Sources and fate of PCDDs and PCDFs in rural and urban ecosystem and food chain in southern Vietnam

Problems related to Dioxins contamination in Southern Vietnam are until now still a hot newsreel and controversy subject in international conferences as well as in many articles. The adverse effects of Dioxins residue from chemicals used by U.S. Army during the Vietnam War (Ranch Hand Operation 1961 to 1971) have caused many consequences not only to Vietnamese ecosystem and people but also to the U.S army veterans that participated in the war at that period as reported in many scientific reports. In Vietnam, since 1980 many studies have been carried out in collaboration with overseas scientists and laboratories on Dioxin contamination levels as well as its influences on ecosystem and human health. However, regardless of many efforts, up to now the problem of Dioxin contamination and its consequences is not yet completely and appropriately solved. There is still relative high Dioxins residue in the areas named as "hot spot" in Southern Vietnam and its contamination is causing the adverse effects on local residents. Nowadays, studies on this subject are relatively difficult to perform due to many factors: over 30 years passed; land use disturbance; degradation and transfer of dioxin into biological food web; population emigration; etc.. In addition, a lack of related documents and military secrets also contributed to this. With the support of SDC in frame of a collaboration project between Vietnam and Switzerland, we have carried out the research named "Sources and Fate of PCDDs and PCDFs in rural and urban ecosystem and food chains of South-Vietnam". Our research has examined integratelly the PCDD/Fs sources with special regard on the PCDD/Fs source from the war, but also consider the others possible sources such industrial and municipal combustions, agricultural used chemicals, etc. The selected locations for our research have been set up based on collected document from Division 10-80 and Office 33 (two responsible organizations for Dioxins and related problems in Vietnam): CamLo District – QuangTri Province; DaNang City; MaDa Forest and BienHoa City (BienHoa Airforce Base and BienHung Lake) – DongNai Province; and industrial zone – Thu Duc – Hochiminh City. Seventeen 2,3,7,8-PCDD/Fs congeners have been chosen for our research due to their high toxicities. Soil, sediment, municipal waste incinerator (MWI) bottom ash, fish tissue, and human adipose are selected as the matrices to examine the PCDD/Fs residue. The result showed that even after more than 30 years, the PCDD/Fs concentration based on i-TEQ value (especially for 2,3,7,8-TCDD) is still higher than guideline values in some countries: very high i-TEQ value in cultivate soil of CamLo district and DaNang City. For the area named "hot spot" such BienHoa Airforce Base and BienHung Lake, the i-TEQ value and 2,3,7,8-TCDD concentration in soil and sediment are superior than values proposed by Agency for Toxic Substance and Disease Registry (ATSDR), Canadian Council of Ministers of the Environment (CCME) and U.S Environmental Protection Agency (U.S. EPA). The PCDD/Fs concentration in fish tissue of BienHung Lake (catfish and snake-head) is superior in comparison with European Council (EC) standard. The dioxin contamination risk for local resident health is very high due to the lack of information and inconsiderable attention of responsible organizations about this problem. As BienHoa was selected as a case-study for PCDD/Fs transfer in food chain, using statistic methods (cluster analysis and Principal Component Analysis - PCA), we have showed a high similarity of PCDD/Fs profiles pattern between BienHoa Airforce Base soils, BienHung Lake sediments and fish tissue. The similarity in PCDD/Fs relative i-TEQ profiles between MWIs bottom ash and soil of industrial zone ThuDuc proves that industrial and municipal combustions are responsible sources for PCDD/Fs in the soil of such these zones. However in comparison with the sites contaminated by Agent Orange (A.O)/Dioxin, the PCDD/Fs contamination level in the industrial soil is lower and mainly dominated by PCDFs than PCDDs. The result also showed comparables i-TEQ values in BienHoa residents with inhabitants of industrial countries, however Vietnam is only developing and agricultural country. Beside that, 2,3,7,8-TCDD has been detected in many tested cases. 2,3,7,8-TCDD is the most toxic compound and related to the A.O/Dioxins from the war, unfortunately we have not enough data to assess the relationship between A.O/Dioxin residue and these samples. To find out this relationship it is necessary an integrated large-scale investigation. Result of our research serves as a base for set-up a reference laboratory for PCDD/Fs and dioxin-liked compounds research in the South of Vietnam. At present time, a small laboratory for PCDD/Fs analysis has been installed in IER and serves as a member of VietNam Dioxin Research Network.

