boil-water advisories Waste water treatment Air pollution Environmental tobacco smoke exposure Health-Based Indicators: Carbon monoxide and other poisonings notification rates Allergic rhinitis prevalence Cancers of interest for environmental health Hospitalization/mortality rates for diagnoses linked to environmental hazards Proposed Indicators: Noise Indoor air Pesticides Climate change (mortality for heat waves, morbidity and mortality linked to extreme weather events)

135

CNR Environment and Health Inter-departmental Project 3.2 Canada In the state of Quebec, the Ministry of Health and Social Services has established a surveillance system regarding environmental hazards, occupational health and infectious diseases. The system is based on 26 of the 41 indicators proposed by a panel of experts (17 refer to environmental data, 9 to health data) (Table 1).

3.3 European Union The ECOEHIS project (European Community Health and Environment Information System), conducted under the leadership of the WHO-European Centre for Environment and Health (22), has developed environmental health (EH) indicators as part of the European Community Health Indicators (ECHI), which would serve as tools to aid in the following: - To measure the health impact of selected environmental risk factors, their determinants and trends therein, throughout the Community; - To facilitate planning, monitoring and evaluation of Community programs and actions; - To provide Member States and international organizations with information to make comparisons and evaluate their policies (22).

The core set of environmental health indicators has been developed within the DPSEEA framework and focuses on the population’s exposure to environmental hazards, their health effects, and policy actions to prevent illnesses, injuries and deaths.

Based on feasibility and usefulness testing and after approval by the EU Member States, the indicators were to be delivered according to the evidence, data and methodological limitations, in one of three 136

categories: - ready and recommended for implementation (these indicators are recommended as ‘core’ European Community Health Indicators) - ready, but not feasible for immediate implementation (these indicators are recommended for WHO projects such as ENHIS) - desirable but requiring further developmental work (these indicators are recommended for further elaboration).

The Institute for Environmental Protection and Research (ISPRA) acting as National Focal Point (NFP) for Italy, has coordinated the national feasibility studies of these indicators. Indicators refer to the following areas: - Air; - Housing and health; - Noise and health; - Traffic accidents; - Water and sanitation; - Chemical Emergencies ; - Radiation.

Based on the pilot project conducted in Italy, ISPRA-APAT has classified the indicators according to availability, data quality and feasibility of their implementation for three environmental sources (air, water, soil) and for each of the five categories of the DPSEEA framework.

Some indicators are not calculated due to the unavailability of data or the gaps in the informative flows.

The selected indicators are considered of national importance, both in terms of comparability and of data quality. They need to be implemented and adapted at local scale for surveillance in areas with different environmental risks.

Environmental Health Surveillance Systems 3.4 Italy In 2001, as part of the Italian Association of Epidemiology, the Environmental Epidemiology Group (GEA) was established (23) in order to coordinate, organize and take over environmental epidemiology and risk assessment activities throughout the country (24).

The environmental protection agencies that have joined the group are: ISPRA, Regional Environmental Protection Agency (ARPA) Marche, ARPA Piemonte, ARPA Emilia Romagna, ARPA Tuscany, ARPA Veneto, ARPA Campania, ARPA Friuli Venezia Giulia, ARPA Umbria, ARPA Lombardia, APPA Bolzano , ARPA Basilicata, ARTA Abruzzo, ARPA Liguria, ARPA Puglia, ARPA Sicily, ARPA Sardegna.

As part of the GEA, the following four subgroups have been formed: - Group 1. Definition of guidelines for environmental epidemiology studies in small areas, in order to collect information and experiences reported in the literature for epidemiology studies in small areas.

- Group 2. Integration between essential health care levels (LEA) and the Essential Levels of Environmental Protection (LETA), in order to send proposals to the Ministry of Health and the Environment on LEA/ LETA related issues.

- Group 3. Realization of a reference network for environmental epidemiology studies, in order to promote organizational proposals to create a network of experts on environment and health.

- Group 4. Environmental and health indicators at the local level (IAS).

It is based on the formulation of a proposal for the definition and testing of environment and health indicators at local level (25).

These groups have produced ideas and documents contained in the acts of the second national workshop Portonovo (Ancona) (26).

4. EPIDEMIOLOGICAL

AND ENVIRONMENTAL

CHARACTERIZATION OF BRINDISI (EXPERIMENTAL SITE)

In the province of Brindisi, an area at â&#x20AC;&#x153;high risk of environmental crisisâ&#x20AC;? has been identified by the Italian Ministry of the Environment (Law n. 305 of 1989), due to the presence of numerous industrial sites.

They produce a remarkable environmental impact and cause serious alterations of every type to the equilibrium of the environment, as well as adverse effects on the health of the population.

In fact, in the province of Brindisi and particularly in the southern area of the main town, on the Adriatic sea, many sources of air pollutants with high environmental impact are located near the urban area.

Next to the petrochemical area (built in 1959), various industries have grown up over the years: three fossil-fuel power plants, among them one of the largest in Europe (Federico II Enel); several chemical, pharmaceutical, metallurgical and manufacturing industries; an airport; an harbour, mainly for passenger traffic to Greece.

In 2002, in seven municipalities including Brindisi, the State Forestry Service has discovered 15 illegal dumps (covering an area of 127,278 m2).

The Federico II Enel plant has the highest record of CO2 emissions in Italy, and in the area designed as Reclamation Sites of National Interest (RSNI) there is a significant concentration of particulate matter as underlined by emissions and concentrations recorded by the Region of Puglia (CORINE-AIR).

137

CNR Environment and Health Inter-departmental Project Southwest of Brindisi there is the province of Taranto, whose industrial area includes steel factories, a refinery and a cement factory and proximity to this border could be a further source of exposure to environmental pollutants.

The town of Brindisi can be selected as a site to test the surveillance system.

This requires epidemiological and environmental characterization 4.1 Epidemiological characterization Between 1990 and 1994, the World Health Organization has conducted an epidemiological study in four municipalities (Carovigno, Torchiarolo, S. Pietro V. and Brindisi) located in the RSNI of Brindisi. Significant excesses of mortality from all causes, all cancers, lung cancers, respiratory and ischemic diseases were observed both for males and females.

In particular, an elevated value of mortality from melanoma was reported (27).

In Brindisi, in the male population, excesses of mortality from all causes and from all cancers were detected, while in the female population excesses were found for digestive system and for psychiatric causes. Different mortality patterns by gender are likely to be caused by professional exposure.

A recent descriptive geographical study of the province of Brindisi estimates the

mortality among residents in the twenty municipalities of the province aggregated in four geographic areas: the one at â&#x20AC;&#x153;high riskâ&#x20AC;? including the main town, and the areas located north, west and south of the Brindisi RSNI area (28).

The analysis was run by gender, specific causes of death, and by two 10-yearperiods between 1981 and 2001. Results for RSNI area confirmed the previous WHO analysis, while ither excesses for specific causes were observed in the remaining areas.

In the province of Brindisi, excess mortality due to cardiovascular disease and cancers is higher than regional levels.

The analysis restructed to working age groups (34-64 years), showed higher rates of mortality than those reported for cardiovascular mortality, among men as well as women; excess mortality for cancer of the prostate and for trauma was higher in men, wheras women show a higher mortality rate for the cancer of the central nervous system. In addition, for Brindisi Municipality, excess mortality for pleural mesothelioma was reported also among women.

Table 2 shows the results of death incidence analyses from all cancers and from specific causes during the period 1999-2001, also compared with the data of the whole province. (the data source is the Jonico-

Table 2. Standardized rates of cancer incidence (x 100,000 inhabitants) and IC 95%.

Site

Risk area of Brindisi

Province of Brindisi

Rate

CI 95%

Rate

CI 95%

Rate

CI 95%

All cancer sites

424,5

391.3-459.9

382,3

356.0-410.2

368,1

353.8-382.8

Lung

88,9

74.1-105.9

83,6

71.5-97.1

77,6

71.2-84.6

Pleural

0,6

0.0-3.8

0,4

0.0-2.7

0,5

0.2-1.4

Urinary Bladder

38,1

28.6-49.8

32,7

25.3-41.6

32,1

28.0-36.6

NH-lymphoma

14,8

9.1-22.7

12,8

8.3-18.8

11,1

8.7-14.0

Soft Tissue

4,2

1.5-9.3

2,95

1.1-6.5

2,4

1.3-4.0

Source:RTJS 1999-2001

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Brindisi

Environmental Health Surveillance Systems Table 3: emissions in atmosphere in Puglia and in each of the five provinces â&#x20AC;&#x201C; 2006.

Province/Pollutant

Emissions

Measure Unite

CO2 Foggia

926.714

Mg / y

Bari

925.379

Mg / y

Taranto

23.492.769

Mg / y

Brindisi

19.799.096

Mg / y

Lecce

1.018.493

Mg / y

Puglia

46.162.451

Mg / y

C6H6 Taranto

237.308

Mg / y

Brindisi

11.000

Mg / y

Puglia

248.308

Mg / y

PAH Taranto/Puglia

32.240

Mg / y

NOX Foggia

1.243

Mg / y

Bari

3.370

Mg / y

Taranto

35.846

Mg / y

Brindisi

11.961

Mg / y

Lecce

2.444

Mg / y

Puglia

54.864

Mg / y

SOX Foggia

252

Mg / y

Bari

1.452

Mg / y

Taranto

53.077

Mg / y

Brindisi

12.697

Mg / y

Puglia

67.479

Mg / y

CO Bari

1.555

Mg / y

Taranto

541.434

Mg / y

Brindisi

2.900

Mg / y

Lecce

1.860

Mg / y

Puglia

547.749

Mg / y

Particulate Bari

57

Mg / y

Taranto

11.805

Mg / y

Brindisi

730

Mg / y

Puglia

12.591

Mg / y

PCDD, PCDF Taranto/Puglia Source: INES registry

92

g/y

Salentino Tumor Register- RTJS).

In 2004 a case-control study was published to investigate mortality from cancer in the areas near Brindisi petrochemical industry.

In period 1996-1997, a moderate excess of mortality, from lung and bladder cancer and lymphohematopoietic system was observed in the population residing in an area within 2 km from the centroide of the petrochemical site, compared to the population residing outside 5 km (29).

A case-crossover study has recently been conducted to investigate the association between daily mortality and hospital admissions data, on the one side, and the daily concentration of atmospheric (PM10 and NO2) pollutants, on the other.

The study population included residents in Brindisi city who died or hospitalized for several diseases during the period 2003-2006 (30).

This study found strong and consistent associations between outdoor air pollution (coming from both industrial emissions and urban traffic) and short-term increases in both mortality and morbidity.

In particular, PM10 was associated with mortality from all natural causes. The risk was more pronounced for cardiovascular mortality.

The association with hospitalization for cerebrovascular diseases was statistically significant for PM10 among females and elderly over 75 years old.

In specific population groups, increased mortality and hospital admissions have been associated with NO2 (31).

4.2 Environmental characterization AIR characterization The Italian Pollutant Emissions Register (INES) registry can be used to gather information about emission in water and air coming from the facilities under 139

CNR Environment and Health Inter-departmental Project Table 4: emissions in atmosphere in Brindisi by industrial complex â&#x20AC;&#x201C; 2006 Industries

Emissions

Meysure Unite

%

Thresold value Kg / year

CO2 POLIMERI EUROPA SPA - Brindisi

481.738

Mg / y

2,4

100

ENIPOWER S.P.A. - Brindisi

2.132.833

Mg / y

10,8

100

ENIPOWER S.P.A. - Brindisi

130.172

Mg / y

0,7

100

POWER PLANT Federico II (BR South)

14.372.364

Mg / y

72,6

100

Power plant Brindisi

2.681.989

Mg / y

13,5

100

Total

19.799.096

Mg / y

42,9

100

11.000

Kg / y

4,4

1

POLIMERI EUROPA SPA - Brindisi

262

Mg / y

2,2

100

ENIPOWER S.P.A. - Brindisi

826

Mg / y

6,9

100

ENIPOWER S.P.A. - Brindisi

310

Mg / y

2,6

100

POWER PLANT Federico II (BR South)

9.282

Mg / y

77,6

100

Power Plant Brindisi

1.282

Mg / y

10,7

100

Total

11.961

Mg / y

21,8

100

ENIPOWER S.P.A.

430

Mg / y

3,4

150

ENEL PRODUZIONE SPA

10176

Mg / y

80,1

150

EDIPOWER

2.091

Mg / y

16,5

150

Total

12.697

Mg / y

18,8

150

2.900

Mg / y

0,5

500

Mg / y

5,8

50

C 6H 6 POLIMERI EUROPA SPA - Brindisi / Brindisi NOX

SOX

CO POWER PLANT Federico II/Brindisi

Particulate POWER PLANT Federico II/Brindisi

730

Source: INES registry

The Integrated Pollution Prevention and Control (IPPC) Directive (2008/1/CE).

The emission concentrations are available on the site: www.eper.sinanet.apat.it In 2006, the Apulia region had the highest emissions of all the pollutants considered, against national data. The emissions could be attributed mostly to the provinces of Taranto and Brindisi (table 3).

CO2, NOx and SOx emissions - typical of 140

energy production - and benzene (C6H6) emissions from the chemical sector mostly originate from the province of Brindisi (Tab. 3-4) .

WATER characterization Table 5 shows the list of pollutants with threshold values for each specific issue and polluting industrial complex.

