Improved and Standardized Methodology for Oil Spill Fingerprinting

The existing Nordtest methodology for oil spill Identification has over the past 10 years formed an important "platform" for solving oil spill identification cases both in the Scandinavian countries as well as other countries in Europe, the USA and Canada. " Revision of the Nordtest Methodology for Oil Spill Identification " is a cooperative project between the National Oil Spill Identification laboratories in Norway, Sweden, Finland, Denmark and the Battelle Memorial Institute (Duxbury) in the USA. The goals of the project are: (1) to refine the existing Nordtest methodology into a technically more robust and defensible oil spill identification methodology with focus on determination of quantitative diagnostic indices (ratios) and (2) to adjust the revised Nordtest methodology into guidelines for the European Committee for Standardization (CEN). This paper presents the recommended methodology for the analytical oil spill identification part. The sampling techniques and handling of oil samples and background (reference) samples prior to their arrival at the environmental forensic laboratory is not covered in this paper. The recommended methodology approach is a result of documented analytical improvements and a more quantitative treatment of analytical data from gas chromatographic-flame ionization detector (GC/FID) and gas chromatographic-mass spectrometer methods (GC/MS-SIM) and the operational experiences over past few years among the participating forensic laboratories. The experience and literature in the field of oil exploration and production geochemistry have also played an important role for the recommended methodology. The results from a recent Round Robin test carried out among 12 laboratories using this new methodology are presented in a separate paper in this issue (Faksness et at ., 2002d).     

Integrated survey on toxic effects of lindane on neotropical fish: Corydoras paleatus and Jenynsia multidentata

We report the effect of lindane on fish experimentally exposed to lindane. Sublethal toxicity was assessed through (a) changes in histopathology; (b) the activity of GST in different organs; and (c) bioaccumulation in exposed fish. We present a survey on toxic effects of lindane at these three levels, proposing a sequence of dose-dependent effects. Physiological damage was reversible at lowest doses, but severe at the highest, including damage consistent with fibrosis in liver and karyolitic nucleus in brain of both studied species. Exposure of Jenynsia multidentata above 6 μg L⁻¹ caused activation a GST in liver and gills, followed by inhibition at 75 μg L⁻¹. Interestingly, the bioaccumulation rate was suddenly increased when GST was inhibited. Corydoras paleatus exposed to 6.0 μg L⁻¹ lindane did not present significant changes in GST activity; however, enzymatic inhibition was observed above 25 μg L⁻¹. The bioaccumulation rate in C. paleatus remained constant throughout the experiments. All in all, these results evidence that C. paleatus is more sensitive to lindane than J. multidentata.     

Altitudinal and thermal gradients of hepatic Cyp1A gene expression in natural populations of Salmo trutta from high mountain lakes and their correlation with organohalogen loads

The biomarker of xenobiotic exposure cytochrome p450A1 (Cyp1A) was used to analyze the biological response to chemical pollution in Salmo trutta (brown trout) from nine high mountain European lakes in Norway, Tatras, Tyrol, and central Pyrenees. Hepatic Cyp1A mRNA levels correlated both with the reciprocal of absolute annual average air temperatures of the sampled lakes and with muscle concentrations of several hydrophobic organohalogen compounds (OC), including chlorinated polychlorobiphenyls (PCB), DDE, and DDT. The correlation between Cyp1A expression and OC content was observed across the whole temperature range (between −0.7 °C and +6.2 °C), but also in the absence of any thermal gradient. We concluded that airborne pollutants accumulate in high mountain lake fish at concentrations high enough to increase Cyp1A expression, among other possible effects. As geographical distribution of semi-volatile OC is strongly influenced by air temperatures, future climate modifications will potentially enhance their physiological effects in lake ecosystems.     