Molecular analysis of the biodiversity of protists in hydrocarbon-polluted and pristine soils

Enrique Lara

Eukaryotic microorganisms are an essential component of the Earth's global diversity. It is believed that the number of protistan taxa far outnumbers the one of viruses and bacteria. The taxonomic classification of these organisms, which also represent the major part of the total eukaryotic diversity, is still not very well known. It is only lately that the main phylogenetically consistent eukaryotic super assemblages (also called "kingdoms") have been defined. Inside these kingdoms, lateral branches represent the main multicellular taxa, the plants, animals and fungi. Protists are present in virtually all environments; in this thesis, attention was focused on the soil as habitat for the eukaryotic microorganisms. The communities inhabiting soil are characterized by adaptations to drought (the capacity to form cysts). Moreover, certain groups are found exclusively in this environment, to which they are morphologically adapted, whereas others are absent and exclusively found in aquatic systems. Detailed studies on the ecology of certain groups have been performed, and it is possible to define amongst them typical fast reproducing, early colonizers for new environments (r-specialists), whereas others can be found only in stable and highly productive habitats (K-specialists). All these organisms are distributed in a heterogeneous manner in the soil, being usually more frequent in the top horizon and around hotspots such as, for instance, root tips. Prokaryotes have drawn most of the attention of microbial ecologists, who almost neglected the eukaryotic microbes with the exception of fungi. Ecologists usually define four categories ("guilds") of protists in soil: Flagellates, naked amoebae, testate amoebae and ciliates. The importance of protistan activity in soil ecosystems has been shown with the concept of microbial loop: nutrients, produced by autotrophic organisms, are taken up by decomposers (bacteria and fungi) to produce their own biomass. Heterotrophic protists, by preying on them, release these compounds in a form which can be directly used by the autotrophs ("mineralization"), thus closing the loop. This phenomenon is of major significance, because protistan grazing controls the composition and activity of the prokaryotic community, and also the growth of the autotrophs. In soil, this phenomenon takes mainly place at the tip of the roots of vascular plants, where exudates are released. When soils are exposed to an organic pollution, the protistan communities and thus also their impact on the microbial loop, are influenced in two different ways: either by the direct toxic effects of the pollutant and or the indirect effects on their bacterial preys. A detailed study on the negative effects of environmental pollution, however, requires reliable methods to study protists. Molecular approaches offer the possibility to study protists in situ without the limitations inherent to cultivation dependent approaches. The aim of this thesis was to develop molecular methods and to apply these in combination with classical approaches for the characterization of protistan communities in a polycyclic aromatic hydrocarbon contamined and a non-contaminated control soil (PAH). The eukaryotic communities in a PAH polluted and a non-polluted control soil were first compared by amplifying total soil DNA with eukaryotic domain-specific primers targeting the 18S rRNA genes, cloning and sequencing the resulting PCR products. Using non-specific eukaryotic primers proved to be non-applicable for a reliable analysis of the 18S rRNA gene pools in soil, since protistan 18S rRNA gene sequences were (with the exception of the genus Acanthamoeba) underrepresented in the clone libraries obtained.. The biased picture obtained in the first chapter of the thesis was clearly shown when protist were cultivated from both soils: enrichment and cultivation of protists confirmed that members of the genus Acanthamoeba were indeed the most common protists in polluted soils, whereas they were completely absent from the control soil. Additionally, a diverse protistan community was recovered, which could not be detected by the molecular 18S rRNA-based approach. Twenty-two isolates of flagellates and naked amoebas were obtained, and characterised morphologically as well as by 18S rRNA gene sequence analysis. The diversity of cultivated organisms from the polluted soil was lower as compared to the control soil; however, the total number of protists was higher. Some of the cultures obtained in this cultivation dependant approach were used for further detailed studies; a jakobid isolate was described using light and electron microscopy, thus providing new insights into the knowledge of a relatively unknown group of excavates, i.e. the group of Jakoba incarcerata. Other cultures belonging to the kineoplastea were used to design a set of 18S rRNA-targeting fluorescently labelled oligonucleotide probes for fluorescence in situ hybridization. These probes are specific for the detection of the class Kinetoplastea and four genera within the class, i.e. Bodo, Parabodo, Rhynchomonas and Dimastigella and were applied to pure cultures and to environmental samples obtained by the so-called coverslip method. The effect of the PAH-pollution on a selected soil protistan guild, the ciliates, was further analyzed by applying ciliate-specific PCR based techniques. A specific primer set was used to amplify ciliate 18S rRNA genes from the environment and to conduct comparative sequence analysis of environmental clone libraries. This study indicated that the ciliate 18S rRNA gene pools present in the polluted soil were less diverse than those of the control soil. They contained mainly representatives of the class Colpodea, a clade of r-selected ciliates, typical for unstable environments. The control soil was characterized by a variety of trophic specialists, including carnivores, cyanobacteria and diatom feeders, in contrast to the polluted soil, which harboured mainly sequences affiliating to bacterivores and generalists. Based on these results, we could deduce that the trophic webs are less complex in a PAH polluted soil in comparison to a pristine control. This observation was further confirmed when we studied a larger number of samples using a fingerprinting technique, i.e. denaturing gradient gel electrophoresis (DGGE) with ciliate specific PCR primers. The profiles obtained showed again a dominance of the members of the class Colpodea in polluted soils.

