Application of human biomonitoring data to support policy development, raise awareness and environmental public health protection among countries within the HBM4EU project.

Most countries have acknowledged the importance of assessing and quantifying their population's internal exposure from chemicals in air, water, soil, food and other consumer products due to the potential health and economic impact. Human biomonitoring (HBM) is a valuable tool which can be used to quantify such exposures and effects. Results from HBM studies can also contribute to improving public health by providing evidence of individuals' internal chemical exposure as well as data to understand the burden of disease and associated costs thereby stimulating the development and implementation of evidence-based policy. To have a holistic view on HBM data utilisation, a multi-case research approach was used to explore the use of HBM data to support national chemical regulations, protect public health and raise awareness among countries participating in the HBM4EU project. The Human Biomonitoring for Europe (HBM4EU) Initiative (https://www.hbm4eu.eu/) is a collaborative effort involving 30 countries, the European Environment Agency (EEA) and the European Commission (contracting authority) to harmonise procedures across Europe and advance research into the understanding of the health impacts of environmental chemical exposure. One of the aims of the project was to use HBM data to support evidence based chemical policy and make this information timely and directly available for policy makers and all partners. The main data source for this article was the narratives collected from 27 countries within the HBM4EU project. The countries (self-selection) were grouped into 3 categories in terms of HBM data usage either for public awareness, policy support or for the establishment HBM programme. Narratives were analysed/summarised using guidelines and templates that focused on ministries involved in or advocating for HBM; steps required to engage policy makers; barriers, drivers and opportunities in developing a HBM programme. The narratives reported the use of HBM data either for raising awareness or addressing environmental/public health issues and policy development. The ministries of Health and Environment were reported to be the most prominent entities advocating for HBM, the involvement of several authorities/institutions in the national hubs was also cited to create an avenue to interact, discuss and gain the attention of policy makers. Participating in European projects and the general population interest in HBM studies were seen as drivers and opportunities in developing HBM programmes. A key barrier that was cited by countries for establishing and sustaining national HBM programmes was funding which is mainly due to the high costs associated with the collection and chemical analysis of human samples. Although challenges and barriers still exist, most countries within Europe were already conversant with the benefits and opportunities of HBM. This article offers important insights into factors associated with the utilisation of HBM data for policy support and public awareness.

Human Biomonitoring Data Enables Evidence-Informed Policy to Reduce Internal Exposure to Persistent Organic Compounds: A Case Study.

Human biomonitoring (HBM) monitors levels of environmental pollutants in human samples, which often is a topic of concern for residents near industrially contaminated sites (ICSs). Around an ICS area in Menen (Belgium), including a (former) municipal waste incinerator and a metal recovery plant, increasing environmental concentrations of dioxins and polychlorinated biphenyls (PCBs) were observed, causing growing concern among residents and authorities. The local community succeeded in convincing the responsible authorities to investigate the problem and offer research funding. Persistent organic pollutants (POPs) were measured in two consecutive HBM studies (2002-2006 and 2010-2011), in the context of the Flemish Environment and Health Study (FLEHS), as well as in soil and locally produced food. Meanwhile, local authorities discouraged consumption of locally produced food in a delineated area of higher exposure risk. Ultimately, HBM and environmental data enabled tailored dietary recommendations. This article demonstrates the usefulness of HBM in documenting the body burdens of residents near the ICS, identifying exposure routes, evaluating remediating actions and providing information for tailored policy strategies aiding to further exposure reduction. It also highlights the role of the local stakeholders as an example of community-based participatory research and how such an approach can create societal support for research and policy.

Biobank@VITO: Biobanking the General Population in Flanders.