Bioaccumulation of chlorinated pesticides and PCBs in the tropical freshwater fish Hoplias malabaricus: histopathological, physiological, and immunological findings

For assessing the impact of chlorinated compounds, such as organochlorine pesticides, polychlorinated biphenyls, chlorotriazines (atrazine, simazine), and chlorinated phenylureas (diuron), on the Ponta Grossa lake South of Brazil, ten freshwater trahira fish (Hoplias malabaricus) were collected in October 2005. The contamination status was evaluated by the energy budget and various histopathological markers. The results showed detectable amounts of persistent organic pollutants (POPs) in the liver and muscle; the bioaccumulation was higher in the liver than in the muscle. The presence of some banned pesticides, such as hexachlorobenzene and dichlorodiphenyltrichloroethane, in the liver suggests an acute exposure to these compounds. Some physiological disturbances and morphological damages found in the liver of H. malabaricus were associated with chlorinated-compound bioaccumulation. The most important alterations in the liver were lesions such as fibrosis, large necrosis area, leukocyte infiltration, and the absence of melanomacrophages (MM). Individuals containing higher concentrations of pesticides, such as aldrin, alachlor, and dichloroaniline (a metabolite of diuron), showed the nonoccurrence of MM in the liver. These data suggest an immunosuppression in the individuals from Ponta Grossa Lake after exposure to POPs. According to the present data, the POPs found in the studied site are bioavailable, induce severe damages in target organs such as the liver, and can disturb the immune system of the trahira. This is the first study of POPs in the Paraná state, and one among the few studies in the south of Brazil. The present data suggest and motivate further chemical and biomonitoring studies in freshwater ecosystems in the south of Brazil.     

Natural product biomarkers as indicators of sources and transport of sedimentary organic matter in a subtropical river

The sources and transformations of sedimentary organic matter along the Harney River, a representative subtropical river of the Florida Everglades, were assessed using a natural product biomarker approach. Sediment samples were collected from the headwaters to the Continental Shelf, with characteristic vegetation dominated by freshwater marsh species, mangrove (middle to lower estuary), and seagrass as the marine end-member. A peat sample was collected inland. All sample extracts were analyzed by GC–MS as underivatized and as silylated compounds. With these total extract analyses, major compound classes can be defined: n-alkanols, n-alkanoic acids, methyl alkanoates, methyl - and ω-hydroxyalkanoates, triterpenoids, phytosterols and saccharides, with traces of hydrocarbons. In general, the peat sample extract has a different overall composition compared to the sediment extracts. The major differences include distinct carbon number maxima for the lipid series (e.g., C_max = 28 for n-alkanols) probably from sawgrass and periphyton biomass, and predominance of phytosterols (sitosterol and stigmasterol) from higher plant detritus. In contrast, river sediment extracts contain biomarkers predominantly from mangrove-derived organic matter, such as the triterpenoids taraxerol and myricadiol. Significant amounts of saccharides and ω-hydroxyalkanoates are also found. Generally, compound concentrations decrease downstream due to dilution, and alteration of organic compounds from plant waxes and coastal vegetation is obvious in both peat and sediment samples. This is confirmed by the significant low abundance of n-alkanes and n-alkenoic acids due to biodegradation, oxidation of -tocopherol to homophytanic acid γ-lactone, and presence of traces of dihydrolacunosic acid, a photochemical alteration product of taraxerol.     

Exotic gene expression in transgenic plants as a tool for monitoring environmental pollution

Environmental monitoring can benefit from the application of molecular and biochemical techniques as biomarkers. In principle, molecular biomarkers may have some advantage over more classical, organismal level markers because of their sensitivity, efficiency and reproducibility. Nevertheless, some disadvantages such as the cost, resource requirements and lack of specificity have limited their widespread use. Molecular genetics and recombinant DNA technologies, however, allow to improve simplicity and cost, together with reliability, efficiency and sensitivity, of molecular biomarkers. The stress inducible promoter of the barley gene Hvhspl7, which is induced by a number of environmental stress (including some heavy metals), has been fused to the reporter gene GUS in a plasmid. This plasmid was used for stable transformation of tobacco. Some of the transgenic plants obtained after selection showed enhanced GUS expression after exposure to some heavy metals. Maintenance of the plants for three generations, and the analysis of the response to treatments, suggest that these transgenic plants may be utilised as an affordable biomarker for monitoring heavy metal pollution.     

Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles

Engineered nano-sized Cu oxide particles are extensively used in diverse applications. Because aquatic environments are the ultimate “sink” for all contaminants, it is expected that nanoparticles (NP) will follow the same fate. In this study, two marine invertebrates Scrobicularia plana and Hediste diversicolor were chosen as ecotoxicological models. The aim was to evaluate behavioural (burrowing kinetics, feeding rate) and biochemical (biomarkers) responses of S. plana and H. diversicolor exposed in the laboratory to Cu (10 μg L⁻¹) added in natural seawater either in the form of engineered nanoparticles (NPs) of CuO or as dissolved Cu in 2% HNO₃. Exposure was characterized by considering (i) the physico-chemical fate of NP (ii) the fraction of labile Cu in experimental media and (iii) Cu bioaccumulation. Results showed high aggregation of CuO NPs in seawater and no additional bioavailable Cu concentrations. Behavioural impairments were observed in S. plana exposed to CuO NPs or soluble Cu whereas in H. diversicolor, only the exposure to soluble Cu led to a burrowing decrease. No obvious neurotoxicity effects were revealed since in both species, no changes in cholinesterasic activity occurred in response to both forms of Cu exposure. Biomarkers of oxidative-stress catalase and glutathione-S-transferase were enhanced in both species whereas superoxide dismutase was increased only in S. plana exposed to CuO NPs. Metallothionein-like protein was increased in bivalves exposed to both forms of Cu. Since, no detectable release of soluble Cu from CuO NPs occurred during the time of experiment, ecotoxicity effects seem to be related to CuO NPs themselves.     

Heat map visualization of complex environmental and biomarker measurements

Over the past decade, the assessment of human systems interactions with the environment has permeated all phases of environmental and public health research. We are invoking lessons learned from the broad discipline of Systems Biology research that focuses primarily on molecular and cellular networks and adapting these concepts to Systems Exposure Science which focuses on interpreting the linkage from environmental measurements and biomonitoring to the expression of biological parameters. A primary tool of systems biology is the visualization of complex genomic and proteomic data using “heat maps” which are rectangular color coded arrays indicating the intensity (or amount) of the dependent variable. Heat maps are flexible in that both the x-axis and y-axis can be arranged to explore a particular hypothesis and allow a fast overview of data with a third quantitative dimension captured as different colors. We are now adapting these tools for interpreting cumulative and aggregate environmental exposure measurements as well as the results from human biomonitoring of biological media including blood, breath and urine. This article uses existing EPA measurements of environmental and biomarker concentrations of polycyclic aromatic hydrocarbons (PAHs) to demonstrate the value of the heat map approach for hypothesis development and to link back to stochastic and mixed effects models that were originally used to assess study results.     

Illicit drugs as new environmental pollutants: cyto-genotoxic effects of cocaine on the biological model Dreissena polymorpha

The increase in global consumption of illicit drugs has produced not only social and medical problems but also a potential new environmental danger. Indeed, it has been established that drugs consumed by humans end up in surface waters, after being carried through the sewage system. Although many studies to measure concentrations of several drugs of abuse in freshwater worldwide have been conducted, no data have been available to evaluate their potentially harmful effects on non-target organisms until now. The present study represents the first attempt to investigate the cyto-genotoxic effects of cocaine, one of the primary drugs consumed in Western Countries, in the biological model Dreissena polymorpha by the use of a biomarker battery. We performed the following tests on Zebra mussel hemocytes: the single cell gel electrophoresis (SCGE) assay, the apoptosis frequency evaluation and the micronucleus assay (MN test) for the evaluation of genotoxicity and the lysosomal membranes stability test (neutral red retention assay; NRRA) to identify the cocaine cytotoxicity. We exposed the molluscs for 96 h to three different nominal concentrations in water (40 ng L⁻¹; 220 ng L⁻¹; and 10 μg L⁻¹).Cocaine caused significant (p < 0.05) primary DNA damage in this short-term experiment, but it also caused a clear increase in micronucleated cells and a marked rise in apoptosis, which was evident in samples from even the lowest environmental cocaine concentration. Because cocaine decreased the stability of lysosomal membranes, we also highlighted its cytotoxicity and the possible implications of oxidative stress for the observed genotoxic effects.