EPFL2005

Endocrine disruption in soil invertebrates

Sophie Campiche

In the past years, it has been observed that some compounds present in our environment can disturb the reproduction and development of animals like fishes, birds, or reptiles by interfering with their endocrine system. Indeed, these endocrine disrupting compounds (EDC) can mimic or antagonize the effects of hormones, alter the pattern of synthesis and metabolism of hormones or modify hormone receptor levels. These substances represent a risk for wildlife, and possibly for humans. Up to now, endocrine disruption was mainly evaluated for vertebrates and aquatic organisms and for oestrogeniclike substances. However, soil invertebrates, which play an important role in soil functioning, have rarely been considered. Moreover, as their endocrine system differs substantially from those of vertebrates (estrogens do not seem to regulate endocrine functions in invertebrates), other substances than can mimic invertebrate hormones should be taken into account. In this sense, insect growth regulators (IGR), which are third generation insecticides specially developed to interfere with insect endocrine system, are interesting compounds. These substances are supposed to have a high specificity for insect pest and a low toxicity for non-target organisms. In the first part of this study, the sublethal effects of six IGR (methoprene, fenoxycarb, precocene II, tebufenozide, hexaflumuron and teflubenzuron) were evaluated on the non-target soil arthropod Folsomia candida. The collembola F. candida represents an integral and beneficial part of the soil ecosystem. It is an euedaphic (subsurface) species which plays an important role in soil respiration and decomposition processes and is therefore vulnerable to the effects of soil contamination. This ecologically relevant organism is one of the most appropriate invertebrate test species for the assessment of environmental quality. It is recommended as test organism by the international standard ISO 11267. The 28-days reproduction tests conducted according to this protocol show that F. candida is affected by the chosen IGR. The most toxic compounds were the two chitin synthesis inhibitors, teflubenzuron and hexaflumuron, with an EC50 of 0.05 mg/kg (dw) for teflubenzuron and an EC50 of 0.6 mg/kg for hexaflumuron. These concentrations are probably environmentally relevant (toxicity/exposure ratios

