Anaerobic reductive dehalogenation of polychlorinated dioxins

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are among the most harmful environmental contaminants. Their widespread distribution due to unintentional or unknown release coincides with environmental persistence, acute and chronic toxicity to living organisms, and long-term effects due to the compounds’ tendency for bioaccumulation and biomagnification. While microbial aerobic degradation of PCDD/Fs is mainly reported for the turnover of low chlorinated congeners, this review focuses on anaerobic reductive dehalogenation, which may constitute a potential remediation strategy for polychlorinated compounds in soils and sediments. Microorganisms in sediments and in microcosms or enrichment cultures have been shown to be involved in the reductive dechlorination of dioxins. Bacteria related to the genus Dehalococcoides are capable of the reductive transformation of dioxins leading to lower chlorinated dioxins including di- and monochlorinated congeners. Thus, reductive dehalogenation might be one of the very few mechanisms able to mediate the turnover of polychlorinated dioxins by reducing their toxicity and paving the way for a subsequent breakdown of the carbon skeleton.     

Degradation of dioxin-like compounds by microorganisms

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF; PCDD/F, dioxins) have not been commercially produced in bulk amounts, as were polychlorinated biphenyls and other haloaromatic organics. Within the past two decades a lot␣of information has accumulated on the biodegradation of PCDD/F and other dioxin-like compounds because of their toxicity and because of significant environmental concern about many congeners of this class of chemicals. PCDD/F are subjected to reductive dehalogenations leading to less halogenated congeners, which can be attacked efficiently by fungal and bacterial oxidases and dioxygenases. In several cases these compounds can be utilized as carbon and energy sources. Pathways for their enzymatic degradation and the organisation of the corresponding degradative genes have been elucidated. Consequently, biotechnological applications will exploit the degradative potential of such microorganisms for bioremediation of contaminated sites. Received: 29 August 1997 / Received revision: 6 January 1998 / Accepted: 8 January 1998     

Toward the bioremediation of dioxin-polluted soil: structural and functional analyses of in situ microbial populations by quinone profiling and culture-dependent methods

In order to obtain basic information toward the bioremediation of dioxin-polluted soil, microbial communities in farmland soils polluted with high concentrations of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were studied by quinone profiling as well as conventional microbiological methods. The concentration of PCDD/Fs in the polluted soils ranged from 36 to 4,980 pg toxicity equivalent quality (TEQ) g(-1) dry weight of soil. There was an inverse relationship between the levels of PCDD/Fs and microbial biomass as measured by direct cell counting and quinone profiling. The most abundant quinone type detected was either MK-6 or Q-10. In addition, MK-8, MK-8(H2), and MK-9(H8) were detected in significant amounts. Numerical analysis of quinone profiles showed that the heavily polluted soils (> or = 1,430 pg TEQ g(-1)) contained different community structures from lightly polluted soils (< or = 56 pg TEQ g(-1)). Cultivation of the microbial populations in the heavily polluted soils with dibenzofuran or 2-chlorodibenzofuran resulted in enrichment of Q-10-containing bacteria. When the heavily polluted soil was incubated in static bottles with autoclaved compost as an organic nutrient additive, the concentrations of PCDD/Fs in the soil were decreased by 22% after 3 months of incubation. These results indicate that dioxin pollution exerted a significant effect on microbial populations in soil in terms of quantity, quality, and activity. The in situ microbial populations in the dioxin-polluted soil were suggested to have a potential for the transformation of PCDD/Fs and oxidative degradation of the lower chlorinated ones thus produced.     

Genomic and mechanistic insights into the biodegradation of organic pollutants

Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.     

Lignin degradation by microorganisms: A review

Lignin is an abundant plant-based biopolymer that has found applications in a variety of industries from construction to bioethanol production. This recalcitrant branched polymer is naturally degraded by many different species of microorganisms, including fungi and bacteria. These microbial lignin degradation mechanisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries. The classes and mechanisms of different microbial lignin degradation options available in nature form the primary focus of the present review. This review first discusses the chemical building blocks of lignin and the industrial sources and applications of this multifaceted polymer. The review further places emphasis on the degradation of lignin by natural means, discussing in detail the lignin degradation activities of various fungal and bacterial species. The lignin-degrading enzymes produced by various microbial species, specifically white-rot fungi, brown-rot fungi, and bacteria, are described. In the end, possible directions for future lignin biodegradation applications and research investigations have been provided.     

