Is There a Link Between Epizootic Lobster Shell Disease and Contaminants?Is There a Link Between Epizootic Lobster Shell Disease and Contaminants?Lawrence A. LeBlancLawrence A. LeBlanc11, L. Brian Perkins, and Deanna Prince, L. Brian Perkins, and Deanna Prince11

11School of Marine Sciences, School of Marine Sciences, 22Department of Food Science and Human Nutrition, Department of Food Science and Human Nutrition, 33Lobster Institute, University of Maine, Orono, Maine, USALobster Institute, University of Maine, Orono, Maine, USA

Determine whether there is Determine whether there is co-occurrence co-occurrence between between contaminant concentrationscontaminant concentrations in in hepatopancreas/hemolymph and hepatopancreas/hemolymph and lobster shell lobster shell diseasedisease

Evaluate the use of Evaluate the use of lobster hemolymph as a rapid lobster hemolymph as a rapid screening toolscreening tool for contaminant exposure for contaminant exposure

Develop a Develop a multiresidue methodmultiresidue method for a wide suite of for a wide suite of organic contaminants suitable for lobster organic contaminants suitable for lobster hepatopancreas and hemolymph “tissues”hepatopancreas and hemolymph “tissues”

HYPOTHESESHYPOTHESES

•There is a relationship between epizootic shell There is a relationship between epizootic shell disease and contaminant body burdensdisease and contaminant body burdens

•Hemolymph can be used as a rapid screening tool Hemolymph can be used as a rapid screening tool for contaminant body burdensfor 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

CANADA

Gulf of Maine

Atlantic Ocean

Canada

Area of Interest

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 analysesICP-AES: inductively coupled plasma atomic emissionICP-MS: 10-100 x greater sensitivity•Mercury•Direct mercury analyzer

OBJECTIVES

CHEMICAL ANALYSESCHEMICAL ANALYSES

Why Hepatopancreas?Why Hepatopancreas?Concentrations of many trace elements are higher Concentrations of many trace elements are higher

than in muscle tissue than in muscle tissue

Elevated concentrations of lipophilic organic Elevated concentrations of lipophilic organic contaminantscontaminants

Why Hemolymph?Why Hemolymph?Easier matrix to work withEasier matrix to work with

Less labor-intensive sample preparationLess labor-intensive sample preparation

Multiresidue analysis

Hexane: Acetonitrile

AluminaColumn

Silica gelColumn

PCBs/OCs OCsAlkylphenolics

C-18 Column

PSA (primarySecondary amine)Column

Bisphenol ASteroidal Estrogens

Organic Contaminant Analysis

Table 1. Hepatopancreas concentrations of different classes of Table 1. Hepatopancreas concentrations of different classes of organic contaminants from lobster 100 samples organic contaminants from lobster 100 samples resulting from the multiresidue methodresulting from the multiresidue method

  Shell Disease Shell Disease

(n = 3)(n = 3)Non- DiseaseNon- Disease

(n = 3)(n = 3)ReferenceReference

(n = 3)(n = 3)

ng/gng/g ng/gng/g ng/gng/g

PCB 19PCB 19 1071 1071 ++ 192 192 1040 1040 ++ 570 570 86.3 86.3 ++ 22.5 22.5

p,p'-DDEp,p'-DDE 91.7 91.7 ++ 17.7 17.7 99.4 99.4 ++ 33.6 33.6 41.5 41.5 ++ 18.0 18.0

n-octylphenoln-octylphenol 171 171 ++ 22.7 22.7 164 164 ++ 48 48 56.7 56.7 ++ 21.1 21.1

n-nonylphenoln-nonylphenol 429 429 ++ 26.6 26.6 247 247 ++ 65.1 65.1 30.0 30.0 ++ 18.0 18.0

PBDEsPBDEs NDND NDND NDND

BPA (mg/g)BPA (mg/g) 9.38, 7.519.38, 7.51 7.77, 7.767.77, 7.76 0.69, 4.590.69, 4.59

PCBs (Boston Hbr)PCBs (Boston Hbr) 1450-35001450-3500 MWRAMWRA

Nonylphenol (Adriatic Sea)Nonylphenol (Adriatic Sea) 270-400270-400 Ferarra (2005)Ferarra (2005)