During the last 15 years, VITO has established an infrastructure for biobanking a collection of biological samples from the general population in Flanders (Belgium). This biobank was set up to contribute to future, yet unspecified, research questions in the field of environment and health. Biobank@VITO is a population biobank in which bio-specimen including human peripheral blood, cord blood, and blood derivatives (e.g., serum, plasma, cells, RNA, DNA), urine, hair, nails, exhaled breath condensate, saliva DNA, and human breast milk collected from non-diseased populations are preserved. Currently, the biobank stores about 70,000 samples from 7,700 individuals. These biospecimen were collected since 2002 in different human biomonitoring studies comprising European (e.g., DEMOCOPHES, HBM4EU), national (e.g., WHO human breastmilk studies), Flemish (Flemish Environment and Health Study (FLEHS) campaigns), and local (e.g., hotspots, 3xG project) well-defined and ethically approved research projects. Participants to the surveys included different age groups (newborns, children, adolescents, and adults) and were representatively selected with regard to gender, age class, residence, and/or socioeconomic status (SES). In each campaign, samples were stored in the Biobank@VITO. The registration, preservation, and management of the samples in the biobank were done in a qualitative and uniform manner which guarantees the traceability of all samples. The samples in the biobank have an extended information backbone on the lifestyle, environment, and health status of the donor. The biological samples in the biobank are an invaluable archive that can be used to address specific policy and research questions in the future, to test old samples with new technology and according to the latest methods and insights or to measure newly identified pollutants in old samples looking for long-term trends.

The added value of a surveillance human biomonitoring program: The case of FLEHS in Flanders (Belgium).

Since 2002, the Flemish Government decided to carry out the Flemish Environment and Health Survey (FLEHS), an extended human biomonitoring (HBM) program, which is integrated in the environmental health policy. Through the FLEHS studies, a vast amount of data such as biomarkers of exposure and effect, exposure-effect associations, time trends and geographical differences, became available to the Flemish policy makers. In order to facilitate the policy interpretation, a phased action-plan was developed collaboratively by FLEHS researchers and policy makers. In this article we look back on more than 15 years of investments of the Flemish government in HBM and reflect on how this large scaled and challenging HBM-initiative contributed to shaping the environmental health policy in Flanders. We used the FLEHS I (2002-2006) and II (2007-2011) results on persistent organic pollutants (POPs) and the resulting policy actions as an example to illustrate the added value of HBM for policy making. Policy measures for POPs, including source-related regulation (e.g. further optimization and tightening of existing Flemish legislation on open fires), investment in monitoring networks and communication and awareness campaigns, are presented and the added value for environmental health policy is discussed. We also reflect on how HBM can support science and innovation in the environmental monitoring context. Finally, we describe what society can gain from HBM in terms of opportunities for (1) feeding the political and societal debate, (2) stimulating community involvement and (3) empowering participants and citizens. All together, the gained insights and phased action plan showed that next to compliance with high scientific standards, results of the Flemish human biomonitoring campaign could be translated in targeted policy actions even for chemicals that have since long been regulated.

Air sampling of flame retardants based on the use of mixed-bed sorption tubes--a validation study.

An analytical methodology using automatic thermal desorption and gas chromatography mass spectrometry analysis was optimized and validated for simultaneous determination of a set of components from three different flame retardant chemical classes: polybrominated diphenyl ethers (PBDEs) (PBDE-28, PBDE-47, PBDE-66, PBDE-85, PBDE-99, PBDE-100), organophosphate flame retardants (PFRs) (tributyl phosphate, tripropyl phosphate, tris(2-chloroethyl)phosphate-, tris(1,3-dichloro-2-propyl) phosphate, tris(2-ethylhexyl) phosphate, triphenyl phosphate, tris(2-chloro-1-methylethyl) phosphate and tricresylphosphate), and "novel" brominated flame retardants (NBFRs) (pentabromotoluene, 2,3,4,5,6-pentabromoethylbenzene, (2,3-dibromopropyl) (2,4,6-tribromophenyl) ether, hexabromobenzene, and 2-ethylhexyl 2,3,4,5-tetrabromobenzoate) in air. The methodology is based on low volume active air sampling of gaseous and particulate air fractions on mixed-bed (polydimethylsiloxane (PDMS)/Tenax TA) sorption tubes. The optimized method provides recoveries >88%; a limit of detection in the range of 6-25 pg m(-3) for PBDEs, 6-171 pg m(-3) for PFRs, and 7-41 pg m(-3) for NBFRs; a linearity greater than 0.996; and a repeatability of less than 10% for all studied compounds. The optimized method was compared with a standard method using active air sampling on XAD-2 sorbent material, followed by liquid extraction. On the one hand, the PDMS/Tenax TA method shows comparable results at longer sampling time conditions (e.g., indoor air sampling, personal air sampling). On the other hand, at shorter sampling time conditions (e.g., sampling from emission test chambers), the optimized method detects up to three times higher concentrations and identifies more flame retardant compounds compared to the standard method based on XAD-2 loading.

