Camp. Eiochem. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Physiol. Vol. IOOC, No. l/2, pp. 21-24, 1991 Printed in Great Britain 0306-4492/91 $3.00 + 0.00 0 1991 Pergamon Press plc DETERMINATION OF BENZO(a)PYRENE IN ISOPODS (PORCELLIO SCABER LATR.) EXPOSED TO CONTAMINATED FOOD T. C. VAN BRUMMELEN, R. A. VERWEIJ and N. M. VAN STRAALEN Department of Ecology and Ecotoxicology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands (Received 1 October 1990) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP Abstract-l. A method is presented for the determination of the polycyclic aromatic hydrocarbon benxo(a)pyrene (BaP) in the isopod Porcellio scaber, using reversed-phase HPLC with fluorescence detection. 2. This technique has a detection limit for BaP in P. scaber of approximately 3.2 ng g-l fresh weight. 3. BaP was assimilated from food by P. scaber. 4. After four weeks of ad libitwn feeding on BaP-contaminated food, concentrations in the isopods were approximately 30-40 times lower than those in the food. 5. Male and female isopods did not differ in BaP concentration. Variation among males seemed to be much higher than among females. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are emit- ted into the environment by a number of anthropo- genie activities, including oil-spills and incomplete combustion of fossil fuels and other organic com- pounds. Aerial transport and subsequent deposition of PAHs in exhausts, are causing areas distant from sites of emission to be increasingly contaminated. In the terrestrial environment PAHs accumulate in the upper horizons of the soil profile (Thomas et al., 1984). Concentrations of total PAHs in soils from rural areas have increased four- to five-fold over the last century, with some compounds, such as benzo(a)- pyrene (BaP), showing increases up to twentyfold (Jones et al., 1989). In The Netherlands, extremely high PAH concentrations have been found in soils from gas works and other sites of industrial activity. BaP and several other PAHs are well-known carcinogens. In particular, the high molecular weight PAHs are lipophilic and are not easily degraded, having half-life values in soils ranging between 100 and 400 days (Park et al., 1990). Little is known about the possible threat of these compounds to life in the soil. This may be due in part to the sophisti- cated techniques used in PAH analysis. BaP is expected to affect energy metabolism: aquatic crustaceans have been shown to metabolize a variety of PAHs (Herbes and Risi, 1978; James, 1989; Lee et al., 1976). Additionally, BaP and its metab- olites will affect the structure and activity of macro- molecules. We have recently reported on the effects of BaP on food assimilation and growth efficiency in the terrestrial crustacean Porcellio scaber (Van Straalen and Verweij, 1991). Growth, consumption, defaeca- tion and respiration were measured for woodlice on an ad libitum diet of ground poplar leaves. The observed effects were not dramatic and occurred at a concentration which can only be found at highly contaminated sites. No effects of BaP were observed on female isopods. At the highest BaP concentration, male isopods showed a significantly higher food assimilation efficiency, but a significantly lower growth efficiency. The higher use of energy was, however, not balanced by increased respiration, These research results raised two questions con- cerning BaP kinetics in the isopod: (l), do isopods assimilate BaP from food and does BaP enter the body? (2), is the differential effect for male and female isopods related to differences in the way they handle BaP? Male and female blue crabs have been shown to differ for BaP metabolism (Singer and Lee, 1977). To investigate this, a method for PAH analysis in woodlice has been developed and is described here. The BaP content of male and female isopods, which have been exposed to BaP in food, has been analyzed and is presented below. MATERIALSAND METHODS Chemicals Benz(a)anthracene (BaAnt, 99%) and benzo(a)pyrene (BaP, 98%) were obtained in powder form from Janssen Chemica. Standard reference material, consisting of 16 EPA priority pollutant PAHs dissolved in acetonitrile, was purchased from NBS (srm No. 1647a). Sodium sulphate anhydrous @.a.) and the organic solvents acetone @a.), n-hexane @a.), dichloromethane (DCM, p.a.) and aceto- nitrile were obtained from Baker. Boiling chips and silica-gel 60G were bought from Merck. To eliminate impurities, the hexane was distilled and the boiling chips were Soxhlet- extracted prior to use. Sodium sulphate and silica-gel were dried overnight at 140°C. The water for HPLC was double- deionized and filtered through a 0.45 pm Millipore filter. Experimental Male and female Porcellio scaber, ranging from 50 to 80 mg fresh weight, were kept on an ad Iibitum diet of ground poplar leaves containing 0, 1, 5, 25 or 125 pg BaP g-r dry weight. The experiment was performed in a climate room at 17°C with 12 hr of dimmed light per day. The 21