Summary paper of the EC Network on trace element speciation for analysts, industry and regulators—what we have and what we need

Introduction

The thematic network ‘Speciation 21’ was funded by the European Commission's Standards, Measurement and Testing Programme and lasted two and a half years, from October 1997 until March 2000. During the consecutive meetings the Network succeeded in creating a completely new and very lively forum. The meetings were attended by analytical chemists working in speciation method development for trace elements, potential users from industry and representatives from legislative agencies.^1,2 The minutes of the meetings are available on our web page.³ Attention was focussed on species of elements in environmental, food and occupational health. The cross-fertilisation of ideas between the diverse groups of participants generated many new insights and innovative ideas for further development in the challenging domain of elemental speciation.

Historical perspectives

The first awareness about the importance of elements speciation is rooted in the realisation of the different degrees of plant and animal toxicity of a number of elements according to their chemical form. Historical milestones were set with arsenic⁴ and mercury.⁵ The methylated forms of arsenic (synthesised in living systems, primarily as a detoxification mechanism) were found to be far less toxic than the inorganic arsenic compounds. Methylated mercury, by contrast, is much more toxic than inorganic mercury. A representative historical example is the story of the Minamata disease in Japan in 1956. Inorganic mercury was released into the environment and microorganisms in the water methylated the Hg²⁺. The ensuing methylmercury then bio-accumulated in the aquatic flora and fauna. As it is very fat soluble, toxic levels were finally reached in fish, with disastrous results for the fish-eating population of the area.

The deleterious effect associated with the widespread use of manmade chemicals, such as tetraethyllead⁶and organotin^7,8 compounds, to name just these two, became all too evident in the second half of the twentieth century. Biomarkers of exposure again served as early indicators. Lead additives in gasoline gave rise to a dramatic increase of Pb in the blood of urban populations. Tributyltin compounds are to be blamed for the most alarming occurrence of male characteristics in some female gastropods as well as other effects such as reduced growth, reproduction and immunotoxicity.

As a result, during the seventies, extensive research in analytical chemistry was initiated to develop highly specific and sensitive methods for measuring these compounds in various matrices. Today this has led researchers to question the relevance of total element determinations and to posit that total element determinations are obsolete. Such considerations are an important incentive for research and development in elemental speciation analysis. The more so as the scientific community would like to find an answer to the many biological, medical and environmental questions linked to trace elements, which implies identifying and measuring their various chemical species.

Recent developments

In order to circumscribe the domain of element speciation, it may be interesting to quote the IUPAC's (International Union for Pure and Applied Chemistry) recommendations for the definition of the terms related to the chemical speciation of elements:⁹ “chemical species”, “speciation analysis”, “speciation of an element; speciation” and “fractionation”.

Chemical species. (Chemical elements) specific form of an element defined as to isotopic composition, electronic or oxidation state, and/or complex or molecular structure.

Speciation analysis. (Analytical chemistry) analytical activities of identifying and/or measuring the quantities of one or more individual chemical species in a sample.

Speciation of an element; speciation. Distribution of an element amongst defined chemical species in a system.

The term fractionation has been defined as follows.

Fractionation. Process of classification of an analyte or a group of analytes from a certain sample according to physical (e.g., size, solubility) or chemical (e.g., bonding, reactivity) properties.

The terms are described in detail in the original manuscript. Methodological approaches for performing speciation and fractionation analyses are also reported.

Analytical chemists have been very resourceful in developing separation and measurement systems for many element species. There remain, however, still many hurdles to be taken.^10–12 When the species are of anthropogenic origin, calibrants are usually at hand. By contrast, when the species are the result of anabolic and catabolic processes in living systems (e.g., occurrence of arsenosugars in algae) or interaction with natural substances (e.g., chromium bound to humic substances) elaborate studies are needed to identify the compounds and when this has been handled successfully, calibrants are usually unavailable or require extensive efforts to synthesise. When species with very low thermodynamic stability are investigated, quite different approaches are needed.

Every time a proper analytical method has been developed, (including correct calibrants), it requires validation, to make sure that the data produced by different laboratories are comparable and traceable.^11,12 One further step is then to produce reference materials certified for trace element species, especially for “difficult” samples such as sediments, soils, dust, plants, organic rich water, food, tissue, biological fluids, etc., the list could become very long.

