Biomarkers for Mercury Biomarkers for Mercury BiomonitoringBiomonitoring

Marcelo Enrique ContiMarcelo Enrique ContiSapienza, University of Rome, ItalySapienza, University of Rome, Italy

E-mail: marcelo.conti@uniroma1.itE-mail: marcelo.conti@uniroma1.it

Proyecto QSP Proyecto QSP “Campaña Regional para la Minimización de las “Campaña Regional para la Minimización de las

Fuentes Domésticas de Mercurio con Acciones de Intervención en la Fuentes Domésticas de Mercurio con Acciones de Intervención en la

comunidad para la protección de la salud del niño y la mujer en la comunidad para la protección de la salud del niño y la mujer en la

Argentina, Chile, Paraguay, Uruguay, Bolivia y Perú”. Buenos Aires, 3 Argentina, Chile, Paraguay, Uruguay, Bolivia y Perú”. Buenos Aires, 3

de Diciembre 2008de Diciembre 2008

People are continuously exposed to thousands of natural and man-made chemicals (i.e. 100.106 – REACH) through the external environment, food habits and lifestyles.

Using modern analytical technology it is now possible to measure a large number of chemicals and their metabolites present in the human organism (in blood, tissues, urine, hair, etc.).

The biomonitoring is a procedure well known since 1927 when the first paper on the use of the analysis of lead in urine in exposed workers was published.

Today biomonitoring is largely used to control the health risk of people occupationally and non-occupationally exposed.

Programmes on the biomonitoring are currently in progress in the USA and in Europe.

The biomonitoring evaluates the exposure by comparison with appropriate reference values and goes by the knowledge of the relationship between environmental exposure and deriving degree of adverse health effects.

When a health risk is revealed, legislators may

decide to ban a product or restrict its usage to

applications with lower risks for human health.

Biomonitoring techniques are becoming, in fact,

common tools for decision-makers in the health

and environmental field.

Valutazione Rischio ChimicoValutazione Rischio Chimico

Qualunque strumento operativo o Qualunque strumento operativo o processo venga utilizzato per effettuare processo venga utilizzato per effettuare la valutazione del rischio degli agenti la valutazione del rischio degli agenti chimici pericolosi chimici pericolosi (misure, stime, (misure, stime, algoritmi)algoritmi) dovrà tenere conto di tutti i dovrà tenere conto di tutti i requisiti minimi della VRC previsti requisiti minimi della VRC previsti dall’art. 223 del Dlgs 81/08.dall’art. 223 del Dlgs 81/08.

a) le loro proprietà pericolose;b) le informazioni sulla salute e sicurezza comunicate

dal produttore o dal fornitore tramite la relativa scheda di sicurezza predisposta ai sensi dei decreti legislativi 3 febbraio 1997, n. 52 e 16 luglio 1998, n. 285 e successive modifiche;

c) il livello, il tipo e la durata dell’esposizione;d) le circostanze in cui viene svolto il lavoro in

presenza di tali agenti, compresa la quantità degli stessi;

Valutazione del Rischio Chimico Valutazione del Rischio Chimico Requisiti minimi Art. 223Requisiti minimi Art. 223

Valutazione del Rischio Chimico Valutazione del Rischio Chimico Requisiti minimi Art. 223 (II)Requisiti minimi Art. 223 (II)

e) i valori limite di esposizione professionale o i valori limite biologici; di cui un primo elenco è riportato negli allegati XXXVIII e XXXIX;

f) gli effetti delle misure preventive e protettive adottate o da adottare;

g) se disponibili, le conclusioni tratte da eventuali azioni di sorveglianza sanitaria già intraprese.

Valore limite di esposizione professionale: è il limite della concentrazione media ponderata nel tempo di un agente chimico nell'aria all'interno della zona di respirazione di un lavoratore in relazione ad un determinato periodo di riferimento;

Valore limite biologico: il limite della concentrazione delrelativo agente, di un suo metabolita, o di un indicatore

di effetto, nell'appropriato mezzo biologico.

Valori limite esposizione professionaleValori limite esposizione professionale

Valore limite biologicoValore limite biologico

Strumenti per la valutazione del rischio Strumenti per la valutazione del rischio chimicochimico

Stime del rischio (valutazioni di tipo cautelativo basate su dati disponibili)

Algoritmi (valutazioni di tipo semiquantitativo basate su parametri indicizzati di calcolo)

Misure (determinazioni degli inquinanti in ambienti di lavoro)

Biomarkers suggest the occurrence of toxicological events much earlier than the emergence of those effects that can be evaluated.