Environmental Health Surveillance Systems Table 5: emissions in water in Brindisi from industry â&#x20AC;&#x201C; 2006 Industries

Emissions Kg / year

%

Thresold value Kg / year

77,5

0,6

20

269,0

0,4

100

108,4

28,5

5

12,1

0,1

5

73,3

0,6

50

2,1

0,5

1

72,2

2,4

20

36,4

4,4

20

7.391

100,0

2.000

Phenols POLIMERI EUROPA SPA - Brindisi Zn and compounds ENIPOWER S.P.A. - Brindisi/Brindisi As and compounds POWER PLANT Federico II/Brindisi Cd and compounds POWER PLANT Federico II/Brindisi Cu and compounds POWER PLANT Federico II/Brindisi Hg and compounds POWER PLANT Federico II/Brindisi Ni and compounds POWER PLANT Federico II/Brindisi Pb and compounds POWER PLANT Federico II/Brindisi Fluorides POWER PLANT Federico II/Brindisi Source: INES registry

SOIL characterization In April 2008, ARPA-Puglia has published a study on pollutantsâ&#x20AC;&#x2122; concentration in soil near the industrial area of Brindisi.(31) Soil samples were collected at five depth strata (0-1, 1-2, 2-3, 3-4, 4-5) in 23 samplig areas near the Federico II ENEL power plant.

Concentrations of arsenic, beryl, tin, cobalt, Chlordane, Vanadium, DDE, DDD and DDT were measured.

The results show excesses of arsenic, beryl, cobalt, tin, DDD in the sampling points between 0 and 1 meter; excess of beryllium and DDE in the top soil (layer between 0-15 cm).

The arsenic concentrations in the five sampling points (0 and 1 m) is between 24 mg/m3 e 53 mg/m3 (the threshold value in residential area is 20 mg/m3). The arsenic concentrations in 11 samples range from a minimum of 2.2 mg/m3 to a maximum

of 3.8 mg/m3. In a top soil point the concentration is 2.1 mg/m3 compared to a limit value of 2 mg/m3 for residential areas.

Cobalt values are always higher than the threshold value with concentrations ranging from 30 to 39 mg/m3 (the threshold value in residential areas is 20 mg/m3).

Tin concentrations in four sampling points (0 and 1 m) range from 1.2 mg/m3 to 5 mg/ m3 (compared to a threshold value of 1 in residential area).

DDD values in the only one sample point (0 and 1 m) are between 1.2 mg/m3 and 5 mg/m3 (the threshold is 0.1 mg/m3).

The concentration (0.63 mg/m3) exceeded the threshold (0.1 mg/m3) in one on the two samples (0-1 meter).

141

CNR Environment and Health Inter-departmental Project 5. PROPOSAL

FOR AN ENVIROMENTAL HEALTH

SURVEILLANCE SYSTEM

A systematic proposal for sentinel events regarding the environment and health (ESAS) was drawn up by a consensus conference organized by the Agency for Toxic Substances and Disease Registry (ATSDR) in early 1990s (32).

There are two types of events: Type 1) Acute conditions as sentinel indicators of environmental pollution, as defined by Rutstein (33): – Intoxication by pesticides, metals or other substances present in refuse sites, such as lead and carbon monoxide, with particular attention to the most vulnerable groups, such as children; – Some tumors, such as pleural mesothelioma, clear-cell vaginal cancer and hepatic angiosarcoma, which although characterized by long periods of induction-latency have been associated with a high degree of specificity for exposure to chemical or physical agents; – Precocious puberty as an indicator of exposure to endocrine disruptors, such as many pesticides, industrial products, and food additives; – Hemoglobinemia as a classic indicator of intoxication; – Neuropathy from exposure to toxic chemical agents (such as methylmercury causing Minamata disease).

Type II) Unusual models of disease incidence or conditions identified by means of surveillance: – Bladder cancer, especially in nonsmokers and in the absence of occupational hazards; – Lung cancer in non-smokers; – Liver cancer in non-drinkers; – Rare tumors having a proven association with environmental exposure, such 142

– –

– -

as rhabdosarcoma, myeloblastic leukemia, acute leukemia in children and granulocytic leukemia in adults; Asthma in children, especially shortness of breath in children in absence of allergies and passive smoke exposure; New environmentally-correlated rare diseases such as eosinophilia-myalgia syndrome, toxic oil syndrome, and Kawasaki disease; Measures of biological markers, such as the concentration in biological fluids of persistent organic pollutants (POPs); DNA or hemoglobin adducts.

6. CONCLUSIONS In environmental health surveillance, especially in the case of small areas, unusual events, low exposures, there is a high statistical probability that many warnings will be revealed by chance (due to the effect of multiple testing). On the other side, in case of alarm signals coming from outside the surveillance system there is a typical risk of an a posteriori selection.

No signals should ever be considered conclusive, since investigation should always be focused on statisticalprobabilistic confirmation and search for the cause. No sign should be neglected since it must be considered anomalous until proven otherwise.

The adoption of a health surveillance protocol for a polluted site is a suitable tool to provide an answer to the legitimate concerns of citizens regarding their environment and the consequent health impacts. The other crucial aspect of such a surveillance system is the ability to transfer reliable information and suitable recommendation to decision-makers in order to allow them to carry out and evaluate political choices and programs on the basis of scientific evidences.

Environmental Health Surveillance Systems The environmental and epidemiological characterization of Brindisi represents the first step to set up environment-health indicators, which represent the core of a site-specific surveillance system.

With the contribution of WG5 participants: M. Amadori (CNR-IGG), F. Bianchi (CNR-IFC), L.Bisceglia (ARPA-Puglia), A. Bruni (CNR-IFC), M.Cervino (CNR-ISAC), F.Cibella (IBIM), P.Comba (ISS), L.Cori (CNR-IFC), G. Latini (Local health Authority Brindisi), P.Lauriola (ARPA-Emilia Romagna), A. Mantovani (ISS), G.Petruzzelli (CNR-ISE), M. Portaluri (Local health Authority Brindisi), M.A.Vigotti (CNR-IFC).

Keywords: surveillance system, Brindisi, environmental risk, health and environment indicators.

8.

9.

10.

11.

12.

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Salute e ambiente – Quaderno di Epidemiologia Ambientale. Osservatorio Regionale Epidemiologico e per le Politiche Sociali della Val d’Aosta. 2006.

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McGeehin MA, Qualters JR, Niskar AS. National Environmental Public Health Tracking Program: Bridging the Information Gap. Environ Health Perspect 2004;112:1409-14013.

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CNR Environment and Health Inter-departmental Project 19. Centers for Disease Prevention and Control (CDC). National Environmental Public Health Tracking Program “Background”. 2006.

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Strategies for Establishing an Environmental Health Surveillance System in California. A Report of the SB702 Expert Working Group. 2004.

21. Centers for Disease Control and Prevention. Environmental Public Health Indicators. 2006:40 pp.

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Development of Environment and Health Indicators for European Union Countries - ECOEHIS. Final report 2004:655 pp.

23. Beccastrini S. Gli avvenimenti che hanno preceduto la nascita del GEA e la stesura del documento «Ambiente e salute», frutto del secondo convegno di Portonovo.

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2007 Jan-Feb;(1 Suppl 2):1-78. Italian. No abstract available. 2007;31(1 Suppl 2):1-78. http://www.epidemiologiaeprevenzione. it/cms/?q=node/47.

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29. Belli S, Benedetti M, Comba P, Lagravinese D, Martucci V, Martuzzi M, et al. Case-control study on cancer risk associated to residence in the neighbourhood of a petrochemical plant.

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Monitoring Contaminants in Food Chain and their Impact on Human Health A. Mupoa, F. Boscainoa, G. Cavazzinib, A. Giarettab, V. Longoc, P. Russoa, A. Siania, R. Sicilianoa, I. Tedescoa, E. Tostid, G.L. Russoa

a. CNR, Institute of Food Sciences (ISA), Avellino, Italy b. CNR, Institute of Geoscience and Georesources (IGG), Padova, Italy c. CNR, Institute of Biology and Agricultural Biotechnology (IBBA), Pisa, Italy d. Zoological Station Anton Dohrn, Naples, Italy annalisa.mupo@isa.cnr.it

ABSTRACT In recent years a great attention has been focused in Europe on the importance of food safety and the relation between diet and health. Moreover, worldwide changes in population lifestyle, together with modifications in food processing, production and distribution contributed to the eating habits of Western populations and to their reaction to recent public health emergencies. As an example, the real or alleged dioxin contamination that affected several industrialized countries has increased the interest of Authorities, producers and consumers on topics such as food safety and risks for human health deriving from contaminated food. The annual report of the European Commission Rapid Alert System for Food and Feed (RASFF) summarizes notifications on food contaminations occurred in different countries. Data analyses provided a useful tool to develop future efficient programs for food control. In this context, a working group of the PIAS project studied how specific classes of environmental contaminants (e.g, pesticides, metals, dioxins) may affect human health through the food chain. Their results are presented in this Chapter. A special section has been dedicated to highlight issues of major interest in this field, such as the determination of heavy metals and dioxins in food matrixes and biological samples; experimental models to assess the harmful effect of contaminants on human reproduction; the role of cytochrome P450 in xenobiotics metabolism. The last section of this Chapter proposes a research programme aimed at integrating aspects already faced in current literature as independent issues, but rarely considered in a holistic approach. The competences needed to pursue this goal are covered by the Italian National Research Council or by the involvement of other Italian or international institutions. The final proposal targets the youth and intends to determine the cause-effect relationship between the presence of contaminants in the diet, their accumulation in humans and the risk of chronic diseases. Key issues, such as bioavailability and adaptive response (hormesis), will be explored using suitable experimental models to suggest a functional link, at molecular level, between the onset of specific diseases and the concentrations of contaminants measured in food.

1. BACKGROUND 1.1 Food safety: focusing on chemical contaminations The introduction of genetically modified food and food incidents in Europe raised the public interest in food safety. An integrated approach to face this problem requires a strong cooperation by the food industry, food distributors, the scientific community, governments, managers and local administrators in order to build consumersâ&#x20AC;&#x2122; trust and confidence. The food

safety certification is achieved assessing the potentially health adverse effects of food contamination. Three main food contamination groups can be identified: i. physical; ii. microbiological; iii. chemical.

Physical contaminations are due to the presence of extraneous bodies in food (plastic, woods, glass and others) as the results, for example, of food packaging and/or transformation and/or storage.

The substances present in those materials are not for human consumption, but

CNR Environment and Health Inter-departmental Project when in contact with food they migrate into it and risk of being ingested (for example, the perfluorinated chemicals used in greaseproof packaging for fast foods). Microbiological contamination refers to the presence of one or more natural biological agents, such as various bacteria, yeasts, mould, fungi, protozoa or their toxins and by-products, which can adulterate food properties and safety.

Microbiological agents are responsible for â&#x20AC;&#x153;food diseasesâ&#x20AC;? such as food borne infections and intoxications (Botulinum, Listeria, Hepatitis A) and epidemic episodes (e.g, Salmonella enteritidis).

Chemical contaminants or xenobiotics can originate from many different sources and include heavy metals, pesticides, phytopharmaceuticals, antibiotics, additives, dioxins and PCBs. Nowadays, chemical contaminants are a major concern for food safety because of the increased role of man-made chemicals due to our modern lifestyles. In fact, despite the fact that the large use of chemicals improved the quality of our lives, many of them have been reported to produce an adverse impact on human populations, animals and plants continuously exposed to a cocktail of potentially hazardous chemicals (2-4).

However, in humans and animals, diet is predominant route of many dangerous chemicals. Food is a crucial link in the chain of events starting from chemical manufacturing and ending with their presence in human blood, tissues and organs. (5). The worldwide observation of such contaminants in food shows the global scale of chemical contamination.

To assess the impact of such substances on food safety, the following question must be answered: Can the quantity and bioavailabilty of an unwanted chemical in food provide a real risk to human health? In the past, just the presence of 146

a hazardous chemicals, whatever their concentration or weight, was considered as unsafe and adverse to health. The presence of a chemical depends on the sensitivity of the instruments used to assess it. Analysis with an increased sensitivity and different techniques showed that some chemicals, previously stated as not present, were instead only undetected.

This implies the detection itself is not necessarily representing a risk: a new approach is needed to provide a riskbased evaluation of the potential exposure, hazard, and toxicity of chemicals detected at a low-level (6).

For this reason, a threshold is needed for non toxic chemical compounds. In past decades, scientists developed different models to address this issue, concluding that the potential human health risk posed by a chemical substance depends on its inherent toxicity and exposure, including route, dose and duration. In the case of substances found in food, at least two elements must be considered. The first is their concentration level in various foods, assessed through chemical analysis. The second element is the consumed quantity of contaminated food. Bioavailability must be also considered as the capability of a dietary chemical to be absorbed and metabolized. Bioavailability is commonly assessed by measuring the amount of the ingested chemical that gets into the systemic circulation, since in most cases the specific targets and the time required to determine an effect on health are unknown.

Thus, although bioavailability is critical in assessing the potential benefits or risks of a compound, it can only be studied on a comparative basis (see section 5).

It is clear that great effort is devoted to improve risk assessment and to develop common methods to be used to guarantee food quality and to protect consumersâ&#x20AC;&#x2122;

Monitoring Contaminants in Food Chain and their Impact on Human Health health. Such assessments are often based on very limited scientific information and a complete and exhaustive data set to be used to provide definitive conclusions on chemical concerns in food does not exist at the moment. In this context, results from innovative research programmes are the main sources of information to establish a common food policy and widen the existing data set.

1.2 Contamination chain: the food link Environmental contaminants are substances present in the environment where food is grown, harvested, transported, stored, packaged, processed, and consumed. This “food chain route” of contamination implies the presence of different food contamination levels (Fig. 1), an important element to be evaluated in food safety. For example, after they are released into the environment (soil, air, water) chemical contaminants can enter plants and animals at the bottom of the food chain, to be then consumed by animals, going up in the same chain. The chemicals contained in animals and plants can enter human bodies through the diet.

This concept is even more important for persistent chemicals (biomagnification) and accumulated chemicals (bioaccumulation) (e.g, pesticides, dioxins or heavy metals).

These compounds are called Persistent Organic Pollutants (POPs) and include all those substances not rapidly degraded that keep their harmful capability towards both the environment and human health.

These substances have the following properties: i. resistance to degradation; ii.

Long time persistence into the environment; iii. toxicity for humans, animals and plants; iv. accumulation in living organisms.

