Environmental Contaminants in Shell Diseased vs Environmental Contaminants in Shell Diseased vs. Non-Diseased American Lobsters (Homarus americanus) Lawrence A. LeBlanc, Deanna L. Prince and Hans Laufer School of Marine Sciences, Department of Food Science and Human Nutrition University of Maine, Orono, ME Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT

Is contaminant exposure correlated to the presence of lobster shell disease? Metals & Organics have a variety of toxic effects: neurotoxicity, endocrine disruption, narcotic toxicity Little is known of the interaction effects of metal and organic contaminants A combination of stressors may lead to compromised immune systems

Why Shell Disease and Contaminants? Shell disease in crustaceans more prevalent in impacted environments Lobster- Massachusetts Bay, Buzzards Bay (Estrella, 1984) Evidence of elevated concentrations of trace metals with shell disease Blue Crabs (Weinstein, et al. 1992) Lobster (Vassiliev, 2004) Alkylphenols are postulated to have a role in shell disease (Laufer et al.)

Two Approaches LeBlanc and Prince: Laufer et al. Compare patterns of trace metal and organic contaminant accumulation in shell-diseased and non-diseased lobsters Laufer et al. Investigations of the effects of alkylphenol compounds on shell thinning

Our Hypothesis: There is a relationship between epizootic shell disease and contaminant body burdens

Project Objectives Determine whether there is a relationship between contaminant concentrations in hepatopancreas/hemolymph and lobster shell disease 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” Examine the partitioning of metals between various component tissues (including gills, ovaries, muscle, exoskeleton)

Project Objectives Why Hepatopancreas? Why Hemolymph? 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

Lobster Collection Lobster collections: Regional: RI, LIS, ME Lobster 100: RI Shell-diseased and healthy lobsters from the same sites Healthy lobsters from a reference site (ME) – lobster 100 study Approximately 185 samples analyzed Hepatopancreas, hemolymph, other tissues

What We Measured: Twenty five different trace elements, including: Contaminant metals Arsenic, cadmium, copper, chromium, nickel, lead, mercury Nutritive trace elements Calcium, potassium, magnesium Other elements Aluminum, iron, manganese

What We Measured: Polychlorinated biphenyls (PCBs) Selected congeners – 19 congeners Legacy organochlorine compounds DDTs, aldrin, dieldrin, endosulfan Polybrominated diphenyl ethers (PBDEs) Selected alkylphenols and bisphenol A See Poster

What We Have Learned: Contaminant Metals Regional Collections: RI, LIS, ME Concentrations are generally similar between diseased and non-diseased lobsters Outliers occurred in all groups Hepatopancreas and hemolymph

No Difference Between Diseased & Non-Diseased (RI Hepatopancreas) 12

What We Have Learned: Lobster 100 Two metals: Cr, Hg Diseased > Non- Diseased and Reference Two-ten fold higher in diseased

Lobster 100 Metal Concentrations Similar to Other Impacted Sites Cd (mg/g) Cr Cu Ni Pb SD (31) 2.8 - 100 (4.3) 0.08 – 35 (4.23) 200 – 4,300 (182) 0.32 – 31 (1.28) 0.04 – 3.3 (0.24) NSD (30) 2.6 – 23 0.08 – 1.2 (0.41) 300 – 3,700 (150) 0.27 – 5.1 (0.96) 0.04 – 0.72 (0.27) Ref (19) 0.40 – 75 (6.23) 0.05 – 1.3 (0.28) 15 – 800 (24) 0.79 – 3.6 (1.39) 0.02 – 0.11 (0.13) Other Studies Boston Harbor 1.2 – 6.6 0.1 – 2.5 37 - 640 0.51 – 0.85 LIS 5.4 – 50.8 0 – 0.75 180 – 3,880 0.90 – 5.97 1.2 – 15 Outfall (GOC) 11.5 + 7.5 0.9 + 0.5 380 + 500 4.4 + 0.7 350 + 77 Maine 1.8 - 59.8 0.07 - 1.3 12 - 500 0.07 - 2.4 0.02 - 0.46

What We Have Learned: Lobster 100 Lobster sex (M/F) did not appear to be a significant factor in contaminant metal concentrations Sediments from the lobster 100 site had metal concentrations approaching levels of concern

Sediment metals from the lobster 100 site approach and exceed levels of concern   As Cd Cr Cu Ni Pb (mg/g) (mg/) Mean (n = 5) 15.3 0.2 104 31 420 106 Std. dev. 1.3 0.1 2.2 2.8 25 82 ERL1 8.2 1.2 81 34 30 47 ERM2 70 9.6 370 270 50 218 1ERL = effects range low = the l10th percentile of the concentration range where biological effects were observed in standardized sediment exposures 2ERM = effects range median = the 50th percentile concentration range where biological effects were observed. 16

What We Have Learned: Metals Partitioning

Hepatopancreas: As, Cd, Cu, Fe Muscle: Gills: Cr Shell: Mn, Ni, Pb Ovary: Hemolymph: Similar partitioning patterns observed between lobsters with and without shell disease Hepatopancreas generally contained the greatest concentration of metal contaminants Exoskeleton had highest tissue burden of Cr, Fe, Mn, Ni, Pb Body burden of some contaminants reduced through molting

Hepatopancreas metal concentrations sometimes exceed FDA action levels Metals Action Level1 (ppm) Hepatopancreas Concentration (ppm) Muscle Concentration (ppm) Arsenic 76 13 (0.75 - 170) 10 (6.9 – 14) Cadmium 3 5.8 (0.08 -51) 0.91 (0 – 8.8) Chromium 12 0.32 (0.03 – 17) 0.01 (0.01 – 0.02) Lead 1.5 0.05 ( 0.01 – 0.11) 0.06 (0.01 – 0.57) Mercury 1.0 (methyl) 0.17 (0.04 – 0.49) (total) 0.07 (0.001 – 0.16) Nickel 70 1 0.82 (0.08 – 15) 1.0 (0.02 – 9.8) 1 FDA Action Levels for Crustacea

Organic Contaminants Developed multi-residue method Hepatopancreas Employed various separation and cleanup technologies Hepatopancreas Very lipid rich Difficult to adequately clean up extract Preliminary Data [See Poster]

Multi-residue Method Hexane: Acetonitrile Alumina Column Gel Permeation Chromatography Silica gel PCBs/OCs OCs Alkylphenolics C-18 Column PSA (primary Secondary amine) Bisphenol A Steroidal Estrogens Multi-residue Method See Poster

Take Home Messages No strong relationship between metal body burden & disease state Regionally, no differences b/w SD & NSD Lobster 100 (RI), 2 metals SD> NSD>Ref Metal concentrations in hepatopancreas are similar to those of other impacted sites Metals in hepatopancreas (tomalley) sometimes exceed FDA action limits