Dioxin/polychlorinated biphenyl body burden, diabetes and endometriosis: findings in a population-based study in Belgium

Dioxins and polychlorinated biphenyls (PCBs) are persistent organic pollutants widely distributed in the food chain, which is the main source of human exposure. Their effects on human health at background exposure levels are still poorly understood. Recent epidemiological evidence suggests a possible association between these pollutants and diabetes. We report here the results of a population-based study in Belgium on 257 (142 women and 115 men) environmentally exposed subjects, including 10 cases of endometriosis and nine cases of diabetes. Seventeen 2,3,7,8-polychlorinated dibenzodioxins/dibenzofurans (PCDD/Fs or dioxins), four coplanar PCBs (International Union of Pure and Applied Chemistry [IUPAC] nos 77, 81, 126 and 169) and 12 PCB markers (IUPAC nos 3, 8, 28, 52, 101, 118, 138, 153, 180, 194, 206 and 209) were quantified in serum fat from fasting blood samples in order to estimate the body burden of these pollutants. Whilst no difference was found between women with endometriosis and their controls, diabetic patients had significantly increased serum levels of dioxins, coplanar PCBs and the 12 PCB markers. After adjustment for age and other covariates, serum total toxic equivalent activity (sum of PCDD/Fs and coplanar PCBs) and 12 PCB marker concentrations in diabetics were 62% (p=0.0005) and 39% (p=0.0067) higher, respectively, than in controls. The risk of diabetes was significantly increased in subjects in the top decile for adjusted concentrations of dioxins (odds ratio 5.1, 95% confidence interval [CI] 1.18-21.7), coplanar PCBs (odds ratio 13.3, 95% CI 3.31-53.2) or 12 PCB markers (odds ratio 7.6, 95% CI 1.58-36.3). These findings warrant further studies to assess the significance of the associations between diabetes and environmental exposure to polychlorinated pollutants.     

Dioxin/polychlorinated biphenyl body burden,diabetes and endometriosis: findings in a population-based study in Belgium

Dioxins and polychlorinated biphenyls (PCBs) are persistent organic pollutants widely distributed in the food chain, which is the main source of human exposure. Their effects on human health at background exposure levels are still poorly understood. Recent epidemiological evidence suggests a possible association between these pollutants and diabetes. We report here the results of a population-based study in Belgium on 257 (142 women and 115 men) environmentally exposed subjects, including 10 cases of endometriosis and nine cases of diabetes. Seventeen 2,3,7,8-polychlorinated dibenzodioxins/dibenzofurans (PCDD/Fs or dioxins), four coplanar PCBs (International Union of Pure and Applied Chemistry [IUPAC] nos 77, 81, 126 and 169) and 12 PCB markers (IUPAC nos 3, 8, 28, 52, 101, 118, 138, 153, 180, 194, 206 and 209) were quantified in serum fat from fasting blood samples in order to estimate the body burden of these pollutants. Whilst no difference was found between women with endometriosis and their controls, diabetic patients had significantly increased serum levels of dioxins, coplanar PCBs and the 12 PCB markers. After adjustment for age and other covariates, serum total toxic equivalent activity (sum of PCDD/Fs and coplanar PCBs) and 12 PCB marker concentrations in diabetics were 62% (p=0.0005) and 39% (p=0.0067) higher, respectively, than in controls. The risk of diabetes was significantly increased in subjects in the top decile for adjusted concentrations of dioxins (odds ratio 5.1, 95% confidence interval [CI] 1.18–21.7), coplanar PCBs (odds ratio 13.3, 95% CI 3.31–53.2) or 12 PCB markers (odds ratio 7.6, 95% CI 1.58–36.3). These findings warrant further studies to assess the significance of the associations between diabetes and environmental exposure to polychlorinated pollutants.     

On the isolation of polychlorinated dibenzo-p-dioxins and furans from serum samples using immunoaffinity chromatography prior to high-resolution gas chromatography–mass spectrometry

Immunoaffinity chromatography (IAC) for the purification of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) from biological samples was explored as a means to simplify the cleanup procedure and thereby decrease the time and cost of dioxin analysis. A monoclonal antibody (DD3) was used to produce IAC columns and to isolate the PCDD/Fs from serum. Native and ¹³C-labeled PCDD/Fs were spiked at the ppq to ppt range into serum. Quantitation of the PCDD/Fs was performed by a standard dioxin analytical method, i.e. high-resolution gas chromatography–mass spectrometry (GC–MS), which was easily compatible with IAC. Five of the most toxic PCDD/Fs consistently showed acceptable recoveries (>25%) and were reliably quantitated. The congeners specifically recovered by this method represent almost 80% of the toxic equivalency of dioxins and furans present in the serum samples. Dioxin-like polychlorinated biphenyls (PCBs) were not recognized by this antibody column. Compared to conventional dioxin cleanup methods, IAC decreased solvent usage by 1.5 l/sample and took only 2 h to process a sample for analysis.     