Bioaccumulation implies that the compound is lipid soluble and, in the absence of an adequate metabolic pathway able to eliminate it from the organism, tends to accumulate in the trophic chain.

For example, polychlorinated biphenyls (PCB) are very stable organic compounds; they are highly persistent and present in air, soil and water; they are lipid soluble and bioaccumulate in animal fat, in meat and in the liver, and are transferred into milk and eggs. More than 90% of human exposure to PCBs derives from food of animal origin (7).

Figure 1. “The Food Chain Route” 147

CNR Environment and Health Inter-departmental Project Biomagnification is the process by which a compound increases its concentration along the food chain. Heavy metals can be bioconcentrated along the trophic chain.

These substances can be involuntarily ingested with food and drinks and, once absorbed, they are distributed in tissues and organs, persisting for years or decades in some storage sites such as liver, bones and kidneys. Inorganic mercury, for example, can be converted by water micro organisms into the organic methyl mercury compound, which is then biomagnificated in higher links of the food chain. Fish, especially tuna or swordfish, can concentrate methyl mercury at high levels (8).

1.3 Chemical contamination and human health A major concern about food contaminants is their possible adverse effects on human health. Reports on human illnesses caused by food toxic contaminants began several centuries B.C, and since then numerous episodes of food diseases have been continuously reported (9).

In recent years, many of these chemicals present in food have been detected in the blood, tissues and organs of children and adults. POPs are responsible for nervous systems syndromes, disruption of infant brain development, immunepathologies, reproductive system abnormalities, cardiovascular diseases, cancers, diabetes and obesity, and some of them can act as endocrine disrupting agents. The effect on human health can be classified according to: i. acute exposure (early effects); ii. chronic exposure (long term); iii. foetal and infants exposure.

Acute exposure implies the exposure to a massive dose of the contaminant and its negative effects on health are immediate (e.g, milk contaminated with melamine).

148

Chronic exposure implies a long term contact with the contaminants before the disease is manifested (e.g, heavy metals).

An issue that has recently become a priority is related to the negative effects on normal development of foetus and infants exposed to contaminants through the food chain (e.g, POPs). According to the U.S. Environmental Protection Agency (EPA) Toxic Substances Control Act list (10), there are more than 75â&#x20AC;&#x2122;000 known chemicals in the environment, many of which may enter the food chain. Due to the complexity and the huge amount of information, this section will be focused on some specific compounds (including some heavy metals, dioxins and pesticides) to evaluate their impact on human health.

1.3.1 Heavy metals: lead, mercury, arsenic and cadmium Metals are natural elements that have been used in human industry and products since millennia due to their chemical and physical properties. Metals can be easily dispersed in the environment, in soil, water and air and can be very toxic even at relatively low levels of exposure; moreover they can accumulate in specific tissues of the human body.

The U.S. Agency for Toxic Substances and Disease Registry (ATSDR) produced a complete list of the hazards present in toxic waste sites according to their prevalence and the severity of their toxicity: â&#x20AC;&#x153;heavy metalsâ&#x20AC;? (lead, mercury, arsenic, and cadmium) are at the top of this list (8).

The lead found in food is present as salt or oxide and only a small fraction is adsorbed by humans (up to 10%). Lead toxicity can be acute or chronic. Acute intoxications are unusual but are responsible for gastrointestinal, hematopoietic apparatus (anemia) and nervous system (convulsions) symptoms. Chronic exposure is generally manifested with anemia, which depends on the direct toxic effect of lead on red

Monitoring Contaminants in Food Chain and their Impact on Human Health blood cells and bone marrow. It can cause toxic effects also on the nervous system (hyperkinesia, parlysis) and renal failure.

Everyone can be affected from lead toxicity, but infants and fetuses are more vulnerable to lead exposure and can suffer serious damage to the development of their nervous system and learning disabilities.

Mercury is a chemical element widely used in scientific equipment (e.g. thermometers, barometers). However, mercurous and mercuric mercury can form inorganic and organic compounds with other chemicals and can be readily absorbed through ingestion. At high levels, mercury poisoning is responsible for injuries to the lungs and the neurologic system. At lower levels, mercury poisoning is responsible for erethism (tremor of the hands, excitability, memory loss, insomnia, timidity, and sometimes delirium).

Exposure to low doses of mercury is of great concern for its effects on the nervous system development in fetuses and infants.

In 1955 after the disaster in Minamata Bay, Japan, local doctors and medical officials noticed for a long time an abnormally high frequency of cerebral palsy and other child disorders in children born in the area (congenital Minamata disease).

Moreover, studies in the Faroe Islands have demonstrated that, even at much lower levels, mercury exposure of pregnant women, through dietary intake of fish and whale meat, is associated with decrements in motor function, language, memory, and neural transmission in their offspring (1112). Organic mercury, the form of mercury bioconcentrated in fish and whale meat, readily crosses the placenta and appears in breast milk.

Exposure to arsenic originates from anthropic industrial activities and the use of products such as wood preservatives, pesticides, herbicides, fungicides, and

paints. In some areas of the world, arsenic is also a natural contaminant of water.

Moreover, arsenic can accumulate in seafood. Once absorbed into the body, arsenic undergoes some accumulation in soft tissue organs such as the liver, spleen, kidneys, and lungs, but the major long-term storage site for arsenic is keratin-rich tissues, such as skin, hair and nails. Acute arsenic poisoning is infamous for its lethality, since arsenic destroys the integrity of blood vessels and gastrointestinal tissue and its effect on the heart and brain are huge. Chronic exposure to lower levels of arsenic results in somewhat unusual patterns of skin hyperpigmentation, peripheral nerve damage, diabetes, and blood vessel damage (13). Chronic arsenic exposure also causes a high risk of developing a number of cancers, in particular skin, liver lung, bladder, kidney and colon cancers.

Cadmium pollution (e.g, the emissions from cadmium smelters or industrial emissions and the introduction of cadmium into sewage sludge, fertilizers, and groundwater) can result in significant human exposure through the ingestion of contaminated foodstuff, especially grains, cereals, and leafy vegetables. Once ingested, cadmium is adsorbed in the gastro-intestinal tract and accumulates in liver and kidneys. Acute high-dose exposures can cause severe respiratory irritation. Lower levels of exposure are worrisome mainly for their kidney toxicity.

Even without causing kidney failure, cadmium effect on kidneys can have metabolic and pathologic consequences. In particular, the loss of calcium caused by the effect of cadmium on the kidneys can be severe enough to lead to bone weakening (osteoporosis, osteomalacia) (8).

149

CNR Environment and Health Inter-departmental Project 1.3.2 Pesticides Pesticides are a class of chemical compounds used in agriculture to fight parasites and other organisms dangerous for plants, animals and humans. They are divided into different classes of molecules according to their properties as inorganic, natural organic and synthetic organics.

Synthetic organic pesticides are the most used and include DDT, DDE, aldrin, dieldrin and others. DDT (diclorofenicloroetan) was synthesized in 1940 and used in agriculture against many insects. Due to its high toxicity and high persistence, the use of DDT is now banned in most countries.

The major concern on the use of pesticides is related to their carcinogenic effect, their activity as endocrine disruptors and their neurotoxic effects. Epidemiological studies on workers (agriculture) in contact with these substances showed an increased risk for their health safety (14).

Scientific evidence showed that many pesticides used today have neurotoxic activity. For example, commonly used organophosphorous pesticides can inhibit the acetylcholinesterase (AChE) function, the enzyme that degrades the acetylcholine neurotransmitter in the central and peripheral nervous system. Acetylcholine can then accumulate in the nervous system producing an unwanted nervous response that can be responsible for paralysis, muscle debility, convulsions and, sometimes, death (15). Moreover, the use of some fungicides (mancozeb, maneb), that are rapidly metabolized in the organism and in the environment, can generate a highly toxic product, etilentiourea (ETU), that interferes with the thyroid functions and can induce malformations in fetuses when exposed to high doses (16).

In Europe, the legislation on the use of pesticides is complex and articulated.

Very recently, the European Commission 150

officially adopted and published a new regulation setting in motion major changes in how plant protection products are placed on the market and how they are used in practice. Essentially, the new regulation will forbid some â&#x20AC;&#x2DC;active substancesâ&#x20AC;&#x2122; in pesticides. In particular, the European Parliament says, the legislation seeks to outlaw highly toxic chemicals, such as those which cause cancer (17).

1.3.3 Polychlorinated biphenils (PCB) Polychlorinated biphenyls (PCB) cover a group of 209 different congeners, classified according to their number and position of their chlorine atom substituents. Most important, PCB are highly persistent, are globally circulated by atmospheric transport and therefore are present in all environmental media. Due to their lipid solubility and the absence of adequate metabolic pathways in the organisms, PCB tend to bioaccumulate along the trophic chains. As a consequence, PCB are major components of POPs together with polychloro-dibenzodioxins and polychloro-dibenzofurans (PCDD/Fs) (7). In general, human exposure occurs trough the diet, particularly through the ingestion of meat, fish, milk and other dairy products, while in industrial areas showing dioxin emissions the inhaled component has a greater importance.

There is a lot of concern on the negative role of dioxins on human health. Dioxins toxicity has been related to different types of cancer, endocrine interference, deficit in the immune response and developmental defects in fetuses. However, studies on children indicate that the exposure of the general population to low levels of polychlorinated PCDD/Fs does not result in any clinical evidence of disease, although accidental exposure to high levels either before or after birth have led to a number of

Monitoring Contaminants in Food Chain and their Impact on Human Health developmental defects (18): Experimental data indicate that the endocrine and reproductive effects of dioxins should be among the most important effects in animal and humans (19,20). Nowadays, the debate is still ongoing on the real toxic effect of dioxins after low-level exposure, an issue that needs to be further investigated.

2. STATE

OF THE ART AND INTERACTION

BETWEEN BASIC AND APPLIED RESEARCH

2.1 Food quality control Quality is defined as any of the features that make something of a degree of excellence or superiority (21). The word “quality” is differently used in food science and technology referring to a complex concept which includes, on one side, characteristics related to nutritional, microbiological and chemical properties as evaluated from food experts (22) and, on the other side, the sensory quality of a food defined as the attributes of the food which make it agreeable to the person who eats it (the consumer). The latter involves positive factors like color, flavor and texture (23). Quality control is the sum of all those controllable factors that ultimately positively or negatively influence the quality of the finished product, e.g, selection of raw materials, processing, packaging, storage and distribution methods. All along the supply chain, food is exposed to numerous hazards. To prevent or mitigate most of them, the risk factors present at each phase of the supply chain must be known and an effective and comprehensive quality system must be in place. The aim of quality control is to achieve good and consistent quality standards compatible with the market for which the product is designed. Food quality control implies the control of different food processing steps

to prevent the adulteration of the final product.

Some of the most important steps that need to be evaluated in food quality control are: i. agricultural materials / ingredients; ii. processing / engineering; iii. additives; iv. packaging; v. finished product inspection (24). Problems may arise in some of those phases, having a negative impact on the finished product: they are critical points (CP).

The first CP in food control concerns soil quality. In fact, food can be contaminated at a very early stage in the food chain and the contamination propagates all the way along. Soil quality can be evaluated in two distinct ways: i. as an inherent characteristic of a soil; ii. as the “health” condition of the soil (25). The former includes some parameters that reflect the potential of a soil to perform a specific function, (i.e.: plant growth and production, quality of the plants and fruits, soil natural resources).

The latter includes agricultural practices that can adulterate ground functions and composition, such as manure, the use of fertilizers and pesticides, but also manmade chemicals or other contamination that usually arises from direct industrial waste discharge into the soil, percolation of contaminated water or wind contamination.

The most common chemicals involved are PCBs, solvents, lead and other heavy metals. Soil quality control is performed by environmental scientists in compliance with generic guidelines that include field measurement, also using computer models, to evaluate the minimal acceptable level of a substance and eventually determine the clean up options for the contaminated soil.

In the “food processing” industry, raw materials are the main source of contamination. Stores and warehouses often make a large use of a wide range of 151

CNR Environment and Health Inter-departmental Project raw materials. Every product has one (or several) dominant raw material on which the quality of the finished product mainly depends (26). Raw materials control is another CP to ensure food quality and to be performed it requires the use of specific sampling. The formulation of the sampling type and test applied must reflect in the finished product and must be fast, simple and suited to the purpose. These tests can be chemical, physical, bacteriological or organoleptic and are usually performed in those specific laboratories that can authorise the factory to use the raw material.

Finally, food packaging control is needed in order to protect consumers from the package to foodstuff migration of harmful substances. Nowadays, packaging is an essential element in food manufacturing processes because it gives food more safety and a longer shelf life. In Europe, the Commission of European Communities (CEC) controls and establishes directives for the use of plastic packaging materials.

In general, these directives are based on analytical test methods to establish the limits of plastic-package migration into food. These analytical procedures are used: i. to identify the potential migrants and their toxicity; ii. To identify the factors responsible for migration: iii. To estimate the intake of food contaminants; iv. To determine the level of contaminants in the packaging materials and in the food they have been in contact with (27).

After the manufacturing process, food quality can not be modified. Thus finished products examination can only grant acceptance to materials reaching the desired standard or rejection to materials failing to reach this standard.

Food quality control is a concept which evolves as experience and knowledge in the field grows. In the modern world, all 152

food processing undergoes quality control, often based on discoveries derived from basic research in the field. From these observations, it can be argued that in the future there will be a possibility to generate a unique control model and, using modern data processing methods, obtain a continuous monitoring of the events during all the phases of the food production flow.

2.2 Dietary exposure to contaminants: total diet studies From the information collected so far, it is clear that there is a general concern on food quality, contaminations, safety and effects on human health. Moreover, there is the need to put together all information coming from different sources (government, food scientists, local agencies and others) in order to define the best approach to prevent food diseases and to establish general rules for a “better food”. A strong contribute in this direction comes from both basic and applied research. There are three key steps that must be considered when defining a scientific approach to food contamination as proposed in Thacker’s model (Table 1) (28).