Although there are several reliable procedures for a number of species, (such as methylmercury, organotin compounds, arsenic species, Cr(III) and Cr(VI) species, to name just these four), major new developments in speciation analysis are required. Isolation and separation of the species leaving the original compounds intact, is a primary task.¹³ Furthermore, a sizeable improvement in sensitivity is needed in the measurement of the trace elements. As the concentration of the different species is far below that of the total concentration (in the environment species typically occur at the ng L⁻¹ and pg L⁻¹ level and below), it is questionable if the existing methods will ever be able to meet the needs. New methods based on new physical principles will have to be designed. The objective has to be developing ‘routine’ analytical procedures with sensitivities that are several orders of magnitude lower than whatever has been achieved hitherto.

Besides the danger of disruption of a trace element that is non-covalently bound (e.g., to proteins, humic acids, sediments), there is also the opposite effect of random trace element impurities being captured by the matrix. As the latter may have so many ligands, they act as scavengers for trace element impurities of the reagents, the column fillings, the inner walls of the apparatus, etc.

These considerations are generating so many difficulties that it is time to think about alternative solutions to do speciation analysis. There are, unfortunately, few hints how this can be done. Perhaps in-situ speciation? Very desirable indeed, and perhaps feasible for specific species, if some microchip could be designed.

The industry, i.e., the manufacturers of products and instruments, are on stand-by. They are waiting for legislation to cause them to act, or for a pollution accident to occur at which time action is taken belatedly. The other side of the coin will be an outspoken demand for species of essential elements, e.g., in the food and/or pharmaceutical industry in the pursuit of creating a lucrative business. The ability to provide “true” speciation data is becoming increasingly important to national and international trade. At the same time there will come the necessity for a greater range of reference materials.

It may be interesting now to gauge the various needs in the fields of environment, food and occupational health and hygiene where elemental speciation and fractionation analysis are expected to bring much needed answers.

Potential applications

The Network meetings revealed a list of topics that need to be dealt with as soon as possible in the areas of the environment, food, occupational health and hygiene. Entwined with these are the requirements for revised risk assessment and legislation based on speciation and fractionation of elements.

Environment¹⁴

Organotin⁸ and alkylmercury¹⁵ compounds are very important in environmental pollution issues. The analytical methodologies for these species are available and the state of the art continues to improve steadily. This would, however, benefit from further validation and improvement to routine measurements. Some regulations are already in operation.

Waste management is an area where speciation analysis will increase greatly in interest, because of economic implications. Clean up procedures dedicated to the toxic species of an element will facilitate remediation and lower costs. Portable instrumentation for in-situ measurements becomes invaluable, because some species are labile and do not survive transfer to the conventional laboratory.

In developing countries contamination of water supplies is still prevalent. In some areas of Asia, (e.g., Bangladesh), a major problem has been created by the distribution of artesian water that contains substantial amounts of inorganic arsenic. Symptoms of arsenic poisoning have become chronic among a large part of the population.

During recent years use of the platinum group metals has increased dramatically,¹⁶ mainly as a catalyst for the reduction of pollutants in automobile exhausts. The question now arises about the fate of the platinum, rhodium and palladium compounds released into the environment. The analytical challenges are huge, because of the problematic “noble metals chemistry” and the extremely low concentrations in which they are emitted. It is already a very difficult task to measure the total amount of these metals in environmental samples. The measurement of the different species is a huge but necessary challenge, for improving our understanding of the accumulation and transport of these metals in the environment.

In all cases it is necessary to learn more about the fate and cycling of the species of all these elements in the environment and especially in the life cycle.^17,18 Development of reliable modelling would be a major asset to gauge the influence of various environmental factors, to learn about fluxes and transformation mechanisms.¹⁹ More knowledge is also needed to achieve a better understanding of the effects of long-term exposure to low levels of the species for all forms of life.

In the context of the environment, it is appropriate to mention the rapid tests for element speciation.²⁰ Such tests differ from those usually associated with metal species, but the compounds measured conform to the definition of elemental speciation and fractionation. For example, today there exist very rapid, inexpensive tests with high sensitivity for ammonium, hydrazine, nitrite, nitrate, orthophosphate, hydrogen sulfide, sulfite, sulfate, chloride, elemental chlorine, Cr⁶⁺ and Fe²⁺ in water and/or soil.

Food²¹

A better understanding of the predominant chemical forms of trace elements in food and their subsequent behaviour in the digestive tract would certainly assist the decision-makers concerned with dietary requirements and related legislation. Here, iron is the best documented element. Its chemical form is of primary influence on its bioavailability. It has long been established that “heme-Fe” (found in red meat) is very well absorbed, as opposed to “non-heme-Fe” (found in fruit, vegetables and cereals). In the latter case the absorption is heavily influenced by the other ligands in the gastro-intestinal tract.