细菌降解萘、菲的代谢途径及相关基因的研究进展

多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是一类在环境中广泛存在的具有毒性的污染物,微生物降解是其在自然界中降解的主要途径,因而尤为重要。随着研究的深入,关于微生物降解PAHs的分子降解机制、途径等的认识逐渐积累。以下对细菌降解萘、菲的研究进展进行了概述,介绍了萘的水杨酸降解途径,菲的水杨酸、邻苯二甲酸及其他降解途径,同时也包括降解过程中涉及的降解基因簇,如nah-like、phn、phd、nid和nag等以及细菌在PAHs胁迫条件下其他相关基因的表达与调节等方面的最新进展。这些进展可为降解菌株的分子及遗传机制研究提供理论依据,将促进通过基因工程优化降解菌、更有效地检测PAHs环境污染及实现PAHs污染的生物修复。     

含氧杂环及其衍生物的生物降解研究进展

含氧杂环化合物（Oxygen heterocycles）是污染环境中常见的一类难降解有机污染物,具有毒性和致癌特性,其所引发的环境问题受到人们广泛关注。本文综述了典型含氧单杂环化合物四氢呋喃、1,4-二氧六环以及含氧稠杂环化合物二苯并呋喃、二苯并对二噁英的生物降解研究进展,主要包括降解菌降解性能、降解途径和降解相关基因。此外,本文还对二噁英类衍生物的研究现状进行了讨论,并展望了含氧杂环及二噁英类衍生物生物降解的未来研究方向。     

Biodegradation of Phenol: Mechanisms and Applications

Phenol, or hydroxybenzene, is both a synthetically and naturally produced aromatic compound. Microorganisms capable of degrading phenol are common and include both aerobes and anaerobes. Many aerobic phenol-degrading microorganisms have been isolated and the pathways for the aerobic degradation of phenol are now firmly established. The first steps include oxygenation of phenol by phenol hydroxylase enzymes to form catechol, followed by ring cleavage adjacent to or in between the two hydroxyl groups of catechol. Phenol hydroxylases ranging from simple flavoprotein monooxygenases to multicomponent hydroxylases, as well as the genes coding for these enzymes, have been described for a number of aerobic phenol-degrading microorganisms. Phenol can also be degraded in the absence of oxygen. Our knowledge of this process is less advanced than that of the aerobic process, and only a few anaerobic phenol-degrading bacteria have been isolated to date. Convincing evidence from both pure culture studies with the denitrifying organism Thauera aromatica K172 and with two Clostridium species, as well as from mixed culture studies, indicates that the first step in anaerobic phenol degradation is carboxylation in the para-position to form 4-hydroxybenzoate. Following para-carboxylation, thioesterification of 4-hydroxybenzoate to co-enzyme A allows subsequent ring reduction, hydration, and fission. Para-carboxylation appears to be involved in the anaerobic degradation of a number of aromatic compounds. Numerous practical applications exist for microbial phenol degradation. These include the exploitation of indigenous anaerobic phenol-degrading bacteria in the in situ bioremediation of creosote-contaminated subsurface environments, and the use of phenol as a co-substrate for indigenous aerobic phenol-degrading bacteria to enhance in situ biodegradation of chlorinated solvents.     

Microbial breakdown of halogenated aromatic pesticides and related compounds

Abstract Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.     

Microbial breakdown of halogenated aromatic pesticides and related compounds.

Considerable progress has been made in the last few years in understanding the mechanisms of microbial degradation of halogenated aromatic compounds. Much is already known about the degradation mechanisms under aerobic conditions, and metabolism under anaerobiosis has lately received increasing attention. The removal of the halogen substituent is a key step in degradation of halogenated aromatics. This may occur as an initial step via reductive, hydrolytic or oxygenolytic mechanisms, or after cleavage of the aromatic ring at a later stage of metabolism. In addition to degradation, several biotransformation reactions, such as methylation and polymerization, may take place and produce more toxic or recalcitrant metabolites. Studies with pure bacterial and fungal cultures have given detailed information on the biodegradation pathways of several halogenated aromatic compounds. Several of the key enzymes have been purified or studied in cell extracts, and there is an increasing understanding of the organization and regulation of the genes involved in haloaromatic degradation. This review will focus on the biodegradation and biotransformation pathways that have been established for halogenated phenols, phenoxyalkanoic acids, benzoic acids, benzenes, anilines and structurally related halogenated aromatic pesticides. There is a growing interest in developing microbiological methods for clean-up of soil and water contaminated with halogenated aromatic compounds.     