Table 1: Thacker’s model Step

Example

Hazard identification

Pesticides, Heavy metals, PCBs

Risk/source of exposure

Diet

Risk evaluation (outcomes)

Effects on human health

First, the contaminant must be identified; then, the source through which the contaminant could reach the consumers must be identified; finally. the adverse

Monitoring Contaminants in Food Chain and their Impact on Human Health effect (hazard) of the contaminant must be determined in order to prevent and/ or protect exposed individuals. In this context, the present paragraph will analyze some examples coming from the scientific literature regarding elegant approaches applied in different countries to estimate human exposure to food contaminants trough the diet (Total Diet Study). In particular, the case of dioxins will be considered. This topic concept will be also discussed in section 5 from a different point of view.

Bilau and co-workers (29) have recently carried out an important study on three age groups of the Flemish population, adolescents (14-15 years), mothers (18-44 years) and adults (50-65 years) to determine the intake of dioxin-like compounds via animal fats or other sources, namely dairy products, added fats, fish and seafood.

The study was performed by assessing the dietary intake of all the participants to the study using a semi quantitative food frequency questionnaire (30). The questionnaire has been used to estimate the daily consumption of fat-containing food items for each participant and, based on their dietary habits, the intake of fat from the different sources (meat, fish, dairy products) was determined.

Contaminant concentration (dioxin and dioxin-like compounds) was measured in food items coming from the Flemish market, via the chemical-activated luciferase gene expression (CALUX) bioassay (31). To estimate the dietary intake of dioxin-like compounds in the studied population a simple approach distribution was used, combining a point estimate for contaminants concentration with the distribution of individual consumption data (32). The result of the studies shows that a large part of the three study groups exceeded the weekly “safe” intake of

dioxin-like compounds and also that this intake decreased with age. Moreover, in the Flemish population fish and seafood resulted as the main source of dietary intake of dioxin-contaminants.

In another study, the same approach was used for Swedish children and adults showing that children are a vulnerable group with a daily over-intake of dioxins from food commodities in particular from fish. For this reason, the authors suggest that it should be useful to perform agespecific dietary intake assessments to protect highly exposed individuals (33).

Similar studies have also been conducted in other European countries with comparing results (see section 5).

However, a key aspect emerging from this scientific work is that in most of the studies only two of the three steps foreseen by the Thacker’s model have been considered: the identification of the contaminant or hazard is fulfilled (e.g, dioxins in foodstuff) and the diet is identified as the source of contamination. It lacks the proof of concept that this low-dose exposure deriving from food and assessed by the Total Diet Study is really responsible for effects on human health (see below on section 5).

In many cases, there is a general assumption that the mere presence of the contaminant will affect or be harmful for consumer safety, now or in the future. It is clear that casual exposure to high doses of contaminants generally represents a threat for human health (e.g, dioxin exposure in Seveso population). However, the low exposure impact of some contaminants, such as dioxins, is still debated. An interesting example on this specific point comes from the Food and Drug Administration (FDA) website regarding questions and answers about dioxins: Q: “What levels of dietary dioxin exposure cause adverse health effects in humans?”; 153

CNR Environment and Health Inter-departmental Project A: “Known incidents of high dioxin levels in humans have resulted from accidental exposures that are not typical with dietary exposures. Despite a large body of research and data collection, there are numerous questions and uncertainties regarding scientific data on and analysis of dioxin risk. These uncertainties are unlikely to be resolved in the near future” (www.fda. gov).

3. CNR SPECIFIC EXPERTISE: QUALIFIED TEAMS, EXTERNAL COLLABORATIONS AND FUNDING

3.1 General overview on food agency: an eye on Europe and Italy “There are certain things only a government can do. And one of those things is ensuring

that the food we eat is safe and does not cause us harm.” (President of United States of America, Barack Obama).

Food always had a strong influence on daily life and production/consumption of food is central to any modern society.

For this reason, at the heart of any foodrelated topic there is the need to consider the citizen/consumer as the final “user” of the total food/feed chain and the one who needs to be protected from any risk of disease. In Europe, the main agency that controls risk assessment regarding food and feed safety is the European Food Safety Authority (EFSA): “EFSA aim is to provide appropriate, consistent, accurate and timely communications on food safety issues to all stakeholders and the public at large, based on the Authority’s risk assessments and scientific expertise” (www.efsa.europe.eu). The main mandate of EFSA is related to risk assessment and risk communication. Risk assessment is a specialized field of applied science that involves the analysis of scientific data and studies in order to evaluate risks associated with certain hazards. This implies scientific 154

data collection and analysis on a wide variety of hazards (e.g, pesticides, PCBs, microbiological agents and others) to gather information on dangers posed from these substances, to develop general methods to assign a date risk for the consumer. One of the key responsibilities of EFSA is to communicate food and feed safety advice to its principal clients, stakeholders and the public in a timely, clear and helpful way, in order to help bridge the gap between science and the consumer.

It is clear that due to the complexity of this issue, and, what is more, the different sources of information ranging from local to international agencies, a key step concerning food safety is the possibility to exchange information among controlling agencies. Nowadays, the Rapid Alert System for Food and Feed (RASFF) in Europe represents a powerful tool to exchange data about measures taken in response to serious risks detected in food or feed. There is a very simple principle at the basis of the RASFF system: “Whenever a member of the network has any information relating to the existence of a serious direct or indirect risk to human health deriving from food or feed, this information is immediately notified to the Commission under the RASFF. The Commission immediately transmits this information to the members of the network” (34) (Fig. 2) .

In Italy, the main actions concerning food quality and control derive from government agencies, local offices and/ or private companies. Most information coming from the government is released by the Ministero delle politiche agricole alimentari e forestali (www. politicheagricole.it), and the National Institute of Health (www.iss.it). At local level, control and communication roles are mainly played by the Environment

Monitoring Contaminants in Food Chain and their Impact on Human Health Protection Regional Agencies (ARPA), the Local Health Authorities (ASL), the National Agrifood Informative System (SIAN) and others, often working in collaboration with the local police. All these agencies develop specific actions and projects to understand, prevent, control and reduce the risk related to food contamination.

Several institutes of the National Research Council (www.cnr.it) are involved in these tasks from different points of view: i. to develop new methods and strategies for analysis; ii. To apply for national and international research projects in the field; iii. To establish collaborations and consultation with official agencies devoted to control activities. In this context, one of the actions in the PIAS project has been to better categorize data in the field of “food chain contamination and effects on human health” originated from the scientific work performed by CNR research groups, an issue that will be further described in the following section.

3.2 CNR research activities: results from PIAS questionnaire The CNR is a public organisation promoting, transferring, communicating and enhancing scientific research in different fields to improve the country’s technological, economic and social activities. The organization is divided into eleven Departments, also in accordance with the research work performed at CNR. All the relevant projects developed at CNR can be viewed surfing the CNR web sites (www.cnr.it; www.cnr.it/progetti/Progetti. html).

Due to the huge amount of information and the many different scientific topics that are part of the research activities performed in the CNR, sometimes it could be very hard to gain data on the field of interest. In this

view, one of the aims of the PIAS project was to clarify, and eventually harmonize, the activities of the different scientists in CNR trough communication, data exchange and eventually collaboration. In our survey, out field of interest was the “monitoring of environmental contaminants in the food chain and their impact on human health.”

To acquire information on the activities related to this topic at CNR, we elaborated a questionnaire which was send to the main Departments and Institutes involved Table 2. Summary of the information obtained from the PIAS questionnaire

Methods applied

Biochemistry, GCMS, HPLC-DAD, analytical chemistry, Gas chromatography, molecular biology, cell biology, bioinformatics, immunochemistry, epidemiology, biomarkers, massspectrometry

Contaminants studied

POPs, pesticides, heavy metals, organic compounds, toxins, hormones

Food

Pasta, bread, milk, cereal, fruit, vegetables, fats

Pathology

Cardiovascular disease, inflammation, reproductive fitness, cancer, neurodegenerative disease, toxinfections, lung disease, genetic disease, immune response

155

CNR Environment and Health Inter-departmental Project in our field of interest. Key points in the schedule were represented by the group composition, expertise, methodologies applied, type of contaminant/food studied, type of pathologies analyzed and main projects developed by the research groups.

The data elaborated from the questionnaire are summarized in Table 2.

From Table 2, we can assume that CNR possesses specific expertise originated by the different groups working on the indicated contaminants, present in food matrices reported in Table 2 by using specific methodologies spanning from cell biology to mass spectrometry. This approach allows the performance of a risk assessment related to specific diseases. It is clear that the data reported in Table 2 probably represent only a fraction of the real competence present at CNR. This underestimation is probably due to an incomplete feedback received from the questionnaire we send. However, the data collected show the existence of the capabilities required to fulfill Thacker’s model, which are already part of CNR scientists’ cultural background. This is a key point, because we can speculate that, in a near future, it will be possible to develop a global, collaborative project in the field of food quality and safety related to the presence of environmental contaminants by merging the different competences coming from the different CNR groups.

4. HIGHLIGHTS 4.1 Heavy metals in food: traditional and innovative detection methods Metals are constituents of the human body and some of them are fundamental for body growth, metabolic reactions and catalysis mechanisms. For this reason they are considered as essential constituents for life, and are distinguished in ‘major’ 156

or ‘minor’ (trace), depending on their concentration levels (35-39).

However, the role that some metals play in the human body is not completely understood. Some metals have been recognized to be certainly dangerous for human health. Cd, Hg, Pb, As, Ni, Al, Cu may cause illness, aging and even genetic defects, and mankind is today exposed to the highest levels of these metals due to their use in industry, to the unrestricted burning of coal, natural gas and petroleum, and to the incineration of waste materials worldwide (40-48).

The term heavy metals is often used in current literature to indicate the toxic metals as a group, although heavy refers to mass (it should be actually used only for elements the atomic weight of which is higher than 200 such as mercury, thallium, lead and bismuth) and mass does not seem directly related to toxicity (49,50).

Plants contamination occurs when heavy metals are present in the soil where they are grown, and animals that are fed with these plants are also contaminated (51,52).

The quantities of the different elements in soil generally vary from place to place, and the amounts absorbed by plants and retained in their tissues can also show large variations (15,53-60). Therefore, there can be considerable variations in concentrations and also in isotopic composition of metals even within the same class of food, depending on its geographical origins and other factors.

For these reasons, concentration and/ or isotopic ratios of metals can be used, sometimes with success, as indicators of food provenance (61-71).

Changes in concentrations and isotopic composition of metals in food may be not confined to primary, geographicallyrelated variations. They also may be due to food manufacture, and in some cases,

Monitoring Contaminants in Food Chain and their Impact on Human Health

Figure 2. Schematic representation of the information flow of the RASFF trace of metals can cause undesirable preceded by exchange chromatography) changes in food during cooking and/or (109-112), with ICP-MS (113,114) and with storage (72-75). All these factors may have GC-MS (115). consequences on the consumers; therefore, In some of these techniques (AAS-F/GF, many government authorities have specific ICP-AES/MS, ID-TIMS/ICP-MS), samples rules for food manufacture. are reduced to a perfectly homogeneous In the last two decades, analytical solution before the instrumental analysis, techniques and instrumentation to with the exception of liquids, such as determine concentrations of metals in beverages (including water) which may foods have been improved.(76-80). A only require dilution. In most foods, limited number of methods are mainly organic matter must be removed by used in this field and they include: oxidation because it would interfere with Atomic Absorption Spectrophotometry the analytical process, either by the use of (AAS) (81-86), Spectrofluorimetry, Gas oxidising acids in a wet digestion or by dry Chromatography-Mass Spectrometry ashing in the presence of air or pure oxygen.

(GC-MS) (87,88), Inductively Coupled Virtually, all organic matrices of food can Plasma-Atomic Emission Spectrometry undergo these two different preparation (ICP-AES) (80,83,89-91), Inductively procedures and the choice depends mainly Coupled Plasma-Mass spectrometry (ICP- on the metal(s) to be determined. Dry MS) (83,89,92-108), Isotope Dilution ashing technique at 550째C causes Hg, Sn combined with Thermal Ionization Source and As loss by evaporation. Thus, for these Mass Spectrometry (ID-TIMS) (usually elements sample digestion with HNO3 157

CNR Environment and Health Inter-departmental Project and H2O2 in closed PTFE vials within a microwave system is the typical preferred procedure(101,104,116-121).

AAS is a very versatile technique which does not need a particular laboratory and instrumental condition, and AAS-GF may allow determinations at ultra-trace concentration levels (<1 ppm). However, in this technique, only one element at a time can be determined, and, due to relatively low operative temperatures, determination of the refractory metals (e.g, REE, Sc and Y) is substantially precluded (83).

Due to definitely higher temperature in the torch, ICP-AES/MS is a powerful tool to determine all the metals, including the refractory ones, combining high sensibility with considerable accuracy and fastness (80,83,89-108). In particular, ICP-MS, by rapid determination of isotope ratios of elements, can be combined with isotope dilution technique in multi-collector instruments (MC-ICP-MS), greatly increasing the precision and the accuracy of the determinations (113,114,122,123).

At present, ICP-MS technique has considerably extended its capabilities, by combining with different separation procedures, as chromatography and electrophoresis, and by developing of methods of sample introduction, such as flow injection, thermal vaporisation and laser ablation (92,95-100,106,124-126).

Unlike the AAS system, however, the ICP systems require quite rigorous laboratory and instrumental conditions to improve the stability of emission (78).

LA-ICP-MS is a recent, very effective method to determine metals in food samples. Since substantial part of the sample preparation is avoided (there is no preparation except that food is dried at 110째C), when compared with classical ICP-MS, this technique is faster, and possible contamination effects due to 158

reagents is greatly limited. Moreover, it allows accurate mapping of the analyzed sample. However, due to kinetic effects in laser ablation and/or in sample transfer to ICP-MS system, elemental fractionation is generated, so that results obtained by LA-ICP-MS technique must be considered semi-quantitative, unless the effects of fractionation can be corrected by instrumental calibration with adequate standards (101,127,128).