Other elements that are documented as “species” in foodstuffs or food supplements are selenium, arsenic and tin. In the case of selenium, species are important to increase their bio-availability and hence their importance as cancer-preventing agents.²² The case of arsenic is quite different: speciation is necessary to discern the toxic inorganic species from the inoffensive organo-arsenicals.^23,24 The tin story is limited to the organotin compounds and is dealt with in greater detail in the paragraph on “legislation”.

It is now evident that the study of human nutrition and health requires more information about trace element species in food.²⁵ Proper dissemination of accurate information will give consumers informed choice, it will allow the industry to make the best choice of foods, supplements and processing (which might alter chemical species), and it will provide governments with the basis for good advice and legislation.²⁶

The food industry has already embarked on speciation measurement, normally specific for one analyte and usually for commercial or functional purposes. Although the analytical methodologies for speciation of trace elements in food have been developed in order to evaluate safety and quality, validated speciation methods and reference materials certified for species of elements are indispensable. Only then can trace element speciation assure food safety and quality.

Occupational health and hygiene^27,28

The Network looked at the implications of element species for health issues, with special emphasis on industrial hygiene. Three major issues emerged: (i) biological monitoring of trace element species by measuring biomarkers of exposure, (ii) kinetics of trace element species in the body due to industrial exposure and the dependency on the chemical form of exposure and (iii) identification of the chemical form of the chemical species that will become the basis of toxicity studies.

Biological monitoring of trace element species by measuring biomarkers of exposure

Up to now workers in the As-, Cr-, Ni-, Pt-, etc., industries are monitored for exposure to one of these metals or their compounds by measuring the total As, Cr, Ni or Pt in their urine. These tests are insufficiently specific for the evaluation of the potential hazard and harm resulting from their occupational exposure. The solution lies in developing a test by measuring a substance that is unique to an individual's exposure to a given species of the element. This is not inconceivable because exposure to different chemical forms of an element will probably be followed by different biological interactions in the body, leading to the production of different metabolites.

The problem of arsenic has been solved to a great extent. Arsenic in fish or seafood is mainly present in four different species: arsenobetaine, arsenocholine, arsenosugars and dimethyl arsinic acid (DMA). It is generally accepted that these compounds are not metabolised in humans. In the case of biological monitoring for exposure to harmful inorganic arsenic compounds (arsenate, arsenite, arsine) these will show up in urine as arsenate, arsenite, monomethyl arsonic acid (MMA) and DMA. It is then sufficient to separate the different As-containing species in urine to identify the cause of exposure. Separation and detection techniques to execute this type of measurement became very reliable during the past few years.²⁹

The problems faced when different species of the element are metabolised to the same compounds are not yet solved. For instance, Cr in urine as a result of inhalation of Cr(VI) or gastro-intestinal absorption of Cr(VI) cannot be distinguished from, e.g., Cr(III) supplements. Urinary excretion in both cases solely contains Cr(III).

Extensive research in biomonitoring of trace element species is needed in order to improve the measurement of potentially toxic substances or their metabolites or biochemical effects in tissues, excreta, expired air or any combination of these. Its outcome will allow the evaluation of occupational or environmental exposure and health risks by comparison with appropriate reference values, based on the knowledge of the probable relationship between ambient exposure and resultant adverse health effects.

The study of trace element kinetics in the body due to industrial exposure and dependency on the chemical form of exposure

For many elements the toxic effect of the element and/or some compounds is known. Only in a limited number of cases has a fairly specific description of how the chemical species exert their deleterious effect been documented. Studies are needed to describe the pathways the trace element species undergo after being inhaled, orally ingested or after dermal contact, including its transformation of species, binding to various ligands, its active role in life sustaining biochemical processes, its storage in certain target organs and its excretion. Such studies require in-situ measurements in cells. This concept is taking off, albeit not yet for trace element species measurements.