Key aspects of a Flemish system to safeguard public health interests in case of chemical release incidents.

Although well-established protocols are available for emergency services and first-responders in case of chemical release incidents, a well-developed system to monitor and safeguard public health was, until recently, lacking in Flanders. We therefore developed a decision support system (DSS) to aid public health officials in identifying the appropriate actions in case of incidents. Although the DSS includes human biomonitoring as one of its key instruments, it also goes well beyond this instrument alone. Also other, complementary, approaches that focus more on effect assessment using in vitro toxicity testing, indirect exposures through the food chain, and parallel means of data collection (e.g. through ecosurveillance or public consultation), are integrated in the Flemish approach. Even though the DSS is set up to provide a flexible and structured decision tree, the value of expert opinion is deemed essential to account for the many uncertainties associated with the early phases of technological incidents. When the DSS and the associated instruments will be fully operational, it will provide a valuable addition to the already available protocols, and will specifically safeguard public health interests.

Differential hepatic metal and metallothionein levels in three Feral fish species along a metal pollution gradient.

The accumulation of cadmium, copper and zinc and the induction of metallothioneins (MT) in liver of three freshwater fish species was studied. Gudgeon (Gobio gobio), roach (Rutilus rutilus) and perch (Perca fluviatilis) were captured at 6 sampling sites along a cadmium and zinc gradient and one reference site in a tributary of the Scheldt River in Flanders (Belgium). At each site up to 10 individuals per species were collected and analyzed on their general condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI). From each individual fish the liver was dissected and analyzed on Cd, Cu and Zn and MT-content. Although not all species were present at each site, hepatic Cd and Zn levels generally followed the pollution gradient and highest levels were measured in perch, followed by roach and gudgeon. Nevertheless also an effect of site was observed on this order. MT-levels appeared to be the highest in gudgeon although differences with the other species were not very pronounced and depended on the site. Significant relationships were found between hepatic zinc accumulation and MT levels. For each species the ratio MT(theoretical)/ MT(measured) was calculated, which gives an indication of the relative capacity to induce MTs and thus immobilize the metals. Perch had the lowest capacity in inducing MTs (highest ratio). Relationships between hepatic metal levels and fish condition indices were absent or very weak.

Opening the research agenda for selection of hot spots for human biomonitoring research in Belgium: a participatory research project.

BACKGROUND: In order to select priority hotspots for environment and health research in Flanders (Belgium), an open procedure was organized. Environment and health hotspots are strong polluting point sources with possible health effects for residents living in the vicinity of the hot spot. The selection procedure was part of the work of the Flemish Centre of Expertise for Environment and Health, which investigates the relation between environmental pollution and human health. The project is funded and steered by the Flemish government. METHODS: The involvement of other actors than merely experts is inspired by the 'analytical-deliberative' approach of the National Research Council in the United States and the extended peer community approach. These approaches stress the importance of involving different expert- and social perspectives in order to increase the knowledge base on complex issues. In the procedure used in the project a combination of expert and stakeholder input was essential. The final decision was supported by a multi-criteria analysis of expert assessment and stakeholder advice. RESULTS: The endeavour was challenging from the start because of the complicated ambition of including a diversity of actors, potential hotspots, concerns and assessment criteria, but nevertheless the procedure proved its value in both structuring and informing the decision-making process. Moreover the process gained the support of most actors participating in the process, even though the final selection could not satisfy all preferences. CONCLUSIONS: Opening the research agenda exemplifies the value of inter- and transdisciplinary cooperation as well as the need for a well-structured and negotiated procedure that combines relevant factors and actors with pragmatism. The value of such a process also needs to prove itself in practice after the procedure has been completed: the tension between an ambition of openness on the one hand and a more closed attitude amongst experts on the other will continue to play a role even after closure.

