Hazard screening of contaminated sites : bioavailable fractions and biological in vitro tools

The environmental bioavailability of contaminants, rather than their total concentrations in the soil compartment play a decisive role for the risks associated with contaminated sites. Various soil constituents and abiotic conditions have strong influence on bioavailability, which may vary substantially between different locations. It is therefore necessary to site-specifically use tools that reflect the fractions of contaminants that are available to biota and pose the highest potential environmental risks. Bioassays provide integrated toxic responses which include effects from unknown contaminants or combinatory toxic effects from mixtures of contaminants. Thus, biological effect data greatly contribute to establish more realistic exposure and risk-scenarios at contaminated sites. The work underlying this thesis presents possible techniques for high capacity screening for site-specific hazards at contaminated areas. By combining rapid water extractions and cell-based in vitro designs measures of the toxic potential in soils was obtained. Toxicologically bioavailable fractions of mixed metal pollution, including arsenic, were primarily investigated in this thesis. In two of the studies, environmental availability and toxicological bioavailability of arsenic was explored in CCA-contaminated soils. Application of cell-based in vitro screening techniques was also conducted at a metal contaminated industrial site to obtain spatial distribution of toxicity. Multivariate association techniques were employed in the interpretation of environmental exposure and cytotoxicity data. It was shown that cell-based in vitro systems for both basal cytotoxicity and specific end-points targeting arsenic could assess the toxic potential from extracts obtained by several water-based extraction techniques including Pressurised Liquid Extraction (PLE). The cell-based in vitro systems were found to add important information on the site-specific differences in arsenics genotoxic potential from CCA-contaminated soils. The results highlight the importance of taking speciation and toxicological bioavailability into account in the risk analysis, rather than to base risk estimates on total load of contaminants. The presented screening approach was successfully applied at a metal polluted industrial site where spatial distribution of toxicity was obtained. PLE extraction also provided means for combined toxicological and chemical screening of explosives in soils from live-fire training ranges. Multivariate association techniques highly facilitated the interpretation of complex environmental data. The PLE was found to be a rapid extraction technique that has sufficient environmental relevance to be used in environmental impact analyses. It was also concluded that other cell-based in vitro systems that target specific toxic effects have large potential for being used in screening for a variety of environmental chemicals. Keywords: Environmental availability, Environmental bioavailability, Toxicological bioavailability, mixture toxicity, hazard screening, contaminated soils, heavy metals, arsenic, CCA, explosives, soil extraction, water extracts, cell-based in vitro tests, cytotoxicity, genotoxicity, PLE, MVDA, PCA, PLS.Föroreningars biotillgänglighet snarare än deras totala koncentration i markmiljön styr den risk som kan förknippas med förorenade områden. Biotillgängligheten är ofta långt från 100% p.g.a. en rad olika bindningsytor och processer i jorden som reducerar biotillgängligheten. Således kan biotillgängligheten variera kraftigt mellan olika förorenade platser och även inom samma plats till följd av de specifika förhållanden som råder på respektive plats. Tillämpning av biologiska indikatorer som ger ett mått på den samlade giftigheten från biotillgängliga föroreningar är därför viktiga verktyg i platsspecifika exponerings- och farobedömningar. Många biologiska tester är ofta laborativt intensiva och dyra och lämpar sig mindre väl i testning av ett stort antal prover vilket är önskvärt om en tillräcklig geografisk täckning ska uppnås över ett förenat område. Testsystem som har kapacitet att hantera många prover till en rimlig kostnad är därför mycket användbart för screening i ett inledande skede av en miljöriskanalys av ett förorenat område. Föreliggande avhandlingsarbete presenterar möjliga lösningar i att kombinera snabb vattenextraktionsmetodik med cellbaserade in vitro system för platsspecifik toxikologisk faroscreening av metallförorenade områden. Metodiken erbjuder hög kapacitet för många jordprover. Tillämpning av metodiken har gjorts mot huvudsakligen metallföroreningar, inklusive arsenik. I två delarbeten studerades två modelljordar från CCA-förorenade fastigheter avseende tillgänglighet och giftighet av framför allt arsenik. Vidare studerades om det med applicerad metodik gick att illustrera geografisk utbredning av toxicitet, mätt i cellbaserade in vitro system, som biotillgängliga föroreningar uppvisar på ett metallförorenat industriområde. Slutligen studerades lämpligheten i att använda PLE för kombinerad kemisk och toxikologisk screening av jordar från militära skjutfält som var förorenade med explosivämnen. Cellbaserade in vitro system för mätning av både generell toxicitet och mer specifika effektmarkörer för arsenik visade sig användbara vid mätning från flera vattenbaserade extraktionsmetoder, inklusive PLE (trycksatt vätskeextraktion). Resultaten visade på PLEs tillämplighet som en snabb extraktionsmetod med bibehållen relevans för miljöanalyser. Applikation av cellbaserade in vitro system på vattenextrakt från förorenad jord gav värdefull information bl.a. om platsspecifik genotoxisk potential där specieringen av arsenik hade avgörande betydelse i en fallstudie med CCA-förorenade jordar. Vattenextraktion av jordprover kombinerat med cellbaserade in vitro system kunde också ge en geografisk bild av den omedelbara faran från biotillgängliga metallföroreningar inom ett industriområde. Vattenextraktion med PLE visade sig även användbart för screening av explosivämnen där extrakten direkt kunde användas för såväl kemisk karakterisering som för toxikologisk analys. Även andra typer av in vitro system än de som användes i detta arbete har stor framtida potential för tillämpning i faroscreening av ett stort antal olika typer av miljöföroreningar