Isotope dilution technique may be of very high sensitivity (< 1 ppb), depending on the isotopes and on their enrichment in the tracer (spike) which is mixed to the sample that must be analyzed. Moreover, precision of the isotope dilution method in determining the concentration of a metal is related to the uncertainty which afflicts the isotopic ratio of the element which is measured for the technique.

This uncertainty is amplified by a sort of magnification factor, the value of which depends on sample/spike weight ratio.

Over- or underspiking of sample must be carefully avoided because they may determine magnification factor values which are significantly higher than 1 (109112).

ID-MC-ICP-MS is greatly faster than ID-MC-TIMS but the disadvantage is the definitely lower precision both in measuring isotopic ratios and in measuring concentrations, so that ID-TIMS technique, when possible, can be considered the most effective method (61,122,129,130).

If metals must be determined at ultratrace concentration levels (<1 ppm), particular care should be taken in avoiding any possible source of contamination during sample preparation. At present, inductively coupled plasma multi-collector mass spectrometry and laser-ablation inductively coupled plasma multi-collector mass spectrometry, because of their dynamic

Monitoring Contaminants in Food Chain and their Impact on Human Health range and capability for multi-element analysis, are the most valuable methods for the analysis of trace elements (78,83, 90,93,101,102,104,107,108,127,128,131).

Investment and operational costs for ICPMS technique are however high, and are not justified if a limited number of elements must be determined for a limited number of samples. In this case, AAS technique or related will be preferred. Instead, for the analysis of several elements in a large numbers of samples the ICP-MS technique is economically the most advantageous.

For this reason this technique is mainly used in the most important government analytical centres, where a large number of elements in a variety of food matrices are routinely determined (78,131,132).

4.2 Monitoring dioxins in food and in biological matrices by high resolution mass spectrometry In recent years, based on a growing body of evidence, there is an increasing concern about the possible health threat posed by substances present in environment, food and consumer products termed endocrine-disruptors (EDs) and defined as â&#x20AC;&#x153;exogenous substances that cause adverse health effects in an intact organism or in its progeny, consequent to changes in endocrine functionâ&#x20AC;? (133,134).

The group of molecules identified as EDs is highly heterogeneous and includes synthetic chemicals used as industrial solvents/lubricants and their by-products [polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), dioxins], plastics [bisphenol A (BPA)], plasticizers (phthalates), pesticides [methoxychlor, chlorpyrifos, dichlorodiphenylt r ichloroethane (DDT)], fungicides (vinclozolin), and pharmaceutical agents [diethylstilbestrol (DES)]. Moreover, some naturally

occurring compounds, present in plants and termed phytoestrogens, have been found to posses estrogenic properties.

The majority of phytoestrogens belongs to the large group of flavonoids. EDs have long environmental half-life resulting in a continue increase of their global concentration in the environment; furthermore, they have very low water solubility and extremely high lipid solubility, leading to their bioaccumulation in adipose tissue. Although several studies have definitively assess the toxic properties of those polluting compounds, conclusive evidences are still lacking on the effect of low doses exposition and on the synergistic effect of complex mixtures of compounds. Different studies have been performed in Germany(135), Belgium (136), Sweden (33) and Japan (137), in order to evaluate the body burden levels of PCDDs/PCDFs and DL-PCBs on general population. However, similar studies have not been performed on general population in Italy. The only data available up to now for Italian population are those regarding Seveso population who experienced the highest levels of TCDD exposure known in a residential population (138,139).

Concerning the chemical properties, PCDDs, PCDFs and PCBs constitute a group of 419 persistent environmental chemicals. Only 17 congeners among PCDDs and PCDFs and 12 congeners among DL-PCBs cause toxic responses similar to those caused by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener within these groups of compounds. PCDDs, PCDFs, and PCBs exist in environmental and biological samples as complex mixtures of various congeners with different rates of degradation due to their different solubility and volatility. Therefore, the relative concentration of congeners differ 159

CNR Environment and Health Inter-departmental Project across trophic levels and the composition of these mixtures is often very different from the one originally released into the environment. The complex nature of these chemicals complicates the health risk evaluation for humans. In order to facilitate risk assessment and regulatory control of exposure to these mixtures the concept of toxic equivalency factors (TEFs) has been developed. TEF values are used to calculate the toxic equivalent (TEQ) concentrations in various matrices (animal tissues, soil, sediment and water). TEFs and TEQs are used for risk characterization and management purposes (140,141).

In the frame of PIAS project, in a tight collaboration with the other groups participating to the project, we propose to monitor the concentration of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin like polychlorinated biphenyls biphenyls (DL-PCBs) in blood samples from non occupationally exposed subjects as well as in selected food matrices, typical of the Mediterranean diets such as milk, mozzarella cheese, meat and fish, which, being particularly rich in the lipids, bio accumulate such molecules. For the different matrices, the concentration of some dioxins congeners (PCDDs, PCDFs, PCBs) will be determined by isotope dilution high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) (142-144).

Experimentally, after adding PCDD/F and PCB congeners isotopically labelled with the isotope 13Carbon, the samples will be submitted to specific extraction and gel clean-up steps. PCDD/F/PCB concentration will be reported as pg/g fat, pg WHO-TEQ/ g fat. The measurements will be performed by means of HRGC/ HRMS: the mixtures will be separated on a Gas Chromatographer, using a 160

DB-5 capillary column, coupled on line with a high resolution double focusing mass spectrometer. The Proteomic and Biomolecular Mass Spectrometry Center is equipped with a Autospec NT instrument (Waters) specifically suitable for the analysis of dioxins and dioxin-like compounds, having a resolution higher that 10.000 FWHM. The use of high resolution capillary gas chromatography and highly selective MS conditions (select ion monitoring, resolution > 10.000, accurate m/z assignment to 0.001 Da) greatly reduces the potential for coextracted compounds to interfere with the measurements of those analytes. Moreover, the use of PCDD/F and PCB congeners isotopically labelled allow the accurate calculation of the analytes concentration.

However, it should be underlined that the determination of dioxins concentration in blood and food samples requires long and laborious analytical procedures, high cost of analysis (about 300 â&#x201A;Ź/sample) as well as the use of dedicated instruments and highly expert operators.

The proposed biomonitoring will provide results of relevant importance for the estimation of the toxic human burden due to both the environmental exposure and the food chain. Information on whether and what extent chemical substances are really taken up from the environment (internal dose) are of fundamental importance for the evaluation of the related risk for human health and to elucidate the effect of low dose exposure.

4.3 Biomarkers to determine dietary exposure to xenobiotics: the case of cytochrome P450 The human cytochrome P450 (CYP) superfamily, containing 57 genes (145), contributes to the metabolism of a variety of xenobiotics including drugs,

Monitoring Contaminants in Food Chain and their Impact on Human Health carcinogens, constituents of food including chemicals present as pollutants (146). The resultant increases in polarity usually facilitate excretion and are considered to be a detoxification process, but in some cases foreign compounds are converted to products with much greater toxicity (147). Chemicals present in the diet may be metabolized by CYPs to non-toxic metabolites and excreted, however the formation of toxic metabolites is possible (148). It was reported that xenobiotics may be substrates, inhibitors or inducers of CYPs. Natural products present in cruciferous vegetables have been shown to selectively up-regulate CYP1A1 and CYP1A2 isozymes on chronic ingestion (148). On the other hand, several natural products selectively inhibit mono-oxygenation, especially in the intestine, and may lead to increased bioavailability and reduced metabolism of dietary components (149).

CYP1A is important as it is involved in bioactivation of ubiquitous environmental contaminants such as polychlorinated dibenzo p-dioxines and in the past much concern has been focused on the induction of CYP1A as sensitive bioindicator for the exposure of fish to these contaminants in the marine environment (150). In this context, we demonstrated that CYP1A can be a useful probe for the exposure of adult sea bass and frog to polycyclic aromatic hydrocarbons (151). β-Naphtoflavone, a typical polycyclic aromatic hydrocarbons resulted in an induction in the liver of CYP1A and the induction was manifested by: i. immunoblot analysis using anti-rat CYP1A1; ii. an increase in CYP1A-mediated methoxyresorufinO-demethylase and ethoxyresorufin-Odeethylase activities.

We also demonstrated that the CYP2Alike inhibition can be used as biomarker of exposure of herbicides, such as

dichlobenil. Expression of CYP2E1 in human circulating lymphocytes has raised clinical interest because it has been proposed as a potential non-invasive bioassay determination of CYP2E1 expression and activity in vivo (152). An elevation of CYP2E1 has been reported in lymphocytes from poorly controlled diabetic patients (153). Considering that cytochrome P450 can be induced by several xenobiotics, we can suppose that components of the diet, mainly those present as pollutants or additives, can modulate the cytochrome P450 isoforms. For this reason we can use this system as biomarker to assess dietary exposure to xenobiotics. The studies could be performed using animal models, by the administration of extract of food to evaluate modulation of CYPs. This aspect can also be investigated in humans using lymphocytes to assess if some CYP isoforms are induced and/or inhibited following ingestion of contaminants present in the diet.

4.4 Marine invertebrate as model to assay the effect of xenobiotics on reproduction In the last decade, the international scientific community has become increasingly concerned that exposure to low levels of synthetic chemicals or xenobiotics may disturb hormone function in man and animals (so-called endocrine disruptors â&#x20AC;&#x201C; Medical Research Council (UK), 1995; Danish Environmental Protection agency, 1995). During the past 50 years, large quantities of diverse xenobiotics have been released into the environment as a consequence of efforts to increase agricultural productivity and as a result of modern manufacturing processes and their by-products. These chemicals include herbicides, pesticides, fungicides, plasticizers, polystirenes, PCBs, polychlorinated dibenzodioxidins, 161

CNR Environment and Health Inter-departmental Project alkylphenolic compounds (154), organotins, and more specifically tributyltins (TBT), used for its biocide properties as the active agent in antifouling paints (155).

There is increasing evidence that these xenobiotics in the environment may disrupt the endocrine systems of aquatic life and wildlife. In addition, EDs and other food-contaminating environmental pollutants represent a high risk factors in animal reproduction (156). Such chemicals are receiving more and more attention, particularly because several compounds not specifically designed to possess endocrine activity have been shown to possess unexpected hormonal activity in a wide variety of organisms.

Reproductive hormone-receptor systems appear to be especially vulnerable; in fact, some EDs can interfere with the normal mechanisms of steroid hormone action and with the embryonic development of the male and female reproductive systems of wildlife and experimental animals which in turn may affect normal reproductive functions in adulthood (154). It has been demonstrated that xenobiotics acting through steroid-dependent mechanisms, interact with estrogen receptors, androgen receptors, or with certain steroid binding proteins (ABP, SHBG). The endocrine and reproductive effects of EDs are believed to be due to their ability of: i. mimicking the effects of hormones; ii. altering the pattern of synthesis and metabolism of hormones; iii. antagonizing the effects of hormones; iv. modifying hormone-receptor levels (157).

In general, the magnitude of the cellular response to hormones is dependent upon the number of receptors occupied by the hormone which in turn is related to hormone concentration. Therefore, EDs could potentially alter endocrine functions by influencing the concentration of hormones through changes in the rates of their 162

secretion or metabolism, or by interfering with hormone action at the receptor or at other sites along the hormone signal transduction pathway. It is well known that many aspects of the reproduction in vertebrates are under the control of hormones and sex steroids and a great deal of evidence has been accumulating, showing that it may also be the case in invertebrates and fish (158-161). Several types of sex steroids have been detected in various species of invertebrates (162-164).

Estradiol-17β and progesterone have been found in the tissue and hemolymph of the American lobster (165,166). In Paeneus monodon estradiol-17β and progesterone levels in the hemolymph, ovaries and hepatopancreas were related to the ovarian stage of development (167). Injections of progesterone and 17αOH progesterone induce ovarian maturation in Metapeneus ensis (168) and stimulate vitellogenin secretion in Paeneus japonicas (169) Estrogens stimulate vitellogenin synthesis in Macrobrachium rosenbergii and in Paeneus monodon (167,170). In the female of Pandalus kessleri the level of estradiol coincides with vitellogenin in the hemolymph (166). In P. monodon estrogen treatment during vitellogenesis may suppress molting, while stimulating vitellogenin production (167). Sex steroid hormones (androgens, progesterone, estradiol-17β) and 3 β-hydroxysteroid deydrogenase, a key enzyme in steroidogenesis, have been reported in the gonad of the male of the cephalopod Octopus vulgaris (171-174).

Many animal models are suitable for comparative studies with mammalian models in particular the marine invertebrate Ciona intestinalis (ascidians) share many common biological mechanisms with vertebrates (175). The effect of compounds deriving from marine diatoms have been

Monitoring Contaminants in Food Chain and their Impact on Human Health already investigated showing an influence at molecular level on the initial mechanism of fertilization. This effect seems also to influence the following embryo development (176). Similarly to ascidians, also the mollusk Octopus vulgaris share basic biological mechanisms with mammals. The germinal vesicle breakdown which is the first event in oocyte maturation appears to be supported by an ion current activity of specific L-type calcium channels occurring also in ascidians and mammals (177-179). At present, a study is in progress on the effects of four different heavy metals lead, cadmium, zinc and copper on the ion currents present on the plasma membrane of the oocytes and on the electrical events involved in the processes of maturation fertilization and embryo development of the ascidian Ciona intestinalis. Data obtained show an inhibition of either plasma membrane currents and the first events of fertilization.

These results suggest a plausible negative impact of the xenobiotics on the early events of reproduction in model animals.

Although the ability of some environmental chemicals to exert toxicity on human health and reproductive fitness remains largely speculative, evidence are accumulating that multiple stressors from contaminated environment may adversely affect populations of marine animals and mammals such as humans by interfering with similar known processes of the reproductive process (58,180).