Brominated flame retardants and perfluorinated chemicals, two groups of persistent contaminants in Belgian human blood and milk.

We assessed the exposure of the Flemish population to brominated flame retardants (BFRs) and perfluorinated compounds (PFCs) by analysis of pooled cord blood, adolescent and adult serum, and human milk. Levels of polybrominated diphenyl ethers (PBDEs) in blood (range 1.6-6.5 ng/g lipid weight, lw) and milk (range 2.0-6.4 ng/g lw) agreed with European data. Hexabromocyclododecane ranged between <2.1-5.7 ng/g lw in milk. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) dominated in blood and ranged between 1 and 171 ng/mL and <0.9-9.5 ng/mL, respectively. Total PFC levels in milk ranged between <0.5-29 ng/mL. A significant increase in PBDE concentrations was detected from newborns (median 2.1) to the adolescents and adults (medians 3.8 and 4.6 ng/g lw, respectively). An identical trend was observed for PFOS, but not for PFOA. We estimated that newborn exposure to BFRs and PFCs occurs predominantly post-natally, whereas placental transfer has a minor impact on the body burden.

Exposure of the Flemish population to brominated flame retardants: model and risk assessment.

Human exposure to brominated flame retardants (BFRs) varies widely throughout the world as it depends on country-related usage, production and legislation of these chemicals. US and UK exposure assessments show very diverse levels and patterns which in turn, are likely to differ from those in background exposed countries such as Belgium, where levels tend to be about an order of magnitude lower. The current study assessed human exposure to BFRs through the indoor and outdoor environment (e.g. dust, soil, and air) and food for all age groups in Flanders, Belgium. Most relevant food groups were identified based on a national food consumption survey and food items with Flemish origin were collected. Dust samples were collected using a standardized protocol in 43 homes and 10 offices throughout Flanders. Food, human milk and dust samples were analysed for their polybrominated diphenylethers (PBDE) and hexabromocyclodecane (HBCD) content using GC/MS and LC/MS-MS. An exposure model was developed including all analysed data, complemented with literature data. The model covered human exposure of infants, children and adults through human milk, food, dust/soil ingestion and air inhalation. Total human exposure was compared to the existing toxicological criteria and previous exposure estimates. In general, the exposure levels through human milk are consistent with those of a background exposed European population, whereas dust and food intake are at the low end of what has been reported in previous European intake assessments. Total average intake of SigmaHBCD and SigmaBDE(5) at 50th percentile (P50) levels by newborns equals 3.1 and 12.0ng/kg body weight (bw) day, respectively. This intake increases to 15.2 and 20.9ng/kgbwday for SigmaHBCD and SigmaBDE(5), for higher exposed newborns (95th percentile=P95 levels). Due to the limited database on health-based limit values for PBDEs and HBCD, it is difficult to assess the immediate health concern for any of the age groups, although the higher intake of newborns indicates the need for ongoing monitoring. For median exposed individuals, the average SigmaHBCD intake peaked at the age 3 to 6years with an intake of 6.59ng/kgbwday and declines to approximately 1ng/kgbwday at later age. SigmaBDE(5) intake exhibited a different profile compared to SigmaHBCD with maximal levels for newborns and a decline to approximately 0.7ng/kgbwday at adulthood.