Identification of the chemical form of the chemical species that will become the basis of toxicity studies

The chemical characterisation of environmental and industrial particulate samples is essential for a proper description of those substances that are liable to cause deleterious effects when inhaled. Up to now, hazard identification concerns stoichiometric substances. Under controlled laboratory conditions their adverse effect, target population and conditions of exposure have been determined taking into account toxicity data and knowledge of effects on human health, other organisms and their environment. In reality, when the particles inhaled in hazardous circumstances (such as indoor working places) are examined by modern techniques, no such stoichiometric compounds of suspected elements can be identified.³⁰ The chemical species (i.e., the specific form of an element as to molecular, complex or oxidation state) occur under a variety of unusual bonding conditions, such as chromate bound to silicate.³¹ This implies that most of the past testing to evaluate the hazards of exposure, should be totally re-evaluated on the basis of the chemical species of the elements as they present themselves when inhaled. The bioavailability of the particulates not only depends on the chemical form but also on the particle size. Exposures are seldom to a single species, as has been documented for nickel and its compounds in occupational health.³²

Today there are very specialised ways for characterising particles relevant to speciation, using a combination of methods for the characterisation of the outer layer of a single particle.³³ The instrumentation is complex and the cost of analysis is very high and can only be used for fundamental research. Developing cheaper routine methods is very much needed.

Besides characterisation of the species relevant for occupational health purposes, it is necessary to make a systematic in-vivo and in-vitro study of the interaction between these species and the cell membranes and the possible transformation inside the cells. Such studies will require a multi-disciplinary approach. Only well-planned cooperation between researchers in metallurgy, pharmacology, physics, toxicology, occupational health and hygiene, medicine, biology, biochemistry and analytical chemistry will allow new and better test methods to be developed for exposure threshold values in metallurgy. This will have to be followed by new risk assessment studies and may lead to implementation of new rules by regulators and legislators. There is a pressing need to accelerate research along those lines for metal species of inhaled particulates. A relevant example concerns chromium. The research data on health effects of chromium in the metallurgical industry are controversial. Some studies indicate respiratory effects while others do not. It is necessary to know more about the surface properties of the particles and the form of the agglomerates in the metal aerosols to understand the harmful mechanisms in the target tissue and to identify those stages in the production processes, which pose an elevated health risk.

Risk assessment^34,35

The key steps in risk assessment are hazard identification, dose-response assessment, and exposure assessment. All these components contribute to risk characterisation, an estimate of the probability of harm to any individual in a population at risk.

Trace elements generally penetrate into cells via membrane transport proteins, which have different binding sites for different chemical species. It is therefore very important to define trace element species interactions with the cells before carrying out risk assessment, because of the profound effects on trace element availability and toxicity. Substantial fundamental research is needed in this field.

Legislation³⁶

One of the aims of the Speciation 21 Network was to give impetus to the refinement of existing legislation concerning elements. This covers not only the polluting effect in all areas of life, but also the essentiality of element species in foodstuffs.

In EU Directives and other Occupational, Environmental, Dietary and Clinical legislation, there are numerous references to the concentration of chemical elements and their compounds. The WHO Drinking Water Guidelines give guideline values for antimony, arsenic, barium, boron, cadmium, chromium, copper, lead, manganese, mercury, molybdenum, nickel, selenium, and uranium without any reference to speciation. The third North Sea Conference (1990) list as priority hazardous substances (again without speciation): mercury, cadmium, copper, zinc, lead, arsenic, chromium, and nickel.

Directive 76/464/EEC on pollution caused by certain dangerous substances discharged into the aquatic environment by the Community (OJ L129 18.5.76) puts in List I (the most harmful substances whose discharge to the aquatic environment must be reduced to a level at which pollution of the aquatic environment is prevented) the following: mercury and its compounds, and cadmium and its compounds. In List II (less harmful substances whose discharge needs to be reduced to a level at which pollution of the aquatic environment is reduced) are: zinc, copper, nickel, chromium, lead, selenium, arsenic, antimony, molybdenum, titanium, tin, barium, beryllium, boron, uranium, vanadium, cobalt, thallium, tellurium, and silver. The fact that List II does not mention compounds suggests that only pure metals need be considered (doubt prevails if that was the legislators' intention or simply an oversight). Directive 80/68/EEC on the protection of groundwater against pollution caused by dangerous substances includes the same lists. Directive 79/923/EEC on the quality required of shellfish waters (OJ L281 10.11.79) establishes environmental quality standards for: silver, arsenic, cadmium, chromium, copper, mercury, nickel, lead, and zinc. Again no mention is made of compounds or ionic species. Directive 80/778/EEC on the quality of water intended for human consumption (OJ L229 30.8.80) gives Guide Level (GL) values for substances including: magnesium, sodium, potassium, aluminium, iron, manganese, copper, zinc and silver; and Maximum Admissible Concentrations (MAC) for: arsenic, cadmium, chromium, mercury, nickel, lead, antimony, selenium, calcium, boron, and barium. Again no speciation was apparently considered or mentioned. The UK Sludge (Use in Agriculture) Regulations 1989 implements EC Directive 86/278/EEC on the protection of the environment and in particular of the soil when sewage sludge is used in agriculture (OJ L181 4.7.86). There are Maximum Permissible Concentrations set for: zinc, copper, nickel, cadmium, lead, mercury, chromium, molybdenum, selenium, arsenic, and fluorine. The order given for the elements is taken from the tables listing them and it is explained what the basis of it is. To be truly effective, this legislation should refer to specific chemical species rather than simply to the element.