A biomarker is defines as: ‘...a change, produced by a contaminant, at biochemical or cellular level of a process, a structure or a function that can be measured in a biological system.

This change provides information (qualitative,

semi-quantitative or quantitative) about the

chemical source, and on the correlation between

the biological effects and the environmental

contamination levels.

A contaminant can cause

...primary toxicity at biochemical and molecular levels (alterations in enzymatic activity, DNA level …)

secondly, through cascade events can cause toxicity at cellular, tissue or organism levels.

It is well-known that lifestyle is implicated in

determining the risks for development of cancers,

circulatory diseases, neurodegenerations,

other chronic diseases.

Similarly, life events and periods, such as

childhood, reproduction and senescence, may

affect the distribution of chemicals within the

body.

During pregnancy, as an example, many chemicals

may pass the placental barrier causing exposure of

the foetus.

Lactation may result in excretion of lipid-soluble

chemicals, thus leading to a decreased retention in

the mother along with an increased uptake by the

infant.

During weight loss or development of osteoporosis, stored chemicals may be released, which can then result in a renewed and protracted “endogenous” exposure of target organs.

Other factors may affect individual absorption, metabolism, retention and distribution of chemical compounds and they have to be taken in account when a biomarker has to be measured.

The most important features of a biomarker are: i), stability (to allow the biological sample preservation); ii), sensitivity (i.e., low probability of false negative); and iii), specificity (i.e., low probability of false positive).

Moreover a biomarker must reflect the interaction (qualitative or quantitative) of the host biological system with the compound of interest and it has to be reproducible qualitatively and quantitatively with respect to time (short- and long-term).

Biomarkers can be classified in:

i) biomarkers of exposure;

ii) biomarkers of effect;

iii) biomarkers of susceptibility.

Biomarkers of exposureBiomarkers of exposure

Biomarker of exposure, the first kind of biomarkers used in human biomonitoring studies, may be an exogenous compound or its metabolite (i.e., a metal or a metal compound) inside the body, an interactive product between the compound (or metabolite) and an endogenous component, or another event related to the exposure.

Often, there is not a clear distinction between exposure and effect biomarkers. For example, adducts formation could reflect an effect rather than the exposure.

However, biomarkers of exposure usually indicate changes in the functions of the cell, tissue or total body.

They comprise measurements of the compounds in appropriate samples, such as blood, serum or urine.

Volatile chemicals concentration may be assessed in exhaled breath, after inhalation of contamination-free air.

Biomarkers of exposure may be used to identify exposed individuals or groups, quantify their exposure, assess their health risks, or to assist in diagnosis of diseases with environmental or occupational etiology.

For example, exposure to a particular solvent may be evaluated from data on the actual concentration of the solvent in the blood at a particular time following the exposure.

This measurement will reflect the amount of the solvent that has been absorbed into the body.

Some of the absorbed amount will be exhaled due to the vapour pressure of the solvent.

Biomarkers of exposure alone do not give information on the sources or levels of exposure; when, where, how, or how many times the exposure occurred; or any relationships between exposure and health effects.

Recent technological advances in genomics, proteomics, and metabolomics are providing new tools for investigating endogenous chemicals that can be used to characterize an individual's exposure to a single chemical or a mixture of chemicals.

Biomarkers of effectBiomarkers of effect Biomarkers of effect are referred to reversible biochemical and functional alterations than can be measured in a target tissue of the organism.

A BEF may be an endogenous component, or a measure of the functional capacity, or a marker of the state or balance of the body or organ system, as affected by the exposure.

It is usually a pre-clinical marker of pathology and can be specific or non-specific. (Alimonti and Mattei, 2008)

Both types of biomarkers of effect are useful biomarkers of early (critical) effects.

For example the detection of early damage to the kidney tubules caused by exposure to Cd using urinary levels of low molecular weight proteins such as β2-microglobulin, protein HC (1-Microglobulin) and the enzyme N-acetylglucosaminidase can be determined.

Technical developments have been occurred with biomarkers of effect to mutagenic chemicals.

These compounds are reactive and may form adducts with macromolecules, such as proteins or DNA.

DNA adducts may be detected in white blood cells or tissue biopsies, and specific DNA fragments may be excreted in the urine.

Other macromolecules may also be changed by adduct formation or oxidation.