Hazard screening of contaminated sites : bioavailable fractions and biological in vitro tools

The environmental bioavailability of contaminants, rather than their total concentrations in the soil compartment play a decisive role for the risks associated with contaminated sites. Various soil constituents and abiotic conditions have strong influence on bioavailability, which may vary substantially between different locations. It is therefore necessary to site-specifically use tools that reflect the fractions of contaminants that are available to biota and pose the highest potential environmental risks. Bioassays provide integrated toxic responses which include effects from unknown contaminants or combinatory toxic effects from mixtures of contaminants. Thus, biological effect data greatly contribute to establish more realistic exposure and risk-scenarios at contaminated sites. The work underlying this thesis presents possible techniques for high capacity screening for site-specific hazards at contaminated areas. By combining rapid water extractions and cell-based in vitro designs measures of the toxic potential in soils was obtained. Toxicologically bioavailable fractions of mixed metal pollution, including arsenic, were primarily investigated in this thesis. In two of the studies, environmental availability and toxicological bioavailability of arsenic was explored in CCA-contaminated soils. Application of cell-based in vitro screening techniques was also conducted at a metal contaminated industrial site to obtain spatial distribution of toxicity. Multivariate association techniques were employed in the interpretation of environmental exposure and cytotoxicity data. It was shown that cell-based in vitro systems for both basal cytotoxicity and specific end-points targeting arsenic could assess the toxic potential from extracts obtained by several water-based extraction techniques including Pressurised Liquid Extraction (PLE). The cell-based in vitro systems were found to add important information on the site-specific differences in arsenics genotoxic potential from CCA-contaminated soils. The results highlight the importance of taking speciation and toxicological bioavailability into account in the risk analysis, rather than to base risk estimates on total load of contaminants. The presented screening approach was successfully applied at a metal polluted industrial site where spatial distribution of toxicity was obtained. PLE extraction also provided means for combined toxicological and chemical screening of explosives in soils from live-fire training ranges. Multivariate association techniques highly facilitated the interpretation of complex environmental data. The PLE was found to be a rapid extraction technique that has sufficient environmental relevance to be used in environmental impact analyses. It was also concluded that other cell-based in vitro systems that target specific toxic effects have large potential for being used in screening for a variety of environmental chemicals. Keywords: Environmental availability, Environmental bioavailability, Toxicological bioavailability, mixture toxicity, hazard screening, contaminated soils, heavy metals, arsenic, CCA, explosives, soil extraction, water extracts, cell-based in vitro tests, cytotoxicity, genotoxicity, PLE, MVDA, PCA, PLS.Föroreningars biotillgänglighet snarare än deras totala koncentration i markmiljön styr den risk som kan förknippas med förorenade områden. Biotillgängligheten är ofta långt från 100% p.g.a. en rad olika bindningsytor och processer i jorden som reducerar biotillgängligheten. Således kan biotillgängligheten variera kraftigt mellan olika förorenade platser och även inom samma plats till följd av de specifika förhållanden som råder på respektive plats. Tillämpning av biologiska indikatorer som ger ett mått på den samlade giftigheten från biotillgängliga föroreningar är därför viktiga verktyg i platsspecifika exponerings- och farobedömningar. Många biologiska tester är ofta laborativt intensiva och dyra och lämpar sig mindre väl i testning av ett stort antal prover vilket är önskvärt om en tillräcklig geografisk täckning ska uppnås över ett förenat område. Testsystem som har kapacitet att hantera många prover till en rimlig kostnad är därför mycket användbart för screening i ett inledande skede av en miljöriskanalys av ett förorenat område. Föreliggande avhandlingsarbete presenterar möjliga lösningar i att kombinera snabb vattenextraktionsmetodik med cellbaserade in vitro system för platsspecifik toxikologisk faroscreening av metallförorenade områden. Metodiken erbjuder hög kapacitet för många jordprover. Tillämpning av metodiken har gjorts mot huvudsakligen metallföroreningar, inklusive arsenik. I två delarbeten studerades två modelljordar från CCA-förorenade fastigheter avseende tillgänglighet och giftighet av framför allt arsenik. Vidare studerades om det med applicerad metodik gick att illustrera geografisk utbredning av toxicitet, mätt i cellbaserade in vitro system, som biotillgängliga föroreningar uppvisar på ett metallförorenat industriområde. Slutligen studerades lämpligheten i att använda PLE för kombinerad kemisk och toxikologisk screening av jordar från militära skjutfält som var förorenade med explosivämnen. Cellbaserade in vitro system för mätning av både generell toxicitet och mer specifika effektmarkörer för arsenik visade sig användbara vid mätning från flera vattenbaserade extraktionsmetoder, inklusive PLE (trycksatt vätskeextraktion). Resultaten visade på PLEs tillämplighet som en snabb extraktionsmetod med bibehållen relevans för miljöanalyser. Applikation av cellbaserade in vitro system på vattenextrakt från förorenad jord gav värdefull information bl.a. om platsspecifik genotoxisk potential där specieringen av arsenik hade avgörande betydelse i en fallstudie med CCA-förorenade jordar. Vattenextraktion av jordprover kombinerat med cellbaserade in vitro system kunde också ge en geografisk bild av den omedelbara faran från biotillgängliga metallföroreningar inom ett industriområde. Vattenextraktion med PLE visade sig även användbart för screening av explosivämnen där extrakten direkt kunde användas för såväl kemisk karakterisering som för toxikologisk analys. Även andra typer av in vitro system än de som användes i detta arbete har stor framtida potential för tillämpning i faroscreening av ett stort antal olika typer av miljöföroreningar