5. FUTURE PERSPECTIVES: THE EFFECTS OF LOWDOSE EXPOSURE TO DIETARY CONTAMINANTS IN CHILDREN AND YOUNG ADULTS: A WORKING HYPOTHESIS

Specific goals of PIAS are to propose new projects at national and European level which may fill some of the gaps in the

literature regarding the complex interaction between contaminants and human health.

This working group identified the need to determine a real cause-effect relation between level of contaminants in the diet, their â&#x20AC;&#x153;realâ&#x20AC;? presence in selected human populations and their effect on health. In current scientific literature, this problem has been successfully approached with excellent studies where measurements of contaminants present in the environment and bio-accumulated in the food chain have been linked to individual consumption extrapolating, from these data, the human intake in specific age groups. As already mentioned above (section 2.2), a significant example comes from the study of Bilau and co-workers (29) who report data on the dietary exposure to dioxin-like compounds in adolescents, their mothers and adults, a result of the Flemish Environment and Health Study (www.milieu-en-gezondheid. be). They demonstrated that in the selected aged groups, the median (95th percentile) estimated daily intakes of dioxin-like contaminants were 2.24 (4.61), 2.09 (4.26), and 1.74 (3.53) pg CALUXTEQ kg-1bwd-1 for, respectively, adolescents, mothers and adults. These values exceed the tolerable weekly intake (TWI) of 14 pg WHO-TEQ kg-1bww-1, as derived by the Scientific Committee on Food (181).

The relative validity and reproducibility of this experimental approach was assessed by the same authors in a different study (182). Here, they concluded that the food frequency questionnaire designed to estimate the intake of dioxin-like contaminants represents a valuable tool for ranking individuals in the study population on the basis of estimated intake of dioxin-like contaminants. However, absolute intakes should be estimated without correction factors and interpreted with caution. In a similar study carried 163

CNR Environment and Health Inter-departmental Project out by a Swedish group within the EU funded CASCADE Network of Excellence (Contract N. FOOD-CT-2003-506319) the dietary intake of dioxin-like pollutants was investigated in children and young adults (33). The results showed that among the selected Swedish population, boys and girls up to the age of ten years had a median TEQ intake that exceeded the tolerable daily intake (TDI) of 2 pg TEQ/ kg body weight. Dairy and fish products were the main sources of exposure. In fact, the individuals most highly exposed were characterized by a high consumption of fish.

Also in this case, exposure was estimated matching the concentration data of dioxinlike compound in food commodities (meat, fish, dairy products, egg, edible fats and other foodstuff) with food intake data.

Similar studies on dioxins exposure via food were performed in several countries, generally showing that estimated dietary intake is above the recommended TDI level ranging from roughly 2–6 pg TEQ/kg bw/ day (183-192). On the opposite, an Italian study established that the mean value of dioxins measured in food of animal origin by isotope dilution method was 0.144 ± 0.266 pg-TEQ/g (range: 0.003–1.655 pgTEQ/g). The average daily food intake was obtained from national data collected by the National Institute of Nutrition, and from a cohort study on diet and cancer including 40,000 Italian subjects. The conclusion was that the major contribution to dioxins intake with food comes from cow milk and fish consumption and were below the limits set by the European legislation (193).

Apparently, the adherence to the limits established by EC (194) was confirmed in parallel studies carried out in different Countries such as Germany (195), Finland (196), Japan (197) and Spain (198).

As discussed by others (193), many of the studies cited suffer from the same 164

limitations: i. the amount of dioxins, or other contaminants, intake with diet was estimated from national surveys or epidemiological studies, without measuring dioxins content of a certain food and the individual intake of that foodstuff; ii. data were obtained through a monitoring program, not as part of a research project; this means that the aim of the monitoring was not to study human exposure through food, but to assess food content of dioxins and other residues; iii. dioxin content was lacking for certain food consumed within the population, making the analyses incomplete.

Based on this preamble, we proposed within the PIAS project a large, multicenter and multidisciplinary study with an extended follow-up which will take into account the missing information existing in the Literature. The target population will be represented by children and young adults. In fact, they constitute a vulnerable group and previous studies suggest that it is essential to perform age-specific dietary intake assessments to more carefully consider, in the risk management processes, sensitive and/or highly exposed individuals in the population.

The general objective of this proposal is: to address the healthy status of a young population determining the concentration of xenobiotics which come to humans through the food chain.

The proposal consists in two phases: Phase I The working hypothesis is illustrated in Fig. 3. The experimental aim is to study populations of children, adolescents and adults living in various Italian regions, including Campania (South Italy) establishing biological banks (mainly blood and urine samples). These individuals will be selected from large Italian cohorts that are at least in part already available

Monitoring Contaminants in Food Chain and their Impact on Human Health from ongoing European projects involving members of PIAS working groups. The Italian study sample should be composed by about 2000 individuals with males and females equally represented. Additional cohorts with similar features from other Countries potentially interested to participate will be enrolled in the study to obtain comparable information at European level. At baseline the following variables will be measured: in complete anthropometry including body composition measures; biochemical measures, including selected hormones; physical activity tests; medical history, behavioral and socio-economic questionnaires; foodfrequency questionnaire and repeated 24h dietary record. All these variables will be measured again in the same population in the follow-up survey.

Biological samples, preferentially blood and urines, freshly collected or, where possible, already available if conveniently stored for the expected analyses, will be employed to measure the presence of those contaminants whose presence was independently and previously verified in food commodities taken directly from or through the food chain. A careful evaluation of different types of contaminants/xenobiotics to be analyzed in the present project is actually under scrutiny from members of PIAS working groups. The selection will certainly include compounds belonging to the following categories: pesticides, dioxins and dioxinlike molecules and heavy metals. For the choice, two main criteria will be followed: i. presence of these compounds in the diet of the selected individuals; ii. availability of official methods to detect them in biological samples and foods.

At the end of this phase of the study, we will relate the level of contaminants present in foods and biological samples

with epidemiological data from the populations under study (dietary habits, health status) to determine the potential association between concentrations of selected contaminants and health effects.

General methods. In order to assess the exposure in children and young adults, the individuals will be stratified by gender and age. Individuals with incomplete information on body weight or food consumption will be excluded. The 100 most commonly consumed food items will be collected and analyzed by standard methodology to assess the presence and concentration of selected contaminants.

Food items will be obtained from producers or purchased from different stores in the cities where the cohorts will be recruited.

Accordingly to reports periodically published by EC (199-201), the food groups chosen for the study will be: i. fish, dairy products, egg, edible fats and other fat-containing products for the presence of dioxins, dioxin-like molecules and selected metals; ii. cereals, fruits, vegetables, beverages, vegetable soups and sugar for the presence of pesticides, biocides and heavy metals. Exposure to different xenobiotics based on consumption of various food items by each individual will be expressed accordingly to international units establish for each specific contaminants. Data will be analyzed by standard statistical methods.

In particular, dioxins concentration in different biological samples and in food groups will be associated with selected health outcomes after adjustment for age and gender. The dioxin levels in different food groups (fish, meat, dairy products, egg, edible fats, other fat-containing products, fruits, vegetables, cereals, etc.) will be compared and related to the individual intake of each food item to assess the principal sources of exposure both at the individual and at the population level. The 165

CNR Environment and Health Inter-departmental Project proposed sample size of 2000 individuals is large enough to allow statistical power for different series of analysis. For instance, it will allow to detect a 5% difference in the amount of ingested contaminants between groups, at alpha =0.05 and beta=80%.

Phase II Information obtained from doses of contaminants in biological samples and above the threshold established by international health agencies will represent the starting point for phase II of the proposal devoted to determine whether and how, from a molecular point of view, exposure to these xenobiotics may interfere with the normal physiological state of the cell/organism resulting in pathological conditions in adults. As schematically represented in Fig. 3, phase II will take advantage of different cellular and animal experimental models suitable to address cause-effect relation in specific chronic diseases potentially associated to exposure and/or accumulation of dietary contaminants. Decision about the diseases on which to focus our attention will strictly depend upon results obtained after phase I. In choosing the research groups to assign these specific tasks, we will primarily consider expertise and competence within CNR, as resulted from the census questionnaires settled down during the course of PIAS program (see section 3.2 above). Phase II will also takes advantage of the work and competence deriving from other working groups within PIAS (e.g, endocrine disruptors). As an example of activity performed during phase II of the project, great importance will be devoted to assess the potential effect of xenobiotic exposure to reproductive fitness and development (see section 4.4) and to the role of cytochrome P450 in metabolizing xenobiotics (see section 4.3).

Key issues to be addressed in order to 166

correctly evaluate and interpret data deriving from the experimental models employed in phase II concern: i. genetic background; ii. adaptive response/ hormesis; iii. bioavailability and metabolism of different xenobiotics.

Risk estimates routinely reflect numerous sources of both uncertainty (which describes the range of plausible risk estimates arising because of limitations in knowledge) and variability (which describes the range of risks arising because of true differences).

Among them, and besides age and gender, genetic differences among members of the population may play a relevant role.

Since the majority of the study on dioxins have been conducted using genetically homogeneous inbred mice to characterize the risk, their conclusions should be taken very cautiously when applied to the genetically variable human population.

Although well-designed occupational and environmental epidemiological studies can yield useful information on human population variability, relatively little quantitative information is available about the potential impact on genetic polymorphisms in the human population that might give rise to differences in susceptibility to the toxic effects of dioxins, and DLCs. As an example of candidate gene, the Aryl hydrocarbon receptor (AhR or AHR) is a cytosolic transcription factor able to bind to chemicals such as TCDD, leading to changes in gene transcription.

A state of the art revision of the literature will be done in the course of this project to identify candidate genes or biological pathways to be explored with genetic studies. The panels of SNPs in selected genes will be genotyped in the laboratories of ISA-CNR, using up-to-date genotyping technologies. At the current status, allelic discrimination will be performed by TaqMan速 genotyping assay. The use of

Monitoring Contaminants in Food Chain and their Impact on Human Health other techniques like SNPs array will be considered taking into account the number of samples/SNPs to be evaluated and the cost of the assay at the time of genotyping. The genotyped SNPs will be uploaded in a central database and linked to the phenotype data. To minimize the population stratification bias, a potential source of false positive associations in genetic population studies (202), genetic analyses will be restricted to individuals of Caucasian origin. Additionally, it is likely that all the cohorts will belong to single countries, with the majority of participants coming from specific delimited geographical areas, thus further reducing the risk of population admixture.

Hormesis is a biphasic dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition resulting in a U- or inverted U-shaped dose response (203). The phenomenon of biphasic dose-response relationship has received considerable attention over the past few years (204). A good example on the application of hormesis phenomenon to human health derives from exposure to heavy metals, such as lead, cadmium, mercury and arsenic. Cadmium is a potent carcinogen in a number of tissues, and is classified by IARC as a human carcinogen (205). Reactive oxygen species (ROS) are often implicated in cadmium toxicology, either in a variety of cell culture systems (206-209), or in intact animals through all routes of exposure (210-213). However, in contrast to acute toxicity, the roles of ROS in chronic cadmium toxicity and carcinogenesis have been controversial depending on experimental conditions.

On the other hand, administration of cadmium to animals at low levels for one year increases hepatic and renal glutathione levels, without elevations in tissue lipid peroxidation levels (214).

A biphasic ROS response to cadmium exposure through the drinking water has also been proposed. ROS and ROS-related gene expression occur right after cadmium exposure, but return to normal levels after 8 weeks of exposure (215). A further example comes from chromium (VI), a well-known mutagen and carcinogen that produces ROS during formation of reactive chromium intermediates (216218) and induces oxidative stress (219).

ROS are known to be generated in various cell types, such as K562 leukemic cells, J774A.1 murine macrophages (220) and human epithelial like L-41 cells (221) when acutely exposed to chromium (VI). A potential adaptive response were obtained when immortalized rat osteoblasts (FFC cell line) and U937 were exposed to 0.050.5 micromolar chromium (VI) for 4 weeks (222). In addition proteomic analysis of both FFC and U937 cells exposed to 0.5 micromolar chromium (VI) resulted in a differential time dependent regulation of glycolytic, stress and cytoskeletal proteins that play an essential role in normal cellular functioning such as energy metabolism, cell signaling and proliferation (223).

Hormetic responses to xenobiotic exposure likely occurring as a result of overcompensation by the homeostatic control systems operating in biological organisms have been discussed in excellent reviews which commented on the economic implications of hormesis (203,224-231).

Bioavailability refers to the extent to which humans and ecological receptors are exposed to contaminants from soil, or sediment directly or through the food chain (an extensive and excellent review on this topic has been published by the Committee on Bioavailability of Contaminants in Soils and Sediments of the National Research Council)(232). Our interest in determining the bioavailability 167

CNR Environment and Health Inter-departmental Project of potentially contaminants adsorbed with the diet is related to their risk assessment for human health. However, the concept of bioavailability has recently been exploited by hazardous waste industry as an important consideration in deciding how much waste can be left in place without creating additional risk if these contaminants are not bioavailable (232).

In terms of effects on human physiology, once absorbed, contaminants may be metabolized, excreted, or they may cause toxic effects. Several levels of uncertainty are associated with bioavailability, including: i. limited knowledge about how biota modify bioavailability of chemicals which come into contact with digestive systems, and whether information obtained for one species is representative of others; ii. the effect of food processing (cocking, pressing, etc.) which makes extremely difficult to precisely calculate the daily intake of xenobiotics, despite the nominal values determined in the single food component; iii. the synergic or antagonistic effect on absorption and metabolism of xenobiotics by other food components, such as polyphenols. In this contest, literature reports examples in both directions: the protective effect of oral resveratrol on the sub-acute toxic effects of TCDD in C57BL/6J mice (233), or the confounding activity of contaminating metals which may interfere with the regulated absorption, distribution, and excretion kinetics of essential metals (234), although this study conclude that food contaminations with metals are too low to have an impact on the bioavailability of essential metals. In general, a higher priority could be given to studies exploring combinations of nutrients, xenobiotics and food contaminants, at realistic intestinal concentrations, with hazardous or beneficial impacts on human health using 168

high throughput in vitro tools (235).