In particular, such reactive compounds may generate haemoglobin adducts that can be determined as biomarkers of effect to these compounds.

For the purpose of occupational health, these biomarkers should be restricted to those that indicate subclinical or reversible biochemical changes, such as inhibition of enzymes.

The most frequently used biomarker of effect is probably the inhibition of cholinesterase caused by certain insecticides, namely, organophosphates and carbamates.

In most cases, this effect is entirely reversible, and the enzyme inhibition reflects the total exposure to this particular group of insecticides.

Some exposures do not result in enzyme inhibition but in increased activity of an enzyme. This is the case of several enzymes belonging to the P450 family.

They may be induced by exposures to certain solvents and polyaromatic hydrocarbons.

Generally, the enzyme activity is determined indirectly in vivo by managing a compound that is metabolized by that particular enzyme, and then the breakdown product is measured in urine or plasma.

Other exposures may induce the synthesis of a protective protein in the body.

The best example is probably metallothionein, which binds Cd and promotes its excretion; Cd exposure is one of the factors that result in increased expression of the metallothionein gene.

Similar protective proteins may exist but have not yet been explored sufficiently to become accepted as biomarkers.

Among the candidates for possible use as biomarkers are the so-called stress proteins, previously known as heat shock proteins.

These proteins are generated by a range of different organisms in response to a variety of adverse exposures.

The urinary excretion of proteins with a small molecular weight, such as albumin, may be used as a biomarker of early kidney damage.

Relating to genotoxic effects, chromosomal aberrations or formation of micronuclei can be detected by microscope observation. Damage may also be revealed by adding a dye to the cells during cell division.

Exposure to a genotoxic agent can then be visualized as an increased exchange of the dye between the two chromatids of each chromosome (sister chromatid exchange, SCE).

Chromosomal aberrations are related to an increased risk of developing cancer.

More sophisticated assessment of genotoxicity is based on particular point mutations in somatic cells, that is, white blood cells or epithelial cells obtained from the oral mucosa (Alimonti and Mattei, 2008)

Biomarkers of susceptibilityBiomarkers of susceptibility

Biomarkers of susceptibility are indices of the individual predisposition (hereditary or acquired) to suffer xenobiotic effects, that is, to be particularly sensitive to the effects of a single compound or of a group of such chemicals.

People working under identical conditions may show interindividual variation in the intensity of the effects of a particular degree of exposure.

Therefore differences are seen in health impairment in workers similary exposed to the same substance.

Genetic screening and genetic monitoring.

A clear distinction must be drown between genetic tests which are intended to detect inherited characteristics, which may point to greater susceptibility to certain conditions (genome screening) and genetic tests which aim to find genetic tests which aim to find changes in the hereditary material, which are the changes in the hereditary material, which are the result of exposure to harmful agents result of exposure to harmful agents (genetic biomonitoring).

Genetic biomonitoring can form part of the

periodic medical examination of employees and is

specially designed to asses the effects of exposure

to carcinogenic or mutagenic agents in the

workplace (somatic mutations, chromosome

aberrations, micronuclei, aneuploidy).

If an individual has become sensitized to a particular exposure, then specific antibodies can be detected in serum.

A major problem is to determine the joint effect of mixed exposures at work. In addition, personal habits and drug use may result in an increased susceptibility.

For example, tobacco smoke usually contains a considerable amount of Cd.

...a heavy smoker who has accumulated substantial amounts of Cd in the body will be at increased risk of developing cadmium-related kidney disease.

In the environment Hg occurs in metallic form, as inorganic Hg and organic Hg.

Generally, environmental levels of Hg are quite low between 10 and 20 ng/m3 of Hg have been measured in urban outdoor air (i.e., hundreds of times lower than safe levels to breathe) or less than 5 ng/L in surface waters (i.e. about a thousand times lower than safe drinking water).

Tossicologia del mercurioTossicologia del mercurio

Hg(0): essendo volatile si deposita nei polmoni attraverso i quali si incorpora nell’organismo (reni e Hg(0): essendo volatile si deposita nei polmoni attraverso i quali si incorpora nell’organismo (reni e

cervello).cervello).

Hg(II): viene incorporato attraverso il tratto gastrointestinale e attraverso la pelle. Gli effetti negativi Hg(II): viene incorporato attraverso il tratto gastrointestinale e attraverso la pelle. Gli effetti negativi

dell’intossicazione acuta si mitigano rapidamente dato che la vita media del catione nell’uomo è di dell’intossicazione acuta si mitigano rapidamente dato che la vita media del catione nell’uomo è di

circa 60 giorni.circa 60 giorni.