Membrane composition and successful bioaugmentation. Studies of the interactions of model thylakoid and plasma cyanobacterial and bacterial membranes with fungal membrane-lytic enzyme Lecitase ultra

Cyanobacterial/bacterial consortia are frequently inoculated to soils to increase the soil fertility and to accelerate the biodegradation of organic pollutants. Moreover, such consortia can also be successfully applied in landfills especially for the biodegradation of plastic wastes. However, the bioaugmentation techniques turn out frequently inefficient due to the competition of the indigenous microorganisms attacking directly these inoculated or secreting to their surroundings cell wall and membrane-lytic enzymes. It can be hypothesized that the resistance of the microbial membrane to the enzymatic degradation is correlated with its lipid composition. To verify this hypothesis glycolipid and phospholipid Langmuir monolayers were applied as models of thylakoid and plasma cyanobacterial and bacterial membranes. Hybrid fungal enzyme Lecitase ultra joining the activity of lipase and phospholipase A1 was applied as the model of fungal membrane-lytic enzyme. It turned out that anionic thylakoid lipids sulfoquinovosyldiacylglycerol and phosphatidylglycerols were the main targets of Lecitase ultra in the model multicomponent thylakoid membranes. The resistance of the model plasma bacterial membranes to enzymatic degradation depended significantly to their composition. The resistance increased generally when the unsaturated lipids were exchanged to their saturated counterparts. However, most resistant turned out the membranes composed of unsaturated phosphatidylamine and saturated anionic phospholipids.     

Dairy ruminant exposure to persistent organic pollutants and excretion to milk

Human activities produce polluting compounds such as persistent organic pollutants (POPs), which may interact with agriculture. These molecules have raised concern about the risk of transfer through the food chain via the animal product. POPs are characterised by a strong persistence in the environment, a high volatility and a lipophilicity, which lead to their accumulation in fat tissues. These compounds are listed in international conventions to organise the information about their potential toxicity for humans and the environment. The aim of this paper is to synthesise current information on dairy ruminant exposure to POPs and the risk of their transfer to milk. Three major groups of POPs have been considered: the polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), the polychlorobiphenyls (PCBs) and the polycyclic aromatic hydrocarbons (PAHs). The results show that contamination of fodder and soil by these compounds is observed when they are exposed to emission sources (steelworks, cementworks, waste incinerators or motorways) compared with remote areas. In general, soil contamination is considered higher than plant contamination. Highest concentrations of POPs in soil may be close to 1000 ng/kg dry matter (DM) for PCDD/Fs, to 10 000 mg/kg DM for PAHs and 100 μg/kg DM for PCBs. The contamination of milk by POPs depends on environmental factors, factors related to the rearing system (fodder and potentially contaminated soil, stage of lactation, medical state of the herd) and of the characteristics of the contaminants. Transfer rates to milk have been established: for PCBs the rate of transfer varies from 5% to 90%, for PCDD/Fs from 1% to 40% and for PAHs from 0.5% to 8%. The differential transfer of the compounds towards milk is related to the hydrophobicity of the pollutants as well as to the metabolic susceptibility of the compounds.     

细菌对环境污染物的趋化性及其在生物修复中的作用

细菌对有机化合物的降解能力是一种利用碳源和能源的优势,这种能力可以用来设计安全、有效和无二次污染的污染物的生物修复系统。趋化性是细菌适应外界化学环境变化而作出的行为反应,是一种寻找碳源和能源的优势。细菌的趋化性能够增强细菌在自然环境中的降解污染物的效果,细菌的趋化性与降解性之间的关系研究已经成为热点。介绍了细菌的趋化性的基本概念和趋化信号转导的机制,重点讨论了细菌对环境污染化合物的趋化性,从基因水平揭示了趋化性与降解性之间的紧密联系,认为趋化性可以有效地促进降解性细菌对污染物的生物修复作用。     