Cytosolic distribution of Cd, Cu and Zn, and metallothionein levels in relation to physiological changes in gibel carp (Carassius auratus gibelio) from metal-impacted habitats.

In the present study the impact of environmental metal contamination in gibel carp (Carassius auratus gibelio) was investigated coupling disturbances in subcellular metal distribution to effect biomarkers. Gibel carp were caught at six different sampling sites in Flanders (Belgium), characterized by different degrees in environmental metal contamination. Tissue Cd, Cu and Zn concentrations and total metallothioneon (MT) levels were determined in gills, liver and kidney. Cytosolic metal distribution was measured in the main accumulating organs, liver and kidney, revealing tissue- and metal-dependent profiles. The MT pool dominated the cytosolic distribution profile. Although the importance of the MT pool increased with increasing environmental and cytosolic metal concentrations, also an undefined fraction of Cu and Cd fractions (probably free metal ions or metals bound to small organic complexes) increased at the most contaminated sampling sites. Disturbances in serum ion concentrations, serum alanine aminotransferase activity (ALT), hematocrite and condition factor were measured, as indicators of toxic effects. At the sampling site with the highest environmental Cd pollution a significant decrease in serum Ca(2+) concentrations and a significantly increased serum ALT activity were measured suggesting incomplete detoxification of Cd. Increased serum ALT concentrations suggested structural liver damage. The fact that the liver tissue, and probably also the kidney, could not cope with this high Cd burden in combination with the increased uptake of dissolved Cd through the gills most probably contributed to the perturbed serum Ca(2+) homeostasis.

A kinetic model for the relative contribution of waterborne and dietary cadmium and zinc in the common carp (Cyprinus carpio).

In this work, the uptake and whole-body accumulation of Cd and Zn by the common carp from water, diet, and a combination of both was studied using the radioactive tracers 09Cd and 65Zn. A three-compartment pharmacokinetic model was constructed and revealed metal- and exposure route-dependent accumulation profiles. Under the tested exposure conditions in water (0.1 microM Cd and 1 microM Zn), only 0.1 and 0.07%, respectively, of the total Cd and Zn load that passed the branchial surfaces was accumulated by the carp. In contrast, the carp accumulated 20 and 29% of the total ingested Cd and Zn load (mean concentrations in food of newly accumulated metals were 2.24 +/- 0.29 and 19.91 +/- 2.89 nmol/g wet weight, respectively). However, the contribution of the waterborne metals to the whole-body metal accumulation was higher than the metal uptake from food. The constructed model was used to simulate the effect of variable assimilation efficiency (AE) values on the relative importance of dietary Cd and Zn exposure to the overall metal accumulation in common carp. This simulation was performed under a realistic exposure scenario and with Cd and Zn AE values ranging from 5 to 95%. Dissolved Zn levels were higher and Cd levels lower compared to the laboratory experiments. Levels in the midge larvae were much higher. The results show that under these conditions, even at the lowest Cd and Zn AEs, almost 40% of the total body Cd and Zn concentrations originated from dietary Cd and Zn exposure. Taking into account the Cd and Zn AE of the laboratory experiments, respectively, 31 and 55%, more than 85% of the total body Cd and Zn was taken up from the food.

Bioaccumulation of micropollutants and biomarker responses in caged carp (Cyprinus carpio).

At four different aquatic sites in Flanders (Belgium) with different types and degrees of contamination, juvenile carp (Cyprinus carpio) were exposed in cages for 4 weeks. After exposure, metals, polychlorinated biphenyls (PCBs), and selected organochlorine pesticides (OCPs) were analyzed in the tissues of the carp. Besides pollutant accumulation, several effects were measured as well. Condition measures such as changes in weight, condition factor (CF), and hepatosomatic index (HSI) were assessed. In addition, activity of acetylcholinesterase (AChE) and a set of blood biochemical parameters were measured. At all sites, accumulation of metals in the fish tissues was observed. Levels of cadmium and lead at some of the exposure sites were higher than the levels at the start and comparable to levels in fish from moderately metal-contaminated sites. For most organic pollutants, however, levels were not significantly higher than at the start. Only for two PCB congeners, levels had slightly increased but were still lower than levels in carp captured at noncontaminated sites. Although food limitation probably caused some of the observed effects, significant relationships were found between metal load in tissues and CF, AChE, plasma osmolality and HSI. This study shows that caged carp might be useful for the assessment of bioaccumulation and some effects of micropollutants in aquatic ecosystems.