MeHg(II): i composti contenenti il catione MeHgMeHg(II): i composti contenenti il catione MeHg++ sono fra le sostanze più tossiche, tanto per i loro sono fra le sostanze più tossiche, tanto per i loro

effetti che per la loro incidenza osservata su intere popolazioni. Questo catione forma composti effetti che per la loro incidenza osservata su intere popolazioni. Questo catione forma composti

liposolubili che possono dare fenomeni di bioaccumulazione (catena alimentare). Inoltre questi liposolubili che possono dare fenomeni di bioaccumulazione (catena alimentare). Inoltre questi

composti possono attraversare facilmente la membrana emato-encefalica agendo come potenti composti possono attraversare facilmente la membrana emato-encefalica agendo come potenti

neurotossici.neurotossici.

A potential source of exposure is Hg released from dental amalgam fillings, which can contain approximately 50 % of metallic Hg.

Some people may be exposed to high levels of methyl Hg if they eat often fish, shellfish, or marine mammals.

Hg is not essential to living cells; its absorption distribution and biotransformation are influenced significantly by its valence state.

Chronic exposure to Hg vapor results in toxicity of the central nervous system including tremors, increased excitability and delirium.

Elemental Hg is eventually oxidized to Hg (II) in the body by the hydrogen peroxidase-catalase pathway and is primarily excreted via the kidneys.

However, a small portion may be exhaled. Ingestion of inorganic, oxidized Hg can result in abdominal cramping, ulceration and renal toxicity.

Inhalation of Hg° vapor is associated with an acute, corrosive bronchitis or pnaeumonitis.

Hg has a strong affinity for sulfur, and Hg primary mode of toxic action in living organisms is thought to be the interference of enzyme function and protein synthesis by binding to sulfhydryl or thiol groups.

Excretion by kidneys is the primary route of elimination of oxidized Hg, and because of its strong affinity for protein, proteinuria is a symptom associated with exposure to Hg(II).

Organic Hg is highly lipophilic and exposure occurs primarily via consumption of contaminated fish.

Both methyl-Hg and Hg° cross the placental (inducing teratogenic effects) and blood-brain barrier where they can be oxidized and accumulated

Methyl Hg can react directly with important receptors in the nervous system, such as the acetycholine receptors in the peripheral nerves.

Carcinogenicity and mutagenicity are not

commonly associated with Hg exposure.

Instead, the IARC have not classified Hg as human

carcinogenicity, whilst EPA has determined that

Hg chloride and methyl-Hg are possible human

carcinogens.

Biomarkers of Hg exposure

Blood and urine Hg concentrations are commonly used as biomarkers of exposure. Urine Hg is a biomarker used for detecting elemental and inorganic forms of Hg.

The reference values, such as the reference dose

(RfD) published by US EPA, or the Provisionally

Tolerated Weekly Intake (PTWI) (WHO-FAO),

reflect the levels of exposure that should prevent

humans from suffering adverse effects of

environmental exposure.

MethylmercuryMethylmercury

  Provisional tolerable weekly intake (PTWI) of Provisional tolerable weekly intake (PTWI) of 1.6

µg/kg bw. The Committee considered this PTWI to be The Committee considered this PTWI to be

sufficient to protect the developing fetus, the most sufficient to protect the developing fetus, the most

sensitive subgroup of the population. The Committee sensitive subgroup of the population. The Committee

also reaffirmed its position that fish are an important also reaffirmed its position that fish are an important

part of a balanced nutritious diet and that this has to be part of a balanced nutritious diet and that this has to be

appropriately considered in public health decisions appropriately considered in public health decisions

when setting limits for methylmercury concentrations in when setting limits for methylmercury concentrations in

fish.fish.

The International Commission on Occupational

Health (ICOH) and the International Union of Pure

and Applied Chemistry (IUPAC) Commission on

Toxicology determined that a mean value of 2 μg/L

was the background blood level in persons who do

not eat fish.

Reference values

Human Biomonitoring Commission of the German Federal Environmental Agency (Wilhelm et al., 2004). Reference values indicate the upper margin of the current background exposure of the general population.

Cd (non smokers): blood - 1.0 μ/l ; urine 0.8 μg/l.

Pb : blood – 70 μg/l (female) ; 90μg/l ( male).