Applying Generic Water Quality Criteria to Cu and Zn in a Dynamic Aquatic Environment—The Case of the Brackish Water Formation Strömmen-Saltsjön

The EU Water Framework Directive stipulates that all EU waterways shall have good chemical and ecological status by 2027. Methodologies are described for how to assess and classify waterbodies and make 7-year management plans. Aquatic risk assessment methodologies and environmental quality standards are defined and a biotic ligand model methodology is available to assess the influence of water chemistry on the ability of aquatic organisms to take up metals. Aquatic status classification practices of naturally occurring river basin-specific metals are discussed, specifically how Cu and Zn water quality criteria guideline values have been adopted and defined for Swedish coastal and estuarine waters and how well they represent possible ecological risks. Calculations of bioavailability and ecotoxicity are conducted using recognised models for the Strömmen-Saltsjön water body in Stockholm, in which naturally occurring metals, especially Cu, have among the highest background concentrations of Sweden. Proposals are made to improve risk assessment methodologies to better reflect the vitality of living organisms, and to what extent current levels of these metals in Swedish waterways may influence their welfare. The study concludes that a more local assessment including, e.g., studies of the benthic fauna would be relevant for ecological status classification

Exposure of <i>C</i>. <i>elegans</i> to metals affects the expression of oxidative stress response genes.

<p>Synchronized <i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vormbäcken stream at Björkås (B) and the exit to Vindelälven (V) were analyzed for the expression of <i>gst-4</i>, <i>sod-1</i>, <i>cat-2</i>, <i>cyp-35A2</i>, <i>daf-2</i>, <i>nnt-1</i>, <i>kel-8</i> and <i>gcs-1</i> using qRT-PCR. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control and # represents significant differences in between sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Effect of metal exposure on <i>C</i>. <i>elegans</i> genes involved in apoptosis and innate immunity.

