Metals and their toxicological effect to both human health and the environment are an emotive subject. Film dramatisations bring the subject to the wider public, spurring interest outside the scientific community. Metals of interest are sometimes termed “heavy metals”, a term which is often misleading as there are a variety of definitions of heavy metals, based on density, atomic number, atomic weight, chemical properties or toxicity. The same metals are also called “trace elements” or “trace metals/metalloids”, however broadly speaking the group of metals of interest to the environmental community are arsenic, beryllium, barium, cadmium, chromium, copper, mercury, nickel, lead, selenium, vanadium and zinc.

Distribution in the biosphere

Air pollution is a major contributor to metal redistribution in the biosphere, although the transfer from air particulates to soil and vegetation is complex with a variety of physiochemical processes involved. Sources include mining, coal and fuel combustion, industrial processing and agriculture to name but a few. An example of air pollution causing a redistribution of metals in the environment can be seen with lead released through the use of lead additives in fuel. The lead used in tetra-ethyl lead additives was predominantly from one source, which has a particular isotopic signature. Lead found in analysis along urban and highway routes has shown a similar isotope
ratio, indicating the likely source to be deposition of atmospheric lead from the use of these fuel additives.

Metals are readily transported by water with industrial sources, agriculture, domestic and agricultural effluent, and transport being major sources. This may be through leaching, atmospheric fall out or even direct emission. The chemical form is also important, and biomethylation occurs readily with mercury, arsenic, tin and lead as the metals reach a sediment layer.

Soils and sediments are often directly contaminated from industrial and agricultural sources. Although the soil acts as a natural buffer, its ability to do this depends on a variety of characteristics of the soil, such as pH and organic matter, and therefore affects the complexation, dispersion and bioavailability of the metals within the soil. Global contamination is slow, with local sources being the predominant source rather than long range transport, and typically only the upper layers of the soil profile are affected.

The species of the metal concerned is key as in many cases these have completely different toxicological effects. These are inherently linked with the background concentrations and whether the source is natural or anthropogenic.

“a typical use of such a test would be evaluating lead concentration in soil based on historical site use as an ammunition firing range”

The toxicity and therefore risk is linked to concentration, species, solubility and bioavailability.
These are defined as follows:

  • • Concentration is the amount present per unit of mass/volume of material analysed
    • Speciation is the distribution of an element amongst defined chemical species in a system
    • Speciation analysis aims to quantify all component species, as fractions of a total – if it is impossible to determine the speciation, it is a useful practice to do fractionation instead
    • Fractionation is the 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
    • Solubility is the readiness of a metal species to dissolve in water
    • Bioavailability is the degree to which a substance is absorbed, and this is covered in more detail below with specific note as to how this differs from bioaccessibility

Analysis

On site analysis of metals is possible in both soil and water matrices, but it should be used to yield indicative data, prior to laboratory analysis. Soil samples are typically examined using hand held x-ray fluorescence instruments. These give a good indication of contamination levels, although on site analysis is taken as a “snapshot” rather than a representative homogenised sample. For water samples, a variety of technologies exist. Traditional “test-kits” based on colorimetric analysis have been used for the analysis of metals in water for many years and, although they can only be used for a few analytes which give good reaction chromophores, they do give good quality indicative results in the right matrices. More recently “lab-on-a-chip”  technology has come to the fore, and anodic stripping voltammetry (ASV) methods being used routinely for analysis of arsenic.

At the laboratory, the analysis of metals in water is a fairly straight forward affair with a filtered sample (either before or after addition of nitric acid) being analysed directly. The most popular techniques employed are inductively coupled plasma – optical emission spectrometry (ICP-OES) or inductively coupled plasma – mass spectrometry (ICP-MS), although other element specific techniques exist.