6. CONCLUSION “We are what we eat,” says Ayurveda, the ancient Indian science of life. This sentence immediately clarifies the perception of how food can influence our lives and the relevance of key issues as food safety on human health. In this Chapter, we reviewed critical aspects concerning food contamination and its impact on health.

Our analyses showed that several gaps and uncertainties remain, despite the great concern for “food integrity” and the variety of scientific contributions from different fields, such as environmental science, food chemistry, human epidemiology. These limits cannot be bypassed only with the efforts of the scientific community, but the contribution of Governments and international environmental and healthy agencies is mandatory to face and rapidly solve these deficiencies. This is a critical issue: in evaluating food safety, no gaps are allowed since bad or incomplete information may result in an enormous hazard for consumer’s safety and for the negative consequences that the presence of a contamination in the food chain may generate to the social and economical life of a region.

To correctly approach the issue of the negative influence (if any) of chemical contaminants reaching people throughout diet, we underlined the need of an integrated approach which considers key factors, such as the genetic background of exposed subjects, adaptive responses, age dependent accumulation and bioavailability of specific compounds. Very recently, the publication of the new European regulation concerning the use of pesticides in agriculture (17), raised the problem of the relation existing between exposure to

Monitoring Contaminants in Food Chain and their Impact on Human Health

Figure 3: Schematic model of research program proposed within PIAS low dose contaminants and their potential harmful effects in developing chronic pathologies. Several validated experimental models exist to study the consequences on human health of acute exposure to elevated concentration of chemical contaminants.

On the opposite, a general consensus is still far to be reached on how to assess the effect of prolonged, low-doses exposure to environmental contaminants. Perhaps, a lessons may derive from studies in the filed of radiation exposure.

In this context, PIAS proposed a large, multidisciplinary research project aimed to fill, at least in part, the lack of information existing in the field. The realization of this study cannot be obtained solely by expertise already present within the CNR, but requires the strong contribution of national and European groups with proved experience in the numerous and different

fields considered by the proposal (Fig. 3).

The realization of an integrated approach to assess the impact of food contamination on human health represents, in our view, the only correct way to increase scientific knowledge and build trust and confidence in the consumersâ&#x20AC;&#x2122; beliefs.

KEYWORDS: heavy metals, pesticides, dioxins, xenobiotics, food chain, human health, bioavailability, hormesis.

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The pilot study on Endocrine Disruptors D.G. Mita CNR, Institute of Genetics and Biophysiscs Adriano Buzzati Traverso (IGB), Naples, Italy mita@igb.cnr.it

ABSTRACT Endocrine disruptors have been described as “exogenous chemical substances or mixtures that alter the structure or function(s) of the endocrine system and cause adverse effects at the level of the organism, its progeny, populations, or subpopulations” (EPA, 1998).

Several experimental studies reported that also very low doses of endocrine disruptors can affect the endocrine system causing diseases and altering the development of mammalian (humans included) and non-mammalian species. Among the diseases associated with the exposure to endocrine disruptors cancer, cardiovascular risk, modulation of adrenal, gonad and thyroid functions, and endometriosis are those that mainly catch the public concern considering their social cost.

This paper describes the research activity planned in the pilot study on Endocrine Disruptors granted by CNR in the general contest of the Environment and Health Inter-departmental Project (PIAS-CNR).

1 INTRODUCTION Over the past 50 years, some chemical pollutants, such as pesticides, flame retardants, alkylphenols, polychlorinated biphenyls, phthalates and metals have been released into the environment in an increasing way. Some of these substances, owing to their ability of interfering with hormonal activity, are called “Endocrine Disruptors” (EDs). According to the definition proposed by the Agency for Environmental Protection (EPA) of the United States, the Endocrine Disruptors are “Exogenous agents that interfere with synthesis, release, transport, binding, action or elimination of natural hormones responsible for the maintenance of homeostasis and the regulation of developmental processes and/or behavioural problems”. The effects of these compounds on endocrine functions

in animals, and hence in humans, result in an increase of the incidence of endocrinerelated cancers, increased risk of cardiovascular diseases, reduced fertility and in the alteration of development processes. Endocrine disruptors reach living organisms through air, soil and water.

The major route of transmission, however, remains the aquatic environment, where these substances bioaccumulate through the food chain. Even at very small doses EDs perform their harmful activity (1).

The concerns regarding the exposure to EDs are mainly due to: 1) the adverse effects observed in some wild animals, fish and ecosystems; 2) the increase of some human diseases related to the endocrine system; and 3) the alterations of the endocrine functions observed in laboratory animals after exposure to some environmental chemical pollutants.

Already in 1996, the USA-EPA identified

CNR Environment and Health Inter-departmental Project the endocrine disruptors as one of six priority areas of research.

Human health effects associated with the presence of environmental endocrine disruptors have been recognized throughout foetal development, loss of reproductive capacity, changes in sexual behaviour, and onset of cardiovascular diseases (through obesity) and endometriosis. In addition, it has been observed an excessive cell proliferation and carcinogenesis as well as effects on the neurological and immune system.

2. STATE

OF THE ART: INTERNATIONAL AND

NATIONAL INITIATIVES

The Endocrine Disruptors are one of the major topic of the International and European research on risk assessment in food and environmental safety. The major international agencies have proposed to study the problems associated with the exposure to Endocrine Disruptors from different points of view. Just to give an example, the International Program for Chemical Safety (IPCS) of the World Health Organization in 2002 published the Global Report Assessment on the endocrine disruptors knowledge (http:// www.who.int/ipcs/publications/new_issues/

endocrine_disruptors/en/index.html), whose main objective was the critical review of the scientific evidence of the association between exposure to Endocrine Disruptors and the damage to human health or ecosystems.

Moreover, the Organization for Economic Co-operation and Development (OECD) has dedicated its attention mainly to the development and harmonization of strategies to identify Endocrine Disruptors and characterize their effects on humans and ecosystems with the establishment of the Working Group of Endocrine 182

Disruptors Testing and Assessment (http:// www.oecd. org/document/62/0,2340,en_2 649_34377_2348606_1_1_1_1,00.html).

Europe has not underestimated the problem of EDS. The first definition of the problem took place during the European Workshop on “The Impact of Endocrine Disruptors on Human Health and Wildlife (Weybridge 2-4/12/1996). Ten years later there was a new European Workshop on the “Impacts of Endocrine Disruptors” (Helsinki, 8-10/11/2006).

Resources devoted to the research on Endocrine Disruptors in the last three European Research Framework Programs have been more significant. Among the major projects, we must remember: INUENDO, ANEMONE, the cluster of CREDO projects and the CASCADE network of excellence.

In Italy, some public research institutions, such as ISPESL and ISS, supported the research on endocrine disruptors with funds from the Ministry of Heath. A recent survey, organized by the Interuniversity Consortium INBB and the ISS, evidenced the existence of more than one hundred research groups actively working in this field. The CNR addressed this issue and several initiatives were promoted in joint action between the Department of Earth and Environment and the Department of Medicine. The most important is the Environment and Health Interdepartmental Project, PIAS-CNR, under the responsibility of Dr. Fabrizio Bianchi.

The final goal of PIAS is to understand the links between pollution sources and their effects on human health, since, as previously reported, the environment directly or indirectly affects human health.

The prevention of environmental origin diseases requires a number of actions either on attitudes and lifestyles or on laws and other institutional measures

The pilot study on Endocrine Disruptors designed to guarantee the safety of the population exposed to environmental hazards. This is the goal of the pilot project on “Endocrine Disruptors”, activated by CNR within the scope of action of PIAS, and concisely described in the following pages. This study represents the logical conclusion of a series of PIAS initiatives taken in this scientific area. The project sees the participation of three research units belonging to three CNR Institutes, namely the Institute of Clinical Physiology (CNR-IFC, Pisa), the Institute of Genetics and Biophysics (CNR-IGB, Naples), and the Water Research Institute (CNR-IRSA, Brugherio).

Many research activities, even very good ones, have been excluded from this project solely for the scarcity of available funds.

We hope that the results that will be produced from this project may serve as a basis and stimulus for future initiatives on this research field.

3. GROUND AND CONTENT OF THE PILOT STUDY

The project is divided into three experimental lines, that at first glance might seem unrelated, but that in reality are converging into a single objective: the study of the epidemiological and experimental links between some social diseases and the exposure to endocrine disruptors. These social diseases are the cardiovascular risk, which is among the principal causes of mortality in Italy, and endometriosis, which affects about 15% of women worldwide.

To better analyze the epidemiological link between exposure to endocrine disruptors and the above mentioned diseases, the population of a territorial district recognized as highly polluted: Gela (Sicily, Italy) has been chosen as to be studied from an epidemiological point of view.

Gela is sadly known for its pollution since air, soil and water are polluted by high concentrations of Endocrine Disruptors (with estrogenic or androgenic or arylic activity) and heavy metals. Perhaps this is why Gela is characterized by a higher level of malformations and cancer, kidney and cardiovascular diseases, as well as diseases of the reproductive tract and thyroid with respect to the national average. So Gela is the ideal place to verify the existence of a direct link between “Environmental Pollution and Health”. Since food is the principal mean by which EDs reach man, and since the population under study lives in a coastal-marine area, attention will be paid to determine the concentration of some EDs in some fish types of larger consumption by the indigenous population. Finally, in vivo experiments of EDS prenatal exposure in mice will be carried out in order to verify the possible occurrence of endometriosis.

We will now describe the experimental approach planned for each of the three research lines.

3.1 Line 1: Endocrine Disrupters and cardiovascular risk, occurrence of endometriosis, modulation of adrenal, gonad and thyroid functions in Gela population.

One hundred adult voluntaries of both sexes and resident in the Gela area will be recruited with the aim of studying the possible relationship between the levels of toxic pollutants in their biological fluids and the risk or occurrence of cardiovascular diseases, as well as alterations of thyroid, gonad and adrenal functions, using exposure biomarkers and responses to a specific questionnaire. In particular, personal, medical history, lifestyle, environmental and professional exposure, will be collected together with the weight, 183

CNR Environment and Health Inter-departmental Project height, waist circumference and blood pressure of each recruited person. Blood analysis will include: blood count, αPTT, PT, fibrinogen, PCRD, total cholesterol, HDL, LDL, triglycerides, blood glucose, uric acid, creatinine, BUN. Hormones dosage will be carried out testing for LH, FSH, PRL, P, E2, DHEA-S, total and free testosterone, 4α androstenediol, Cortisol ACTH, Δ4-androstenedione, 17α-OH Progesterone, TSH, FT3, FT4 , aldosterone, PRA. A 24 hours urine collection is planned for the determination of creatinine clearance, electrolytes, and cortisol.

Women recruited into the study for indirect signs of endometriosis will fill in an additional questionnaire on their pregnancy, offspring and ovarian cycle, and will also undergo blood sample to determine peripheral blood markers of endometriosis such as leukocytes, macrophages, TNF1alfa, CD3, CD 25, IL1, CA125.

On the basis of the number of adults recruited and the analysed biological samples, the research can improve knowledge about the possible correlation between the exposure to some environmental pollutants, typical of industrial and urban areas, and health outcomes, with particular reference to cardiovascular diseases, different forms of cancer and endometriosis.

3.2 Line 2: Endometriosis and Endocrine Disruptors: an in vivo experimental study Endometriosis is among the diseases supposed to be associated with exposure to EDs. Endometriosis is a recurrent and benign gynaecological disorder characterized by the presence of endometrial tissue outside the uterine cavity. Endometriosis tissues are found on the peritoneal surface in the female pelvis, on the ovaries, on the recto-vaginal 184

septum, rarely in the pericardium, pleura, and even in the brain (2). Recent statistics report a prevalence of 6-10% among Italian women, but in patients with pain and/or infertility the prevalence rises to 35-60% (3). Several epidemiological data link the occurrence of endometriosis with exposure to various types of endocrine disruptors (4). Reproductive effects were found in monkeys, mice or rats, exposed during foetal life to polychlorinated biphenyls and dioxins (5-7). The possible occurrence of endometriosis in animals exposed during the foetal period to Bisphenol A (BPA), one of the most abundant endocrine disruptors in the environment, is still unknown.

The aim of this research line is to experimentally verify the onset of endometriosis in the offspring of mothers exposed to endocrine disruptors during the prenatal and perinatal life. As reported above, this link has been sufficiently studied in higher animals and in mice or rats, but only in connection to dioxin or dioxinlike compounds exposure (5-7), never in connection to BPA. BPA, on the contrary, has been used to verify the adverse effects on male fertility. There are many scientific papers on BPA estrogenic action (8-12). It has been reported that BPA exposure causes diseases in the developing foetus. It was also found that low levels of BPA exposure during foetal development, for instance, induces earlier puberty (9) and affects the prostate size (13). For this research we will use BALB-C mice exposed to BPA from the beginning of gestation, during lactation and in their early stage of life. The dose-response dependence will be determined together with: a) the morphological and functional changes in the uterus; b) the presence of endometrial tissue outside the uterus in exposed offspring; c) BPA concentrations in some target tissues: muscle, brain, liver, etc.

The pilot study on Endocrine Disruptors The involvement in this research of the Italian Endometriosis Foundation will ensure the transfer of the results to the medical community. The Foundation will also permit the access to its national registers in order to allow the comparison between the epidemiological data of the national population and those of the Gela area.

3.3 Line 3: Determination of Endocrine Disruptors concentration in fish of wide consumption coming from an area of high environmental risk It is well documented that in more heavily populated areas or industrial places, contamination by endocrine disruptors affects not only the system of surface and profound waters (14-17) and the surrounding lands, but also the health of the animals and plants there living (18,19).

It must be remembered that in some animal species living in these environments serious diseases and malformations, attributable to contamination by substances that affect the hormone system, have been found (20,21). Many of these substances are characterized by high lipophlicity and resistance to degradation. This means that many endocrine disruptors accumulate in living organisms and increase their concentration along the food chain in the ecosystem (21,22). Fish consumption is one of the major routes by which endocrine disruptors reach the humans (23,24).

