1.1 Scope and purpose of the document: The purpose of this document is to assess, evaluate, and give guidance on the role of elemental speciation and speciation analysis in hazard and risk assessment, rather than to present a review of each element and its speciation. The effects on the environment are not considered in this document, as this has been the topic of a recent conference and associated documentation (SGOMSEC, 2003). However, exposure of the human population through environmental routes is considered. This document is directed at risk assessors and regulators, to emphasize the importance of consideration of speciation in their deliberations. Until now, this issue has not been a part of most hazard and risk assessments. Further, one of the aims of the document is to encourage the analysis of speciation of elements to increase knowledge on the effect of speciation on mode of action and understanding of health effects. The emphasis is not on nutritional requirements, but on the toxicity of elements to humans. Consideration is made not only of consumer/general exposure but also of occupational exposure. 1.2 Definitions: A chemical "species" is the "specific form of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure". "Speciation" can be defined as the distribution of an element among defined chemical species in a system, and "speciation analysis" as the analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample. 1.3 Structural aspects of speciation: The definitions of species and speciation of elements are based on several different levels of atomic and molecular structure where species differences are manifest. Here, we consider differences at the levels of 1) isotopic composition, 2) electronic or oxidation state, 3) inorganic and organic compounds and complexes, 4) organometallic species, and 5) macromolecular compounds and complexes. Some of these structural levels are more important for risk assessment than others. Thus, stable isotope composition, while important both from a theoretical point of view and in physical and environmental chemistry, is generally of minimal importance in risk assessment concerning human health. Likewise, elemental speciation at the macromolecular level has biological significance in physiology, biochemistry, and nutrition, but its importance in occupational or environmental toxicity is less well understood. Organic complexation is of intermediate importance; as most chelates are labile relative to covalent complexes, they influence bioavailability and cellular uptake. However, they form and exchange in relation to the availability of ligands in the local milieu, and their trafficking to cellular targets is somewhat unpredictable. On the other hand, valence state and inorganic and covalent organometallic speciation are of great importance in determining the toxicity of metals and semi-metals. 1.4 Analytical techniques and methodology: Remarkable advances in the performance of elemental speciation analysis have been made during the past 20 years. Speciation analysis can now be performed for nearly every element, but not for every species of every element. Insight has been acquired into sample collection and storage so as to avoid contamination and to preserve the species intact. Available knowledge allows for sample preparation in order to identify and quantify species in biological fluids, tissues, water, and airborne dust. Sample preparation may include an additional cleanup step, extraction procedures, or preconcentration and derivatization of the species, prior to their separation. The most widely used separation techniques are liquid chromatography, gas chromatography, capillary electrophoresis, and gel electrophoresis. If the species are too complex, groups of species can be isolated by applying sequential extraction schemes. This is most used in the fractionation of sediments, soils, aerosols, and fly ash. The detection is usually that of th element, although molecular detection is gaining ground, especially in clinical and food analysis. Commonly used elemental detection methods are atomic absorption spectrometry, atomic fluorescence spectrometry, atomic emission spectrometry, and inductively coupled plasma mass spectrometry. Additionally, plasma source time-of-flight mass spectrometry and glow discharge plasmas can be used as tunable sources for elemental speciation. Electrospray mass spectrometry and matrix-assisted laser desorption ionization mass spectrometry are ideal to obtain structural information about the molecular species. Electrochemical methods are further powerful tools for speciation analysis. Calibration in elemental speciation analysis still remains challenging, especially so in the case of unknown species. There exists a limited choice of reference materials for elemental speciation. A growing number of them are certified. Direct speciation analysis of elements in particles is of great interest in assessing environmental health hazards. It provides valuable information on the elemental species in the superficial layers of the particles, allowing deductions about the origin, formation, transport, and chemical reactions. In most cases, it necessitates highly sophisticated apparatus. 1.5 Bioaccessibility and bioavailability: Substances must be bioaccessible before they can become bioavailable to human beings. A substance is defined as bioaccessible if it is possible for it to come in contact with a living organism, which may then absorb it. Bioaccessibility is a major consideration in relation to particulates, where species internal to the particles may never become bioaccessible. Elemental species that are accessible on the surface of particles or in solution may be bioavailable if mechanisms exist for their uptake by living cells. The rate of this uptake into cells is usually related to the external concentration of either free ions with appropriate properties or kinetically labile inorganic species (free ions plus inorganic complexes). Organic complexation and particulate binding often decrease elemental uptake rates by decreasing the concentrations of free ions and labile inorganic complexes. However, in certain circumstances, organic complexes of an element may facilitate its uptake. In addition, the site at which particulates have prolonged contact with tissues, such as lung alveolar epithelia, may become a focus of chronic exposure and toxicity. Uptake systems are never entirely specific for a single element, and these systems often show competition between similar chemical species of different elements, resulting in inhibition of uptake of essential elements and uptake of competing potentially toxic elements. Because of these competitive interactions, ion ratios often control the cellular uptake of toxic and nutrient elements. Such interactions also result in inherent interrelationships between toxicity and nutrition. It is important to define chemical species interactions clearly before carrying out risk assessment because of such profound effects on availability and toxicity. 1.6 Toxicokinetics and biomonitoring: 1.6.1 Toxicokinetics: Various aspects of speciation of the elements (e.g. the unchanged forms, the biological mechanisms changing species, the different valence states, and the metal-ligand complexes) must be considered when evaluating absorption, mechanisms of binding to proteins, distribution, storage, metabolism, excretion, reactivity, and toxic activity of the metallic elements themselves. Absorption through the respiratory tract is conditioned by size, solubility, and chemical reactivity of elemental species inhaled as particles. The absorption of elemental species in the gastrointestinal tract varies depending on their solubility in water and gastrointestinal fluids, their chemical and physical characteristics, the presence of other reacting compounds, and the period of ingestion (fasting, for instance). The skin may also be an important absorption route for some elemental species. After absorption, the elemental spe es can form complexes with proteins, including enzymes, such as the essential elements associated with ferritin (iron, copper, zinc), -amylase (copper), alcohol dehydrogenase (zinc), and carbonic anhydrase (copper, zinc). In general, the removal of electrons from or addition of electrons to the atom influences the chemical activity and therefore the ability of metallic elements to interact with tissue targets (ligands). Examples of charge relevance in crossing lipid barriers are represented by chromate/dichromate, Fe2+Fe3+, and Hg +/H0 passages. Among the other metabolic transformations, the most important is bioalkylation, which, for example, mercury, tin, and lead undergo in microorganisms, whereas arsenic and selenium are additionally bioalkylated as part of their metabolic pathways in higher organisms. Alkylation produces species at a higher hydrophobic level, leading to an increased bioavailability, cell penetration, and accumulation in fatty tissues. Bioalkylation is important for some metals, since the alkylated metal species also interact with DNA. Alkylated metal species penetrate the blood-brain barrier more readily, and it is for this reason that such alkylated species are important neurotoxicants. Metallic elements may be stored in tissues/organs as both inorganic species or salts and species chelated or sequestered to proteins and other organic compounds. Excretion depends on the speciation, the route of absorption, and other toxicokinetic phases. The excreted species are either inorganic or organic and frequently at the lowest oxidation state.