Hg : (consumption of fish ≤ 3 x a month) blood – 2 μg/l (no amalgam fillings) urine – 1.0 μ/l.

As : ( no fish consumption) urine – 15.0 μg/l.

Pt : (no dental inlays, crowns, bridges) urine –0.01μg/l.

The American Conference of Governmental Industrial The American Conference of Governmental Industrial Hygienists Hygienists (ACGIH) (ACGIH) and the Deutsche and the Deutsche Forschungsgemeinschaft Forschungsgemeinschaft (DFG), (DFG), the two main the two main organizations involved in the setting of BM reference organizations involved in the setting of BM reference values differ their approach to and definitions of these values differ their approach to and definitions of these values.values.BEIs BEIs are understood as are understood as advisory levels advisory levels that may be that may be exceeded by individuals in the observed group. exceeded by individuals in the observed group.

ACGIH ACGIH has already published BEI values for 37 has already published BEI values for 37 substances or groups of substances.substances or groups of substances.

The DFG BAT values are defined as "the maximum

permissible quantity of a chemical substance or its

metabolites, or the maximum possible deviation from the

norm for biological parameters induced by these

substances in exposed humans. The BAT values are

considered the ceiling values for healthy individuals".

They are intended to protect the workers from work-

related health impairments. DFG has so far determined

BAT values for 50 substances or groups of substances.

Occupational limits are a ACGIH-BEI of 15 µg/l and a BAT of 25 µg/l.

But blood Hg levels peak quickly soon after short-term exposures, so measurements should be made soon after exposure.

Human Biological Monitoring Values (HBM) recommended by Human Biological Monitoring Values (HBM) recommended by the German Commission on Human Biological Monitoring the German Commission on Human Biological Monitoring

(March 1999) (March 1999) (Jakubowski and Trzcinka-Ochocka, 2005)

HBMI HBMII HBMI HBMII

Mercury in urine Children and adults 5μg/g creat. 20 μg/g creat.

Mercury in blood Children and adults 5μg/l 15 μg/l

HBMIHBMI- The concentration of an environmental toxin in human - The concentration of an environmental toxin in human biological material, below which there is no risk of advance biological material, below which there is no risk of advance health effects. health effects.

HBM IIHBM II- The concentration above which there is increased risk - The concentration above which there is increased risk of adverse health effects in susceptible individuals in the of adverse health effects in susceptible individuals in the general population.general population.

A strong correlation has been found among the amount of fish swallowed, the Hg fish level and the Hg hair level.

Expired air samples have been considered as possible biomarkers of exposure for Hg, but results showed that expired air can only be used soon after short-term exposure to Hg vapours.

MethylmercuryMethylmercury

The effects of methylmercury on the adult differ both in quantitative and qualitative terms from the effects observed after prenatal or postnatal exposure. The critical organ is the nervous System and the critical effects include developmental neurologic abnormalities in human infants, and paraesthesia in adults. The foetus is at particular risk. Prenatal exposure leads to psychomotor retardation in infants. Developmental neurologic abnormalities are considered the critical effects in the infant population (Jakubowski and Trzcinka-Ochocka, 2005)

Hair is a biomarker of long-term exposure to methylmercury. Once mercury is incorporated into hair, it remains unchanged. The level of mercury in hair (Hg- H) is dependent on fish consumption

The dose-response relationship between maternal hair concentration and the frequency of health effects in children was used by the IPCS for the purpose of risk assessment. At peak mercury levels in maternal hair at above 70 μg/g, there is a high risk (more than 30%) of neurological disorder in the children, and a 5% risk may be associated with a peak mercury level of 10-20 μg/g in maternal hair.

The present The present background level of Hg-H, associated with , associated with no or low fish consumption or a low fìsh methylmercury no or low fish consumption or a low fìsh methylmercury concentration, amounts to from concentration, amounts to from 0.25 to 0.8 μg/g0.25 to 0.8 μg/g. .