In order to answer to this concern, the aim of this research activity is to determine the concentrations of some pollutants, known for their ability to interfere with the endocrine system, in fish of wide consumption and catch in the Gela Sea. The content of Bisphenol A, Octylphenol and Nonylphenol and two ethoxylates of Nonylphenol ( mono and diethoxylated) will be determined. The concentration

levels of polychlorinated biphenyls (PCBs), organochlorine pesticides, arsenic, cadmium and mercury will be also determined.

4. EXPECTED RESULTS From the results that will be reached during this research activity we hope to obtain indications on: 1)- the link between exposure to endocrine disruptors and some social diseases; and 2)- how the environment and the diet operate synergetically in promoting some severe pathologies in wildlife and humans.

We hope also to goad our legislator into achieving a greater consciousness on the danger of these invisible killers so that they can take useful prevention initiatives.

With the contribution of: A. Baldi (Fondazione Italiana Endometriosi); E. Fommei (Pisa University and IFC), G. Iervasi, A. Pierini (IFC); S. Maffei, C.Vassalle (Fondazione “G. Monasterio” CNR-Regione Toscana, Pisa); C. Roscioli, S.Valsecchi, L. Viganò, D. Vignati (IRSA, Brugherio).

Keywords: Endocrine Disruptors, Endometriosis, Cardiovascular Risk, Fish’s Contamination, Bisphenol A.

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vom Saal FS & Hughes C. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environmental Health Perspectives (2005)113:926-933.

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The Lancet (2004) 364: 1789-1799.

Baldi A, Campioni M & Signorile PG. Endometriosis: pathogenesis, diagnosis, therapy and association with cancer.

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Bioscience 13:1578-1593.

Newbold RR, Jefferson WN & PadillaBanks E. Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract (2007) Reproductive Toxicology 24: 253-258.

Foster WG. Endocrine toxicants including 2,3,7,8-terachlorodiben zo -p - dioxin (TCDD) and dioxin-like chemicals and endometriosis: is there a link? (2008) J Toxicol Environ Health B Crit Rev. 11:177-87.

Rier S & Foster WG. Environmental dioxins and endometriosis (2003) Semin.

Reprod Med. 21:145-54.

Palanza, PL, Howdeshell KL, Parmigiani S & vom Saal FS. Exposure to a low dose of bisphenol A during fetal life or in adulthood alters maternal behavior inmice (2002). Environmental Health Perspectives. 110: 415-22.

Markey CM, Luque EH, Muñoz de Toro M, Sonnenschein C & Soto AM. In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland (2001).

Biology of Reproduction 65: 1215-1223.

Sakaue M, Ohsako S, Ishimura R, Kurosawa S, Kurohmaru M, Hayashi Y, Aoki Y, Yonemoto J, & Tohyama C. Bisphenol-A affects spermatogenesis in the adult rat even at a low dose (2001) J. Occup. Health. 43:185-190.

Howdeshell K, Hotchkiss AK, Thayer KA, Vandenbergh JG & vom Saal FS. (1999). Exposure to bisphenol A advances puberty. Nature 401: 762-764.

Takahashi O & Oishi S. Disposition of orally administered 2,2-Bis(4hydroxyphenyl)propane (Bisphenol A) in pregnant rats and the placental transfer to fetuses (2000) Environmental Health Perspectives 108:931-935.

Ramos JG, Varayoud J, Sonnenschein C, Soto AM, Muñoz de Toro M & Luque EH. Prenatal exposure to low doses of bisphenol A alters the periductal stroma and glandular cell function in the rat ventral prostate (2001) Biology of Reproduction 65: 1271-1277.

14. Furuichi T, Kannan K, Giesy JP & Masunaga S. Contribution of known endocrine disrupting substances to the estrogenic activity in Tama River water samples from Japan using instrumental analysis and in vitro reporter gene assay (2004) Water Research 38: 491–501.

15. Patrolecco L, Capri S, De Angelis S, Pagnotta R, Polesello S & Valsecchi S. Partition of nonylphenol and related compounds among different aquatic compartments in Tiber river (central Italy) (2006). Water, Air, and Soil Pollution 172: 151–166.

16. Salste L, Leskinen P, Virta M, Kronberg L. Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast assay (2007) The Science of the Total Environment 378: 343-351 17. Viganò L, Benfenati E, van Cauwenberge A, Eidem JK, Erratico C, Goksøyr A, Kloas W, Maggioni S, Mandich A & Urbatzka R. Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays (2008) Chemosphere 73: 1078-1089.

18. Latorre A, Lacorte S & Barcelo D. Presence of nonylphenol, octyphenol and bisphenol a in two aquifers close to agricultural, industrial and urban areas.

(2003) Chromatographia 57: 111–116.

19. Hewitt M & Servos M. An overview of substances present in Canadian aquatic environments associated with endocrine disruption. (2001) Water Quality Research Journal of Canada 36:191–213.

20. Viganò L, Farkas A, Guzzella L, Roscioli C & Erratico C. The accumulation levels of PAHs, PCBs and DDTs are related in an inverse way to the size of a benthic amphipod (Echinogammarus stammeri Karaman) in the River Po (2007) The Science of the Total Environment 373: 131-145.

21. Naert C, Van Peteghem C, Kupper J, Jenni L & Naegeli H. Distribution of polychlorinated biphenyls and

The pilot study on Endocrine Disruptors polybrominated diphenyl ethers in birds of prey from Switzerland (2007) Chemosphere 68: 977-987.

22. Hinck JE, Blazer VS, Denslow ND, Echols KR, Gale RW, Wieser C, May TW, Ellersieck M, Coyle JJ & Tillitt DE. Chemical contaminants, health indicators, and reproductive biomarker responses in fish from rivers in the Southeastern United States (2008) The Science of the Total Environment 390: 538-557.

23. Dougherty CP, Holtz SH, Reinert JC, Panyacosit L, Axelrad DA & Wood TJ. Dietary exposures to food contaminants across the United States (2000) Environmental Research 84:170-185.

24. Turyk ME, Persky VW, Imm P, Knobeloch L, Chatterton Jr. P & Anderson HA. Hormone disruption by PBDEs in adult male sport fish consumers. Environmental Health Perspectives (2008) 116: 16351641.

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Pilot study for the assessment of health effects of the chemical composition of ultrafine and fine particles in Italy M.C. Facchinia, F. Cibellab, S. Baldaccic, F. Sprovierid

a. CNR, Institute of atmospheric sciences and climate (ISAC), Bologna, Italy b. CNR, Institute of biomedicine and molecular immunology (IBIM), Palermo, Italy c. CNR, Institute of Clinical Physiology (IFC) Pisa, Italy d. CNR, Institute of Atmospheric Pollution Research (IIA) Monterotondo St. (Roma), Italy mc.facchini@isac.cnr.it

ABSTRACT Endocrine disruptors have been described as â&#x20AC;&#x153;exogenous chemical substances or mixtures that alter the structure or function(s) of the endocrine system and cause adverse effects at the level of the organism, its progeny, populations, or subpopulationsâ&#x20AC;? (EPA, 1998).

Several experimental studies reported that also very low doses of endocrine disruptors can affect the endocrine system causing diseases and altering the development of mammalian (humans included) and non-mammalian species. Among the diseases associated with the exposure to endocrine disruptors cancer, cardiovascular risk, modulation of adrenal, gonad and thyroid functions, and endometriosis are those that mainly catch the public concern considering their social cost.

This paper describes the research activity planned in the pilot project on Endocrine Disruptors granted by CNR in the general contest of the Environment and Health Inter-departmental Project (PIAS).

1. BACKGROUND A number of epidemiological studies have shown a correlation between fine particle concentration and increased mortality or morbidity. At the same time, due to the complex chemical composition and varying size-distributions of PM10 and PM2.5, a clear explanation of the mechanisms underlying the toxic effects of atmospheric particulate matter is still elusive (1).

Inhalation of particulate matter leads to pulmonary inflammation and reduction in lung function (2) with secondary systemic effects or, after translocation from the lung into the circulation, to direct toxic effects on cardiovascular function (3) and on the coagulation pathway thus contributing to the onset of coronary events (4). Through the induction of cellular oxidative stress and proinflammatory pathways (4), particulate matter augments the development and progression of atherosclerosis (5). The

main factor of these adverse health effects seems to be combustion-derived nanoparticles that incorporate reactive organic and transition metal components.

An important source of these particles is new diesel cars with oxidizing converters, such as modern taxis in North Europe.

Many epidemiological, human clinical, and animal studies showed that ultrafine particles (UFPs) penetrate deeply into the lungs initiating an inflammatory response leading to respiratory diseases and may be absorbed directly into the circulating blood, causing cardiovascular diseases (6).

Recent studies highlighted the importance of identifying susceptible sub-populations and mechanisms of involved effects.

Several chronic clinical conditions are good candidates to define the population susceptible to UFP acute effects, while elevated levels of oxidatively altered biomolecules are important intermediate endpoints that may be useful markers in

CNR Environment and Health Inter-departmental Project hazard characterization of particulates (7). At present, UFPs are not usually monitored by air quality stations. Thus, current epidemiological studies have to rely on PM10 data.

Previous studies have pointed to chemical species occurring in trace amounts, having known carcinogenic and mutagenic effects like PAHs (8,9,10) or “heavy” metals (11). Others have focused on the peculiar properties of ultrafine particles (with a diameter below 0.1 μm) to penetrate biological membranes (12).

Overall, despite the increasing amount of data provided by both laboratory and field studies, the nature of the aerosol particles fraction inducing health effects is still a matter of debate. This issue is important, because the different aerosol constituents exhibit distinct sources and emission/ formation processes (13,14). Therefore, linking toxicological and epidemiological impacts of atmospheric particulate matter to their chemical composition is a key to evaluate effective pollution abatement strategies (15,16).

The existing networks of stations monitoring particulate matter concentration, usually PM10 or PM2.5 mass, are not designed to provide chemical composition and size-distribution data.

In Italy, only at the two EMEP stations of Ispra (VA) and Montelibretti (RM), the chemical composition of PM10 and PM2.5 is routinely measured. At the same time, an increasing series of data on the aerosol chemical composition and size-distribution have been provided by short-term intensive field studies performed in the frame of national and European research projects (17). During these experiments, state-of-the-art instrumentation has been deployed for aerosol characterization.

For instance, multi-stage impactors were used to provide size-resolved chemical 190

composition data, down to the ultrafine or quasi-ultrafine size range. At the same time, the chemical analysis of fine particulate samples has shown that even in urban areas the water-soluble fraction of the aerosol contains large amounts of poorly characterized organic compounds (WSOC, “water-soluble organic carbon”), in contrast to the paradigm of many toxicological studies which attributes the organic-soluble and water-soluble fractions of the aerosol to organic and inorganic compounds, respectively. On the contrary, recent findings point to WSOC as a major agent for aerosol toxicity and oxidizing properties (18,19).

In summary, by examining the priorities for the evaluation of upcoming research activities of the Italian National Research Council (CNR) to link atmospheric aerosols composition and properties to their health effects, at least two specific key issues can already be addressed and dedicated to a) ultrafine particles and b) WSOC. This pilot study will combine the results of two advanced activities in the field of atmospheric ultrafine particles composition and their toxicological properties, carried out by CNR-ISAC (CNR Institute of Atmospheric Sciences and Climate) and CNR-IIA (CNR Institute of Atmospheric Pollution Research) (WP1, WP2) with two new advanced health studies carried out

by CNR-IFC (CNR Institute of Clinical Physiology) and CNR-IBIM (CNR Institute of Biomedicine and Molecular Immunology) (WP3, WP4) aimed at exploring short-term effects due to air pollutants exposure in subjects with preexistent arrhythmia and lung diseases.

The results of the specific advanced environmental and health activities will be evaluated and integrated in WP5 with the final aim of designing an integrated Italian

Health effects of the chemical composition of ultrafine and fine particles in Italy research activity for future projects to be presented in the frameworks of regional and national projects funded by European Union Structural Funds (PON, POR) or EU Research Funds (EC-FP7).

2. OBJECTIVES 1. To collect and compile the available chemical composition data of fine and ultrafine particles in urban and rural sites in Italy; 2. To test the oxidative potential of organic compounds in the watersoluble fraction of submicron aerosol; 3. To evaluate the feasibility of performing epidemiological studies assessing short-term effects of exposure to air pollutants in subjects with pre-existent arrhythmia in Italy; 4. To evaluate the feasibility of epidemiological studies assessing short-term effects of exposure to air pollutants in subjects with pre-existent lung diseases in Italy; 5. To provide the background knowledge to design an integrated research project aimed at assessing the effects of fine and ultrafine particles on human health (to be presented within the framework of PON, POR and EU-FP7).

Work-Packages: WP1 Assessment of the chemical composition of ultrafine particles and its variability in urban and rural sites in Italy based on available multi-stage impactor data and initial measurements using Aerosol Mass Spectrometers (AMS).

(CNR-ISAC, IIA) WP2 Evaluation of methodologies to measure the oxidative potential of the water-soluble organic fraction (WSOC) of the aerosol. (CNR-ISAC, CNR-IIA) WP3 A pilot study to assess short-term

effects of exposure to air pollutants in subjects with pre-existent arrhythmia.

(CNR-IFC) WP4 A pilot study to assess short-term effects of exposure to air pollutants in subjects with pre-existent lung diseases in Italy. (CNR-IBIM) WP5 Critical evaluation of the current proposal results and design of a common experimental strategy for an integrated future project on the health effects of fine and ultrafine particles (CNR-ISAC, CNR-IIA, CNR-IBIM, CNR-IFC).

With the contribution of PS2 participants: F. Bianchi (CNR-IFC), S. Decesari (CNR-ISAC), G. Viegi (CNR-IBIM), R. Sicari (CNR-IFC).

Keywords: Ultrafine particles (UFPs), water-soluble organic carbon (WSOC), cardiopulmonary diseases.

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