1. Is There a Link Between Epizootic Lobster Shell Disease and Contaminants? Lawrence A. LeBlanc 1, L. Brian Perkins, and Deanna Prince 1 1 School of Marine Sciences, 2 Department of Food Science and Human Nutrition, 3 Lobster Institute, University of Maine, Orono, Maine, USA Determine whether there is co-occurrence between contaminant concentrations in hepatopancreas/hemolymph and lobster shell disease Determine whether there is co-occurrence between contaminant concentrations in hepatopancreas/hemolymph and lobster shell disease Evaluate the use of lobster hemolymph as a rapid screening tool for contaminant exposure Evaluate the use of lobster hemolymph as a rapid screening tool for contaminant exposure Develop a multiresidue method for a wide suite of organic contaminants suitable for lobster hepatopancreas and hemolymph “tissues” Develop a multiresidue method for a wide suite of organic contaminants suitable for lobster hepatopancreas and hemolymph “tissues” HYPOTHESES There is a relationship between epizootic shell disease and contaminant body burdens There is a relationship between epizootic shell disease and contaminant body burdens Hemolymph can be used as a rapid screening tool for contaminant body burdens Hemolymph can be used as a rapid screening tool for contaminant body burdens THE PROBLEM Alkylphenolics Laufer et al. (2005) postulate that alkylphenolic compounds may be a causative agent in the onset of lobster shell disease: High concentrations of alkylphenols detected in sediments, lobster hemolymph and other tissues (Biggers and Laufer (2004). Alkylphenolic compounds have endocrine-disrupting properties, including interference with juvenile hormone in invertebrates. Contaminant Trace Metal Effects e.g., As, Cd, Cu, Cr, Hg, Ni, Pb, Se, Zn Lethal to invertebrates at high concentrations Sublethal effects at lower doses Affect immune & endocrine functions (Correa et al., 2005) Provoke oxidative stress (Valko et al., 2005) Organic Contaminant Effects Several classes of organic compounds are known to interfere with hormonal regulation PCBs, OCs (legacy pesticides) PBDEs Alkylphenols Estrogens (synthetic and natural) Lethal to invertebrates at high concentrations Sublethal effects at lower doses Interference with reproduction, thyroid functions Lobsters with severely compromised exoskeletons are being found from New York to Nova Scotia with the greatest incidence in Southern New England The effects of combined stressors (e.g. metals + organics) are unknown Is contaminant exposure correlated to the presence of lobster shell disease? BACKGROUND APPROACH Lobster Collection & Tissue Sampling Lobsters collected throughout the northeast Sea Grant State Agencies Industry contacts Matched collections of shell-diseased and healthy lobsters Approximately 185 samples analyzed Solvent extraction of tissue Accelerated solvent extraction Acetonitrile extracts analytes while leaving behind lipids Partition against hexane Removal of lipids Alumina column Gel Permeation Chromatography Primary secondary amine (PSA) Separation of compound classes Solvent:Solvent partitioning: Acetonitrile:Hexane Silica Gel Chromatography Instrumental Analysis Gas chromatography/mass spectrometry GC/ECD, GC/MS Ion specific monitoring High performance liquid chromatography HPLC, LC-MS The Lobster Institute Trace Metal Analysis Acid digestion of tissue: Microwave Acceleration Reaction System (MARS) Instrumental Analysis: Metals:multi-elemental analyses ICP-AES: inductively coupled plasma atomic emission ICP-MS: 10-100 x greater sensitivity Mercury Direct mercury analyzer OBJECTIVES CHEMICAL ANALYSES Why Hepatopancreas? Concentrations of many trace elements are higher than in muscle tissue Elevated concentrations of lipophilic organic contaminants Why Hemolymph? Easier matrix to work with Less labor-intensive sample preparation Organic Contaminant Analysis Table 1. Hepatopancreas concentrations of different classes of organic contaminants from lobster 100 samples resulting from the multiresidue method Shell Disease (n = 3) Non- Disease (n = 3) Reference ng/gng/gng/g  PCB 19 1071 + 192 1040 + 570 86.3 + 22.5 p,p'-DDE 91.7 + 17.7 99.4 + 33.6 41.5 + 18.0 n-octylphenol 171 + 22.7 164 + 48 56.7 + 21.1 n-nonylphenol 429 + 26.6 247 + 65.1 30.0 + 18.0  PBDEs NDNDND BPA (mg/g) 9.38, 7.51 7.77, 7.76 0.69, 4.59  PCBs (Boston Hbr) 1450-3500MWRA Nonylphenol (Adriatic Sea) 270-400 Ferarra (2005)