The analysis of soils is more complex. Soils first need to be dried and homogenised (ground), and this dried sample either digested with acid or analysed directly (when using laboratory XRF instrumentation). The subject of sample preparation has been covered in previous issues of this magazine. The analysis of “total metals” is actually very difficult to achieve, very strong acids or microwave extraction techniques must be used, these are not routine in most environmental laboratories. The most common extraction technique is aqua regia digestion, which breaks down the soil matrix efficiently, and then the subsequent extract is analysed by ICP-OES, although it should be noted that aqua regia does not fully breakdown silicate matrices.

Physical fractionation

It may be useful to determine the physical fractionation of a metal in the environment where the source is anthropogenic. A typical use of such a test would be evaluating lead concentration in soil based on historical site use as an ammunition firing range. Such data can inform a site clean-up or remediation strategy whereby physical processing of the material could be carried out.
Below is an example of data for lead distribution in a range of soil samples:

Speciation

Historically, legislation governing the maximum permissible levels of a polluting element in an environmental sample refers to total concentrations rather than the chemical form of that element. However, this total concentration provides no information concerning the fate of the element in terms of its interaction with sediments, its bioavailability, or its subsequent toxicity. Changes in the chemical speciation may dramatically affect the toxicity of a metal. Over time, the importance of speciation has had greater understanding and as a consequence has been built into more recent legislation and guidance.

One example is chromium. Chromium in its trivalent form (CrIII) is essential for human life and has key roles in biological functions such as sugar and lipid metabolism. Hexavalent chromium (CrVI) however is known to be toxic and carcinogenic. It is therefore clear that in terms of risk assessment, these forms of chromium cannot be treated in the same way. CrVI can be analysed colorimetrically by a reaction with diphenylcarbazide solution, the intensity of the resultant purple colour is directly proportional to the concentration of CrVI present.

“the importance of speciation has had greater understanding and as a consequence has been built into more recent legislation and guidance”

Another example is mercury. Mercury can be found throughout the environment at trace and ultra-trace levels. It is most commonly encountered in the environment in elemental, inorganic (HgII) and mono-methyl mercury forms. Mercury is toxic, with the primary effects being on the central nervous system (CNS), brain and kidneys. Elemental mercury is volatile and easily absorbed via the inhalation pathway, which mainly affects the CNS. Inorganic mercury exposure is typically via ingestion and is linked to kidney damage, where as mono-methyl mercury has been shown to have the most toxic effects on the brain. For these reasons, not only must the
total concentration of mercury be considered and analysed, but some care should be taken in assessing the form of mercury. To this end, the revised soil guideline values for mercury detail elemental, inorganic and methyl mercury. Analytically, the analysis of these forms is challenging, but development of chromatographic separation and ultra sensitive atomic fluorescence detection means that many laboratories are able to perform this analysis and report not just total mercury, but also the breakdown of the species.

Bioaccessibility and bioavailability

Bioavailability can alter health risk estimates, consequently its importance in risk assessment cannot be underestimated. Bioavailability is defined as the degree to which a substance is absorbed and becomes available to the target tissue (without first being metabolised) – determined as in in vivo measurement (performed in a living organism). The bioavailable concentration of a metal does not correlate with the total concentration, it is a function of a variety of factors, including species, geochemical fraction and sample matrix.

Bioavailability is not to be confused with bioaccessibility, which is the degree to which a chemical is released from soil into solution (and thereby becomes available for absorption) when that soil is ingested and undergoes digestion. Bioaccessibility of metals in soil can be measured in vitro (in the laboratory) using methods which have been proven to yield data which correlates to bioavailability.

Bioaccessibility testing is carried out in the laboratory to simulate the effect of the human digestive system on a contaminated soil and determine how much of a toxic contaminant is accessible to the body.

The maximum Bioaccessible fraction (%) is calculated as follows:
Bioaccessible Concentration (chyme with highest concentration) / mg kg-1 x 100 %
“Total” Concentration / mg kg-1 >

The sample preparation method for bioaccessibility testing is critical. Samples are dried and passed through a 250 µm sieve; the sample is not crushed. All bioaccessibility testing methods are based on the physiology of a small child and it is critical that the preparation method replicates the manner in which they may ingest the soil, and therefore the finest fraction which is likely to adhere to their hands is used for testing. A crushed sample may yield false data for a variety of reasons, including the crushing action yielding surfaces for interaction which may not normally be accessible, the dilution of a high metal concentration in the finer fraction with crushed stones, or even vice versa. It is therefore also important that the “total” measurement is undertaken on the same portion of the sample as the bioaccessibility testing.