城市污泥中邻苯二甲酸酯(PAEs)的厌氧微生物降解

邻苯二甲酸酯(PAEs)是城市污泥中普遍存在的一类具有内分泌干扰性作用的有机污染物.研究污泥厌氧生物处理过程中PAEs的微生物降解对保障污泥农用的安全性十分必要.本文以污泥中两种主要的PAEs——邻苯二甲酸二丁酯(DBP)和邻苯二甲酸(2-乙基己)酯(DEHP)为研究对象,通过比较PAEs在污泥厌氧消化系统与发酵产氢系统中降解过程的差异及系统污泥特性的变化,分析不同污泥厌氧生物处理系统中影响PAEs降解的可能因素.结果表明: 在污泥厌氧发酵系统中,DBP在6 d内降解率达99.6%, DEHP在整个14 d的培养期间也降解了46.1%;在发酵产氢系统中,在14 d培养过程DBP的降解率仅为19.5%,DEHP则没有明显的降解.与厌氧消化系统相比,PAEs在发酵产氢系统中的降解受到明显抑制,这与发酵产氢过程中微生物量下降、革兰氏阳性菌/革兰氏阴性菌(G⁺/G^-)和真菌/细菌变小及挥发性脂肪酸(包括乙酸、丙酸及丁酸)浓度升高有关.     

Phylogenetic characterization of a polychlorinated-dioxin- dechlorinating microbial community by use of microcosm studies

Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, "Dehalococcoides," and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical "Dehalococcoides" group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the "Dehalococcoides"-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.     

White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition

White lupins (Lupinus albus L.) respond to phosphate deficiency by producing special root structures called cluster roots. These cluster roots secrete large amounts of carboxylates into the rhizosphere, mostly citrate and malate, which act as phosphate solubilizers and enable the plant to grow in soils with sparingly available phosphate. The success and efficiency of such a P-acquisition strategy strongly depends on the persistence and stability of the carboxylates in the soil, a parameter that is influenced to a large extent by biodegradation through rhizosphere bacteria and fungi. In this study, we show that white lupin roots use several mechanisms to reduce microbial growth. The abundance of bacteria associated with cluster roots was decreased at the mature state of the cluster roots, where a burst of organic acid excretion and a drastic pH decrease is observed. Excretion of phenolic compounds, mainly isoflavonoids, induced fungal sporulation, indicating that vegetative growth, and thus potential citrate consumption, is reduced. In addition, the activity of two antifungal cell wall-degrading enzymes, chitinase and glucanase, were highest at the stage preceding the citrate excretion. Therefore, our results suggest that white lupin has developed a complex strategy to reduce microbial degradation of the phosphate-solubilizing agents.     

Atmospheric and riverine inputs of metals,nutrients and persistent organic pollutants into the lagoon of Venice

Atmospheric deposition in the lagoon of Venice and river inputs from the watershed were collected and analysed from 1998 to 1999 using the same analytical methods. The input from riverine sources largely prevails (>70%) over that from the atmosphere for As, Cr, Fe, Mn, Ni, nitrogen and phosphorus. Equivalent amounts of Hg, Pb, PCBs, HCB are discharged into the lagoon from the two sources, whilst atmospheric inputs prevail for Cd, ammonia and dioxins. A comparison with figures of maximum allowable discharges (MAD) for various compounds, recently set by the Italian Ministry for the Environment, showed that total inputs (riverine + atmospheric) of trace metals were below the MAD thresholds only for Cr, Cu, Ni and Zn. The total inputs of Cu and Ni, and Cr and Zn were approximately 20 and 40% of the MAD limit, respectively. The total phosphorus input of 284 t was close to the imposed limit, whilst the inorganic nitrogen load alone (>4000 t) was much higher than the MAD for total nitrogen. For those metals (As, Cd, Hg and Pb) and persistent organic pollutants, such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) where the MAD states that the load should tend to ‘0’ (no discharge), the measured inputs of 4.8 (As) and 5.1 t (Pb), 151 (Cd) and 39 kg (Hg), 18 g (PCDD/Fs) and 440 mg (Toxicity Equivalents, TEQs, of PCDD/Fs) are by definition ‘above’ the MAD. The principal component analysis (PCA) of loading data and input profiles (markers) of production typologies showed that river and atmosphere contributions can be easily separated and recognised due to their different fingerprints. Riverine inputs were similar to chemical and glass work production markers, whereas atmospheric loadings were mainly influenced by chemical industry (PVC and VCM production), metallurgy and paper-mill.