Metallothioneins (MTs) and delta-aminolevulinic acid dehydratase (ALAd) as biomarkers of metal pollution in great tits (Parus major) along a pollution gradient.

Biomonitoring allows an integrated evaluation of different aspects of exposure, accumulation and effects to environmental pollution, simultaneously accounting for the natural variety between individuals in an ecosystem. In this study, the effects of increased metal accumulation were evaluated at the biochemical level in terms of two biomarker responses in the great tit (Parus major), a small insectivorous songbird, along an established metal pollution gradient. Metal concentrations in internal tissues (liver and kidney) and blood indicated that lead and cadmium were the most important metals in the pollution gradient under study. At the biochemical level, induction of metal binding protein metallothionein (MT) in liver and kidney reflected cadmium concentrations in these tissues (R(2)=0.42 and R(2)=0.94 respectively, n=19), although in kidney, MT induction was not sufficient to complex all cadmium present. Secondly, the activity of the enzyme delta-aminolevulinic acid dehydratase (ALAd) in blood decreased exponentially in response to increased lead accumulation (R(2)=0.70, n=18) and represents therefore a specific effect marker for lead exposure. In the highest polluted area, an ALAd inhibition of 85% was reported. Since a higher metal exposure resulted in an increased metal accumulation and subsequent biomarker responses in a dose-dependent way, this study indicates the applicability of ALAd and MT levels in great tits for biomonitoring responses to heavy metal pollution.

A field survey of metal binding to metallothionein and other cytosolic ligands in liver of eels using an on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS).

The effect of metal exposure on the accumulation and cytosolic speciation of metals in livers of wild populations of European eel with special emphasis on metallothioneins (MT) was studied. Four sampling sites in Flanders showing different degrees of heavy metal contamination were selected for this purpose. An on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS) was used to study the cytosolic speciation of the metals. The distribution of the metals Cd, Cu, Ni, Pb and Zn among cytosolic fractions displayed strong differences. The cytosolic concentration of Cd, Ni and Pb increased proportionally with the total liver levels. However, the cytosolic concentrations of Cu and Zn only increased above a certain liver tissue threshold level. Cd, Cu and Zn, but not Pb and Ni, were largely associated with the MT pool in correspondence with the environmental exposure and liver tissue concentrations. Most of the Pb and Ni and a considerable fraction of Cu and Zn, but not Cd, were associated to High Molecular Weight (HMW) fractions. The relative importance of the Cu and Zn in the HMW fraction decreased with increasing contamination levels while the MT pool became progressively more important. The close relationship between the cytosolic metal load and the total MT levels or the metals bound on the MT pool indicates that the metals, rather than other stress factors, are the major factor determining MT induction.

Induction and metal speciation of metallothionein in wood mice (Apodemus sylvaticus) along a metal pollution gradient.