<p>Apoptosis marker genes <i>ape-1</i> and <i>hus-1</i> and innate immunity modulators <i>tir-1</i>, <i>clec-60</i>, and <i>bar-1</i> were analysed in <i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vombäcken stream at Björnås (B) and the exit to Vindelälven (V). One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control and # represents significant differences in between the sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Effect of metals exposure on nuclear hormone receptors and developmental genes in <i>C</i>. <i>elegans</i>.

<p><i>C</i>. <i>elegans</i> treated with the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vombäcken stream at Björnås (B) and the exit to Vindelälven (V) were analyzed for the expression of genes associated with hormone receptors (<i>fshr-1</i>, <i>nhr-8</i> and <i>nhr-14)</i> and development (<i>vit-2</i>, <i>daf-12</i>, and <i>apl-1)</i> were tested using qRT-PCR. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control whereas # represents significant differences in between the sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Comparative Analysis of Stress Induced Gene Expression in <i>Caenorhabditis elegans</i> following Exposure to Environmental and Lab Reconstituted Complex Metal Mixture

<div><p>Metals are essential for many physiological processes and are ubiquitously present in the environment. However, high metal concentrations can be harmful to organisms and lead to physiological stress and diseases. The accumulation of transition metals in the environment due to either natural processes or anthropogenic activities such as mining results in the contamination of water and soil environments. The present study used <i>Caenorhabditis elegans</i> to evaluate gene expression as an indicator of physiological response, following exposure to water collected from three different locations downstream of a Swedish mining site and a lab reconstituted metal mixture. Our results indicated that the reconstituted metal mixture exerted a direct stress response in <i>C</i>. <i>elegans</i> whereas the environmental waters elicited either a diminished or abrogated response. This suggests that it is not sufficient to use the biological effects observed from laboratory mixtures to extrapolate the effects observed in complex aquatic environments and apply this to risk assessment and intervention.</p></div

Metal exposure alters the expression of heat shock response genes in <i>C</i>. <i>elegans</i>.

<p>Synchronized young adult <i>C</i>. <i>elegans</i> were exposed for six hours to the metal cocktail (MC) or waters from Lake Hornträsket (H) and Vormbäcken stream at Björkås (B) and the exit to Vindelälven (V). qRT-PCR was performed for genes <i>hsp-16</i>.<i>1</i>, <i>hsp-16</i>.<i>2</i>, <i>hsp-16</i>.<i>48</i> and <i>hsp-70</i>. One way analysis of variance (ANOVA) followed by Tukey’s post-test was used for multiple group comparison where * refers to <i>p</i><0.01 as compared to control, and # represents significant differences between the environmental sites H, B and V. The dotted line represents the expression level normalized to that of the control (K-medium prepared with Milli-Q water).</p

Chemical analysis of water samples from Lake Hornträsket and the Vormbäcken stream.

<p>^aThe following substances were below detection limits: aliphatics >C10-C12, >C12-C16, >C16-C35; chlorobenzine; total PCB; naphthalene; acenaphthylen; acenaphthene; fluorene; phenanthrene; anthracene; fluoranthene; pyrene; benzo(a)anthracene; chrysene; benzo(b)fluoranthene; benzo(k)fluoranthene; benzo(a)pyrene; benzo(ah)anthracene; benzo(ghi)perylene; indeno(123cd)pyrene; total PAH.</p><p>Chemical analysis of water samples from Lake Hornträsket and the Vormbäcken stream.</p

Principal component analysis.

<p>The PCA analysis was based on the gene expression pattern in <i>C</i>. <i>elegans</i> exposed to water samples from lake Hornträsket (H), Vormbäcken stream at Björkås (B), Vormbäcken stream at the outlet to Vindelälven (V), K-medium prepared with Milli-Q water (C) and metal cocktail (MC). A. Score plot showing the distribution following exposure to environmental waters and control water based on gene expression. The ellipse shows Hotellings T² (0.05). B. PCA loading plot showing the distribution of the measured variables following exposure to the environmental waters and the K-medium control. C. Score plot showing the distribution following exposure to the MC, environmental waters and control water based on gene expression. The ellipse shows Hotellings T² (0.05). D. PCA loading plot shows the distribution of the measured variables following exposure to the MC, environmental waters and the control (K-medium prepared with Milli-Q water).</p