The same reasoning is applicable to the workplace. There again lack of specificity prevails. One of the few exceptions concerns chromium, where the threshold limit values for Cr(VI) are much lower than for Cr(III). The need for speciation analysis of dust particulates in metallurgy has been made very well by Williams and Huvinen for Ni and Cr.^32,31

Alongside regulations for the workplace and the environment, food is another area where legislation of species must be urgently considered both for the bio-available species that are present as for the relevance of food additives.

Tributyltin (TBT) has become a common seafood impurity.³⁷ The daily tolerable intake (DTI) has been estimated at 0.25 µg TBT kg⁻¹ body weight.³⁸ The intake appears to be exceeded in many regions. Large variations have been documented according to the fish and shellfish species, between various fish tissues and the region of origin. Overall, countries claim that levels of organotin compounds in seafood pose a negligible risk to humans, but this is without scientific basis. There is a need for maximum residue limits for TBT and other organotin compounds in seafood for each country as well as for regular monitoring.

There are regulations for toxic elements in food. For instance, the UK sets a general limit of 1 mg kg⁻¹ arsenic in foodstuffs. As most foodstuffs yield concentrations far below this threshold, these samples comply with the existing legislation. Therefore there is no compulsive reason for industry to conduct speciation analysis.^22,26,36 It can be postulated that speciation legislation is inevitable, although, unless there is a sudden change in knowledge on toxicity or benefit, it may be many more years before it happens.

There is already some evidence of speciation measurements in food control (Fe species in bread and flour, and nitrates and nitrites in cured meats and cheese as they have the potential to form nitrosamines).

The business of food supplementation is closely related to chemical form. The element selenium is a good illustration of how the bioavailability and the beneficial effects are governed by the species. Although essential for good health in trace amounts, the element is extremely toxic and has a narrow tolerance window. The body processes certain organoselenium compounds differently to the inorganic forms. Research has shown these organic selenium compounds to have some cancer preventing properties.

It is evident that there is a need for many speciation measurements in, for example, water, foodstuffs, agricultural products, the environment, the pharmaceutical industry, and clinical measurements. Very few regulations, however, already refer to the chemical form of the elements. As a rule, an element and its compounds are placed in the same toxicity and bioavailability class, which is clearly erroneous. Legislators should rapidly become more familiar with advances in the area of elemental speciation and should be trained in current scientific thinking, to build this new concept into regulations. As most laws concern a given element and its compounds, they impose restrictions that may be too strict for a number of harmless species and too lenient for very toxic compounds.

Conclusions

The only way that elemental speciation will be successfully achieved is through a multi-disciplinary approach. Further developments will depend upon international collaboration between researchers and regulators.³⁹ The EU Speciation 21 Network will have facilitated this process. All through the discussions at the Network meetings it became evident that the manufacturers of chemicals have become conscious of the responsibility they carry, not only for their workers but also for the environment and for the fate of their products “from cradle to grave”.³² Today there are strict rules for the launching of a new pharmaceutical compound. It is not inconceivable that in the next decades similarly strict procedures will be imposed before any new substances can be marketed. Elemental speciation must be part of this undertaking.

It is highly desirable that this network should continue. For that purpose a collective action by representatives from the industry together with academics and legislators is necessary. They should apply for new EC funding with the objective being to create a self-sustainable Institute for Speciation of Elements.

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Footnotes

† Presented at the Whistler 2000 Speciation Symposium, Whistler Resort, BC, Canada, June 25–July 1, 2000.

‡ The opinions expressed in the following article are entirely those of the authors and do not necessarily represent the views of The Royal Society of Chemistry, the Editor or the Editorial Board of JEM.

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