We investigated the binding of Cd, Cu, and Zn to metallothionein (MT) and other metal-binding proteins in free-living wood mice (Apodemus sylvaticus L.) captured in four areas along a metal pollution gradient. We measured total and cytosolic Cd, Cu, and Zn concentrations in mouse liver and kidney by means of inductively coupled plasma-mass spectrometry (ICP-MS). Total (Cu, Cd, Zn)-MT levels were determined in the same tissues by means of the cadmium thiomolybdate saturation assay. Metal speciation of metalloproteins was studied by means of size-exclusion high-performance liquid chromatography-ICP-MS. Liver and kidney of wood mice from the site adjacent to the pollution source showed the highest Cd and Zn concentrations (total and cytosolic) and (Cu, Cd, Zn)-MT levels compared to the other sites farther away from the pollution source. No or only small site differences in tissue Cu concentrations were observed. Almost all the variation (85-95%) in hepatic and renal (Cu, Cd, Zn)-MT levels was explained by the total or cytosolic hepatic Zn and Cd concentration or the renal Cd concentration, respectively. An analysis of the cytosolic metal speciation showed that the Cd-MT, Cu-MT, and Zn-MT fractions in liver and kidney increased significantly with increasing cytosolic metal concentrations. Metals associated with the other cytosolic protein fractions did not increase with increasing exposure. These results illustrate the important role of MT in metal homeostasis and detoxification processes. We conclude that MT is a useful biomarker for environmental metal contamination in free-living wood mice.

Dynamics of cadmium accumulation and effects in common carp (Cyprinus carpio) during simultaneous exposure to water and food (Tubifex tubifex).

In this study, the influence of exposure time and concentration on the accumulation of cadmium and the occurrence of adverse effects was investigated when carp were simultaneously exposed through contaminated water and food. As exposure concentrations increased (9, 105, and 480 microg/L through water and 9.5, 122, and 141 microg/g dry weight through food) the accumulation pattern of cadmium changed, with progressively more cadmium being accumulated in the liver and especially the kidney and less in the gills and the intestine. A strong concentration- and time-dependent effect of cadmium exposure on plasma calcium concentrations was observed, leading to a decrease, with approximately 16% in the mid group and up to 50% in the group exposed to the highest cadmium concentration. Also at the highest exposure concentrations, sodium and chloride levels as well as plasma osmolality were significantly reduced, indicating a disturbed ion homeostasis. Furthermore, an increase in plasma alanine transaminase activity indicated liver damage caused by the cadmium accumulation in this organ. At the end of the experiment, general stress responses such as decreased hematocrit, growth, and mortality (17%) also were observed. All together, these results suggest that plasma hypocalcemia as an ion-disruptive phenomenon is the most sensitive effect during cadmium exposure, although the occurrence and magnitude both depend on exposure time and concentration.

Anion-exchange high performance liquid chromatography hyphenated to inductively coupled plasma-isotope dilution-time-of-flight mass spectrometry for speciation analysis of metal complexes with metallothionein isoforms in gibel carp (Carassius auratus gibelio) exposed to environmental metal pollution.

The capability of post-column isotope dilution (ID) combined with anion-exchange HPLC-ICP-time-of-flight (TOF)-MS was for the first time investigated for environmental quality assessment through metal speciation analysis of metallothionein (MT) isoforms in cytosols of gibel carp (Carassius auratus gibelio), used as biomarkers for environmental metal exposure. A full spectral scanning of the biological sample (with 50 microl injection volume) using ICP-TOF-MS in transient mode allowed fast multi-isotope screening of cytosolic metal-containing fractions and to investigate the presence of matrix-induced interferences. The MT cytosolic fraction of liver and kidney of the carp, sampled at three different sampling sites in Belgium, was partially purified using size-exclusion (SE) HPLC. Quantification of the elements Cd (toxic) and Zn and Cu (essential) associated with MT isoforms in this fraction was addressed using an hybrid approach based on post-column addition of the enriched isotopes 65Cu, 67Zn, 106Cd and monitoring on-line the isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd by ICP-MS with a time of flight instrument, which was coupled to anion-exchange HPLC. With this separation method, baseline separation of up to five MT isoforms, which is required for quantitative metal speciation by HPLC-ICP-IDMS, was achieved within a run of 15 min. The MT fraction of the cytosols was also analysed for the total metal content using IDMS with size-exclusion HPLC-ICP-MS and species-unspecific calibration. Results showed significant differences between speciation results and total MT concentrations of control fish and fish from the most contaminated sampling sites, revealing the potential of gibel carp MT for sequestering excess intracellular free-ions (essential and toxic elements) and for its protection against metal toxicity. Preferences for metal sequestration of metal complexes with MT isoforms were also found to be tissue-specific: excess of Cd was found preferably bound to a major MT isoform (tR = 8.0 min) in kidney, whereas excess intracellular Zn appeared to be mostly sequestered by four MT isoforms (tR=7.3, 8.0, 12.2 and 14.4 min) in liver, the MT form with tR = 8.0 min being the main Zn scavenger form. Such kind of quantitative speciation information on the preferences of MT isoforms in different fish organs for sequestering heavy metals, reported here for the first time, is important to elucidate the role of isoform-specific induction of vertebrate fish MT in metal detoxification and the use of MT as biomarker.

