Bich-Thuy L. Eberhart

Diarrhetic Shellfish Toxins and Other Lipophilic Toxins of Human Health Concern in Washington State

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 μg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

Exposure of two species of deposit-feeding amphipods to sediment-associated [3H]benzo[a]pyrene: Uptake, metabolism and covalent binding to tissue macromolecules

Abstract Two species of deposit-feeding marine gammaridian amphipods, Rhepoxynius abronius and Eohaustorius washingtonianus, were exposed to sediment-associated [3H]benzo[a]pyrene (BaP) at 12±1°C. Concentrations of BaP-derived radioactivity increased with time in both E. washingtonianus and R. abronius, and the levels of radioactivity were similar in both species after 7 days of exposure. A significantly (P

Using cytochrome P450 to monitor the aquatic environment: Initial results from regional and national surveys

Abstract We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAAs Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for II species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.

Rapid enzyme-linked immunosorbent assay for detection of the algal toxin domoic acid

Abstract Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia, which is responsible for causing amnesic shellfish poisoning (ASP) in humans. ASP symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even coma or death. This paper describes the development and validation of a rapid, sensitive, enzyme linked immunosorbent assay test kit for detecting DA using a monoclonal antibody. The assay gives equivalent results to those obtained using standard high performance liquid chromatography, fluorenylmethoxycarbonyl high performance liquid chromatography, or liquid chromatography—mass spectrometry methods. It has a linear range from 0.1–3 ppb and was used successfully to measure DA in razor clams, mussels, scallops, and phytoplankton. The assay requires approximately 1.5 h to complete and has a standard 96-well format where each strip of eight wells is removable and can be stored at 4°C until needed. The first two wells of each strip serve as an internal control eliminating the need to run a standard curve. This allows as few as 3 or as many as 36 duplicate samples to be run at a time enabling real-time sample processing and limiting degradation of DA, which can occur during storage. There was minimal cross-reactivity in this assay with glutamine, glutamic acid, kainic acid, epi- or iso-DA. This accurate, rapid, cost-effective, assay offers environmental managers and public health officials an effective tool for monitoring DA concentrations in environment samples.

Screening tests for the rapid detection of diarrhetic shellfish toxins in Washington State.

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test “pre-harvest” shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer’s recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.

Enhancing Shellfish Safety in Alaska through Monitoring of Harmful Algae and Their Toxins

ABSTRACT Harmful algal blooms cause serious problems to public health and the economic viability of shellfish industries in Alaska. The most common phycotoxins in this region are saxitoxin and its congeners, collectively referred to as paralytic shellfish toxins, the causative agents of paralytic shellfish poisoning (PSP) in humans. The illness of 26 and death of 2 people in southeast Alaska as a result of PSP in 2010 through 2012 illustrates the need for change in the way paralytic shellfish toxins are monitored and managed in Alaska. The implementation of a scientific monitoring partnership, the Alaska Harmful Algal Bloom monitoring network, designed to provide an early warning of harmful algal blooms—in particular, those that cause PSP—is described. The program includes a tiered sampling approach, and weekly microscopic observation of seawater samples for the presence of the causative toxic dinoflagellate Alexandrium, followed by rapid toxin testing using a high-throughput antibody-based test as a complement to the standard regulatory testing performed by managers at the Alaska Department of Environmental Conservation. Partners in the Alaska Harmful Algal Bloom monitoring network began monitoring in 2008 and thus were able to provide an early warning of a widespread PSP event near Ketchikan in 2011, and the environmental conditions preceding a shellfish closure resulting from paralytic shellfish toxins near Mosman Island in southeast Alaska in 2012. A comprehensive and integrative communication network between Alaska Harmful Algal Bloom monitoring network personnel and state managers, health professionals, shellfish growers, and the general public is proposed to protect human health and promote safe shellfish harvest in Alaska.