The general rule of thumb is that the bioaccessible fraction is greater than the bioavailable fraction, but less than the acid extractable and total concentrations of a metal. The bioaccessibility also relates to the fraction of the soil to which the metal is bound.

There are a variety of methods available to indicate bioaccessibility, one of which is the Physiologically Based Extraction Test (PBET). This is an established technique for evaluating the bioaccessibility of heavy metals, in particular arsenic. The method simulates digestion of a soil sample, with bioaccessibility being evaluated in the simulated stomach
and simulated intestine solutions. The test method is in the un-fed state, which is recognised to yield the most conservative estimates of bioaccessibility for metals.

A Simplified Bioaccessibility Extraction Test (SBET) is applied specifically for assessing lead bioaccessibility in waste materials. Lead is known to be most bioaccessible in the stomach phase (lower pH value) and this is reflected in this test which only simulates digestion by stomach acids, intestine phase extractions are not performed. This test is carried out for the fasted state for the same reason as the PBET.

The PBET method has largely been superseded in use by the BARGE UBM method. This has been developed by the Bioaccessiblity Research Group Europe (BARGE), and has led to the development of the Unified Bioaccessibility Method (UBM). This collaborative venture combines many established method protocols and has been evaluated for both heavy metals and organic contaminants. As with the PBET and SBET, metals are assessed in the fasted state. The method simulates both the stomach digestion and intestinal digestion of soil. This method has been validated using both bioavailability data (in vivo) and also inter-laboratory comparisons within the BARGE community.

This type of testing is becoming increasingly popular, and reference materials are available to ensure good high-quality data is produced. There are clear recommended guidelines on minimum reporting requirements in order to reassure the end user of the data of the quality of information. With these recommendations adhered to, along with laboratories using the standard methods (rather than in-house variations); bioaccessibility testing could become more widely accepted and recognised as a viable laboratory method for enhancing site investigations and risk assessments.

Chemical fractionation

Chemical fractionation is another arm of testing which can be used to give additional information about where within a soil matrix a metal is bound. There are a range of different methods available, but they all essentially work in a similar manner, looking at metals that are readily released from soil (water soluble), those that are loosely bound to soil material, those that are bound to organic matter for example, and then looking at residual concentrations in the soil after each extraction is performed.

“developments for the future of metals analysis and monitoring are likely to see more on-site laboratories and testing facilities”

The future

Developments for the future of metals analysis and monitoring are likely to see more on-site laboratories and testing facilities. New miniaturised instruments and “lab on a chip” devices for a variety of contaminants, not just metals, are constantly being brought to the market, the next challenge will be to ensure these are fit for purpose and the technicians using them are capable of producing reliable results in the same way as a laboratory analyst using established instrumentation. The availability of portable microwave plasma (rather than ICP) spectrometers mean that laboratory equipment can more easily set up in a mobile laboratory, which should serve well to compliment on site XRF surveys.

“the availability of portable microwave plasma spectrometers mean that laboratory equipment can more easily set up in a mobile laboratory”

In terms of speciation and risk assessment, arsenic is a particular problem in the south west of the UK, where the natural background levels of arsenic in the soil often exceed the soil guideline values. Arsenic and its compounds are known to be toxic and, in some cases,
carcinogenic, and like other metals, the toxicity depends heavily on the chemical form. Inorganic arsenic species (arsenite, arsenate) are particularly toxic, whereas organic arsenic species (mono- and di- methyl arsenate) are common metabolites found in the human body and are much less toxic. Other forms of arsenic found in the marine environment (arsenosugars etc.), are completely non-toxic. Consequently, development of methods for arsenic speciation in environmental samples is an increasingly popular research topic, it is only a matter of time before this reaches the commercial sector.