Perfluorooctane sulfonic acid and organohalogen pollutants in liver of three freshwater fish species in Flanders (Belgium): relationships with biochemical and organismal effects.

A perfluorooctane sulfonic acid (PFOS) assessment was conducted on gibel carp (Carassius auratus gibelio), carp (Cyprinus carpio), and eel (Anguilla anguilla) in Flanders (Belgium). The liver PFOS concentrations in fish from the Ieperlee canal (Boezinge, 250-9031 ng/g wet weight, respectively) and the Blokkersdijk pond (Antwerp, 633-1822 ng/g wet weight) were higher than at the Zuun basin (Sint-Pieters-Leeuw, 11.2-162 ng/g wet weight) and among the highest in feral fish worldwide. Eel from the Oude Maas pond (Dilsen-Stokkem) and Watersportbaan basin (Ghent) had PFOS concentrations ranging between 212 and 857 ng/g wet weight. The hepatic PFOS concentration was significantly and positively related with the serum alanine aminotransferase activity, and negatively with the serum protein content in eel and carp. The hepatic PFOS concentration in carp correlated significantly and negatively with the serum electrolyte concentrations whereas a significant positive relation was found with the hematocrit in eel. Although 13 organochlorine pesticides, 22 polychlorinated biphenyl (PCB) congeners and 7 polybrominated diphenyl ethers (PBDEs) were also measured in the liver tissue, only PCB 28, PCB 74, gamma-hexachlorocyclohexane (gamma-HCH) and hexachlorobenzene (HCB) were suggested to contribute to the observed serological alterations in eel.

Polybrominated diphenyl ethers (PBDEs) in freshwater mussels and fish from Flanders, Belgium.

The levels and distribution of PBDEs in zebra mussels and several freshwater fish species (eel, carp and gibel carp) were investigated for different sites in Flanders, Belgium. In parallel, other organohalogenated contaminants, such as polychlorinated biphenyls (PCBs), p,p[prime or minute]-DDE and hexachlorobenzene (HCB) were also measured and their relationship with PBDEs was investigated. At most sites, individual PBDE congeners were present at detectable levels in mussel tissue, with the mean [summation operator]PBDE concentration ranging from 0.15 to 1.8 ng g(-1) wet weight (ww). The PCB concentrations in mussels ranged from 6.2 to 102 ng g(-1) ww. HCB and p,p[prime or minute]-DDE could be measured in mussels from most sites, mean values ranging from below the limit of quantification (LOQ) to 0.58 ng g(-1) ww and from 0.66 to 6.5 ng g(-1) ww, respectively. Except for one site (Blokkersdijk, Antwerp) where PBDEs were below the LOQ in carp muscle, all fish samples from other sites contained detectable PBDE levels, with the highest concentrations (14 +/- 14 ng g(-1) ww) being measured in eel liver from Watersportbaan (Ghent). The sampled sites covered a broad concentration range of organohalogenated pollutants with the highest values being consistently measured in eel liver. With few exceptions, all correlations between PBDEs and organochlorine pollutants for each species were low (r < 0.50) and most were statistically not significant (p > 0.05). This suggests that the exposure to contaminants arises from local sources possessing different signatures of PBDEs and organochlorine pollutants.