Much higher Hg-H levels result from the consumption of Much higher Hg-H levels result from the consumption of large amounts of fish or sea mammals. The mean Hg-H large amounts of fish or sea mammals. The mean Hg-H levels in the Faroe Island population amounted from levels in the Faroe Island population amounted from 1.6 μg/g (one fish meal per week) to (one fish meal per week) to 5.2 μg/g 5.2 μg/g (four fish (four fish meals per week). In the Madeira fishermen and their meals per week). In the Madeira fishermen and their families, it amounted to families, it amounted to 38.9 μg/g38.9 μg/g in men and in men and 10.4 μg/g 10.4 μg/g in women in women (Jakubowski and Trzcinka-Ochocka, 2005)

Despite the numerous long-term studies and considerable efforts of the researchers, the so-called 'health-based' reference values have been proposed and validated only for several chemical substances or groups of substances. These recommendations are of great value to health These recommendations are of great value to health professionals because the health effect of exposure can professionals because the health effect of exposure can be predicted directly from the determination of a be predicted directly from the determination of a biomarker of exposurebiomarker of exposure. It is possible to predict early . It is possible to predict early direct health effects of lead based substances on direct health effects of lead based substances on blood blood lead levels.lead levels.Such measurements can be interpreted Such measurements can be interpreted without knowing without knowing the results of environmental monitoring.the results of environmental monitoring.

Biomarkers of effect

A number of possible biomarkers of effect have been investigated, especially for neurological and renal dysfunctions.

For example, the toxic effects observed at kidney level have been well correlated with blood and urine levels.

Biomarkers for decreased kidneys function include increasing in urinary proteins and elevation of serum creatinine or β2-microglobulin.

Biomarkers for biochemical changes include eicosanoids, fibronectin, kallikrein activity, and glycosaminoglycans in urine.

Glomerular changes have been reported as increased high-molecular weight proteinuria.

Tubular changes in workers include an increasing urinary excretion of NAG, β-galactosidase, and retinol binding protein. (Alimonti and Mattei, 2008)

But toxic kidneys parameters are not specific markers for Hg exposure and may be a consequence of other concurrent chemical exposures and they can’t be assessed as biomarker of effect still now.

The neurophysiological and neuropsychological health effects of Hg have been extensively studied in occupationally exposed individuals.

Neurological changes induced by Hg may look like exposure to other chemicals that can cause damage to the brain. Some studies have examined the relationship between nerve function and Hg levels in blood, urine, and tissue.

Tissue levels of Hg have also been found to correlate with impaired nerve function. But also in this case no biomarkers of effect are established.

Potential biomarkers for the autoimmune effects of mercury have been examined and they include measurement of antiglomerular basement membrane antibodies, anti-DNA antibodies, serum IgE complexes, and total IgE (Alimonti and Mattei, 2008).

Biomarkers of susceptibility

Various factors affect the absorption, distribution,

biotransformation, excretion and, consequently,

toxicity of Hg. In the case of methyl-Hg, reduced

glutathione and γ-glutamyl transpeptidase are

involved in the excretion of methyl-Hg.

The glutathione S-transferase (GST) gene family is involved in the detoxification of electrophilic compounds by conjugation and (a study conducted by

Brambila et al.) showed that various GST genes are activated in rats exposed to Hg, indicating that individuals with specific genotypes could be better protected against the cytotoxicity of Hg. (Alimonti and

Mattei, 2008).

Conclusions

BM of exposure and early health effects of exposure should be considered as the prophylactic activity. BM has an important role to play in both health surveillance and exposure assessment in occupational settings and in identifying hot spots and developments in trends of exposure in the general environment.

There are important discrepancies in the approach towards the role of BM between Europe and the USA (occupational medicine or occupational hygiene). (Jakubowski and M. Trzcinka-Ochocka, 2005)

More attention should be paid to the development of the truly health-based biomarkers of exposure based on the dose-effect and dose-response relationships.

The practical implementation of BM as well as the ethical problems can be solved in enterprises where a close cooperation between the health service, management and employees is a routine activity.

References: Biological monitoring of exposure. Trends and Key Developments

M. Jakubowski and M. Trzcinka-Ochocka. J. Occup. Health, 2005, 47, 22 – 48.

Revised and new reference values for some trace elements in blood and urine for human biomonitoring in environmental medicine

M. Wilhelm, U. Ewers, C. Schulz, Int J of Hyg and Env Health, 2004, 207, 69-73.

Large-scale biological monitoring in Japan.

M. Ogata, T. Numano, M. Hosokawa, H. Michitsuji, Sci Total Env,1997,199, 197-204

Biomarkers for human biomonitoring (Chapter 6)

A. Alimonti, D. Mattei (2008) in: M.E. Conti (Ed.) Biological Monitoring: Theory And Applications. The Sustainable World, 17, 163-211, WIT press, Southampton, ISBN: 978-1-84564-002-6.