Urinary PAH metabolites as biomarkers of exposure in aquatic environments.

While immunoassays have been extensively applied to evaluate environmental contamination, to date they have rarely been used for the analysis of biological fluids outside of human medicine. These media are important because pollutants such as polycyclic aromatic hydrocarbons (PAHs) and their metabolites become concentrated in tissues, body fluids, and excreta, thereby offering a measure of exposure to biologically available contaminants. Such analyses also provide a nondestructive tool for monitoring exposure. Crabs (Carcinus maenas) were exposed to phenanthrene and pyrene (separately) at concentrations ranging from 0 to 200 microg L(-1). After 48 h, urine samples were taken and analyzed by immunoassay and UV-fluorescence spectrophotometry. Urinary levels (calibrated against hydroxylated metabolites) proved to be dose dependent for both compounds, and good agreementwas demonstrated between the immunoassay and the fluorescence techniques. The cross reactivity of the immunochemical technique (ELISA) for pyrene and hydroxy-metabolites was lower than for phenanthrene. HPLC analyses demonstrated that urine from the crabs exposed to pyrene contained mainly conjugate PAH metabolites whose concentrations (the sum of the four main pyrene metabolites/conjugates) showed very good agreement with the ELISA (r2 > 0.94) and fluorescence (r2 > 0.91) data. Environmental samples were also analyzed by ELISA and UV-fluorescence, and both techniques detected PAH (mainly petrogenic) contamination in the urine samples from a polluted harbor. These data demonstrate the potential of urine analyses by ELISA and UV-fluorescence to measure exposure of crabs to PAH.

Rapid assessment of polycyclic aromatic hydrocarbon (PAH) exposure in decapod crustaceans by fluorimetric analysis of urine and haemolymph.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and potentially harmful contaminants of the coastal and marine environment. Studies of their bioavailability, disposition and metabolism in marine organisms are therefore important for environmental monitoring purposes. Detecting PAH compounds in the biological fluids of marine organisms provides a measure of their environmental exposure to PAHs. In the present study, the shore crab Carcinus maenas was exposed to waterborne pyrene for 48h. Urine and haemolymph samples were analysed by direct fluorimetry utilising both fixed wavelength (FF) and synchronous scanning fluorescence (SFS) techniques. Samples from exposed crabs exhibited fluorescence due to 1-OH pyrene equivalents, whilst samples from control crabs did not. Levels of equivalents were exposure dependent. Urine was shown to be a more suitable medium for the analysis of PAH equivalents. In a separate experiment, depuration of pyrene equivalents in urine was monitored over time. Urinary levels reached a maximum 2-4 days after initial exposure and decreased steadily thereafter. No unchanged parent pyrene was detected in samples from exposed crabs. While fluorimetric techniques could discriminate between 1-OH pyrene equivalents and parent pyrene, identification of specific metabolites was only possible with HPLC/F analysis. This revealed crabs had bio-transformed pyrene into 3 major conjugates of 1-OH pyrene, which were excreted in the urine. While such biotransformation of PAH is well documented in fish and several crustaceans, this is the first study to use direct fluorimetry to detect PAH equivalents in exposed crustacean urine. Fluorimetric results correlated well with those obtained by HPLC/F and ELISA techniques. The technique has great potential as a rapid, inexpensive and non-destructive technique for field biomonitoring of PAH exposure in crustaceans.