EPFL2006

Organic pollutants in Swiss compost and digestate

Rahel Christine Brändli

Composting (aerobic treatment of organic wastes) and digestion (anaerobic treatment of organic wastes combined with biogas production) are important waste management strategies with increasing significance in the European Union and Switzerland. Most of the compost produced is applied to agricultural soils, which hereby recycles nutrients and influences soil properties beneficially. However, compost can contain pollutants that may be hazardous for the soil ecosystem. The problem related to heavy metals had been recognised and measures for reduction were taken. Regarding organic pollutants, the current knowledge is insufficient for quality control and risk assessment. This thesis provides a comprehensive overview on organic contaminates in compost, digestate, and presswater and describes factors that may influence them. In the beginning an extensive literature review was carried out to summarize the current data. Compound classes to be analysed in Swiss composts and digestates were prioritized and analytical methods established. Polychlorinated biphenyl concentrations (∑ of PCB 28, 52, 101, 118, 138, 153, 180) were significantly higher in urban (median: 30 μg/kgdry weight (dw), n=52) than in rural samples (median: 14 μg/kgdw, n=16), which points – together with low concentrations in general – to aerial deposition on feedstock material as major input pathway to compost. Median polycyclic aromatic hydrocarbon (PAH) concentration was 3010 μg/kgdw (∑ of 16 PAH defined by the US EPA except dibenzo[a,h]anthracene, n=69). PAH levels were influenced by the organic matter degradation process (higher levels in digestate than in compost), the season of input material collection (spring/summer>winter>autumn), the particle size (higher concentrations in unsieved or sieved >20mm than in sieved to ≤ 20mm products), and maturity (lower concentrations in more mature composts). One fourth of the samples exhibited PAH concentrations above the Swiss guide value for compost (Ordinance on the Reductions of Risks linked to Chemical Products). These elevated concentrations can lead to considerable input of PAH to soil by compost application. To assess the major contributors of PAH in compost, characteristic PAH ratios and some molecular markers were considered, which pointed mainly to combustion origin of these contaminants. Multifactor statistical analysis indicated traffic emission, straw combustion and some asphalt abrasion as potential additional sources. Concentrations of other organic pollutants determined (dibenzo-p-dioxins and -furans, dioxin-like PCB, brominated flame retardants (BFR), perfluorinated alkyl substances (PFAS), di(2-ethylhexyl)phthalate, nonylphenol and chlorinated paraffins) were mostly above levels found in background soil, except for nonylphenol, which was not detected. Out of 269 pesticides analysed, 30 fungicides, 14 herbicides, eight insecticides and one acaricide were detected. For the first time, the fate of organic pollutants during full-scale composting and digestion was assessed. Concentrations of low chlorinated PCB increased during composting (about 30 %), whereas a slight decrease was observed for the higher chlorinated congeners (about 10%). Enantiomeric ratios of atropisomeric PCB were close to racemic and did not change. Levels of low molecular weight PAH were reduced during composting (50 to 90% reduction), whereas heavier compounds remained stable. However, as indicated above, conventional composting does not reduce PAH concentrations sufficiently to comply with Swiss guide values (see above). Further research is needed to i) identify measures to reduce PAH concentrations in digestate and compost, ii) to monitor organic pollutants that are still increasing in other environmental matrices (e.g. BFR and PFAS) or identify new compounds which have not been detected in compost yet, iii) to evaluate potential risk of compost application to soil by assessing the bioavailable fractions of organic pollutants and iv) to examine possible new input materials and co-substrates for composting and digestion.

EPFL2006

Molecular analysis of the biodiversity of protists in hydrocarbon-polluted and pristine soils

Enrique Lara

EPFL2005

Endocrine disruption in soil invertebrates

Sophie Campiche

EPFL2006

Organic pollutants in Swiss compost and digestate

Rahel Christine Brändli

EPFL2006