Grantee Research Project Results
Final Report: Domoic Acid Kinetics and Trophic Transfer in Shellfish: An Integrated Laboratory and Estuarine Mesocosm Study
EPA Grant Number: R831703Title: Domoic Acid Kinetics and Trophic Transfer in Shellfish: An Integrated Laboratory and Estuarine Mesocosm Study
Investigators: Schultz, Irvin R. , Skillman, Ann D. , Woodruff, Dana
Institution: Pacific Northwest National Laboratory
EPA Project Officer: Packard, Benjamin H
Project Period: January 1, 2005 through December 31, 2007 (Extended to December 31, 2008)
Project Amount: $449,735
RFA: Ecology and Oceanography of Harmful Algal Blooms (2004) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Water
Objective:
The marine algal toxin domoic acid (DA), produced primarily by several species of pennate marine diatoms of the Pseudo-nitzschia (PN) genus is the causative agent of amnesic shellfish poisoning. The detection of DA in Western U.S. shellfish fisheries has resulted in significant adverse economic and cultural impacts on coastal communities. High levels of DA have been periodically found in razor clams Siliqua patula and Dungeness crabs Cancer magister, which feed on razor clams along the Pacific Coast of Washington. As a result, both recreational and commercial shellfish harvesting has been closed for time periods lasting more than a year and a half. The overall objective of this project is to improve the understanding of the physiological processes that control the bioaccumulation of DA in razor clams and crabs and compare findings to shellfish such as mussels (Mytilus galloprovincialis), which rapidly excrete the toxin. As part of this objective, we are developing and applying more advanced toxicokinetic analysis methods that use clearance-volume compartmental models and physiologically based toxicokinetic models (PBTK).
Summary/Accomplishments (Outputs/Outcomes):
Overview of Experimental Approach: The toxicokinetics of DA was studied using a variety of dosing procedures that were: direct injection into the hemolymph compartment, termed intravascular (IV) dosing, water exposure (static exposure to DA) and oral dosing to crabs, where the DA has been incorporated into a razor clam homogenate (to simulate a natural food source). After dosing, repetitive hemolymph samples are removed from individual clams, mussels and crabs to characterize the DA concentration-time profile. In separate studies, an additional, larger group of crabs were orally dosed then serially euthanized at various times for collection of hepatopancreas, kidney, intestine, brain, muscle and other tissues for characterization of tissue concentration-time profiles of DA. The purpose of these initial studies was to develop a detailed data set on the uptake, tissue distribution and elimination of DA in shellfish, which could be analyzed using toxicokinetic models. Subsequently, follow-on studies were performed (described below) to better establish the initial findings from the kinetic analysis.
Animals – Adult male crabs and mussels were collected from Sequim Bay, WA, USA. Razor clams were collected from various sites along the WA coastline. After collection, all shellfish were kept in separate 100-300 L fiberglass tanks filled with sand to a depth of 8” obtained from Sequim Bay and maintained as single pass flow thru with unfiltered Sequim Bay seawater. After acclimation for at least 2 weeks, individuals were transferred into indoor holding tanks under otherwise similar conditions.
Domoic acid dosing – Shellfish were injected IV with DA (0.1 and 1.0 mg/Kg; dissolved in an invertebrate saline, pH 7.4). Crabs were injected by inserting a dose-filled syringe tipped with a 25 g needle through the arthroidal membrane and into the pereiopod artery of the fourth leg at the third joint position. Subsequent hemolymph samples were removed from different legs by inserting the sampling syringe into the pereiopod arteries and removing approximately 0.1-0.2 mL of hemolymph. Bivalves were similarly injected at a point posterior from the gills. Three additional crabs were also IV dosed by intra-cardiac injection. Here, a 1/8” hole was made in the carapace above the heart and sealed with dental dam glued to the shell. This allowed insertion of the syringe needle through the shell and directly into the heart for injection of DA or hemolymph sampling. This was done to investigate any differences in DA hemolymph levels between peripheral samples (from the pereiopod arteries) or central vascular samples (intra-cardiac). Oral dosing of crabs was done by mixing DA with homogenized razor clam meat and then administering the dose to the crab (while under restraint) by oral gavage. The latter was achieved using a 3 mL syringe fitted with a curved, blunt tipped gavage needle that was directly inserted into the crab’s stomach.
Chemical analysis – DA was measured by three different methods, depending on required analytical sensitivity: HPLC using UV detection at 242 nm (for most routine measurements where DA levels were > 50 ng/g or mL; HPLC using fluorescent detection after derivitization with NBD-F (for water and hemolymph samples) or by immunoassay using ELISA kits purchased from Biosense (Bergen, Norway).
Toxicokinetic analysis – The hemolymph concentration-time profiles were fit to a clearance-volume compartmental model using WinNONLIN, an iterative, nonlinear least squares computer program. The toxicokinetic parameters of primary interest calculated by the WinNONLIN program are the apparent volumes of distribution (V1, V2), total body clearance (Clb) and the elimination half-life (t½). The volume of distribution is a parameter that describes the distribution of a chemical within the animal relative to a reference region or tissue. In our studies, the reference tissue was hemolymph. Total body clearance is a parameter that represents the sum of all excretion pathways for DA from the animal.
Conclusions:
Razor clams and mussels –The results indicate that in both mussels and razor clams, DA has a similar pattern of extravascular distribution that is approximately 10 times the vascular fluid volume. The primary difference in the toxicokinetics is total body clearance, with mussels having a much higher clearance, as expected. Combined, these kinetic results suggest interspecies differences in DA retention among bivalves and are more attributable to differences in the physiological mechanism of excretion as opposed to specific binding of DA (or lack thereof) within the bivalve.
Dungeness Crabs – These results are particularly interesting in that they reveal a strikingly different pattern of disposition depending on whether the DA dose is administered orally or by IV injection. When crabs are orally dosed with DA, essentially the entire dose is absorbed from the stomach within 2 hours and deposited within the hepatopancreas. The DA concentration in other tissues such as muscle, kidney, gonads and gills are more than 100x lower than in the hepatopancreas. Thus, the hepatopancreas has an enormous capacity to absorb and retain DA. The hepatopancreas:hemolymph partition coefficient was estimated to be approximately 1000:1. This extraordinarily high partitioning indicates the capacity of the hepatopancreas to sequester DA. In contrast, when DA is administered by IV injection, the majority of the dose appears to remain within the hemolymph compartment and is slowly eliminated with a t½ of 220 hrs. Toxicokinetic analysis of the IV data after both the 0.1 and 1 mg/kg doses, provided a similar set of results, with the steady-state volume of distribution (V1 + V2) being approximately 280 mL/kg. This value is very similar to the hemolymph volume in crabs and is consistent with little of the injected DA distributing outside the vascular fluid compartment. Additional studies using intra-cardiac dosing also provided similar results, thus indicating the unusual findings from IV dosing are not an artifact of sampling from the peripheral periopod arteries. Collectively, these results suggest a diffusional barrier exists that prevents DA exchange between the hepatopancreas and the hemolymph. If no such barrier exists, then it would be expected that DA would rapidly distribute into the hepatopancreas after IV injection due to the high tissue: hemolymph partition coefficient was observed after oral dosing. This diffusional barrier is what apparently prevents distribution of DA to edible tissues (e.g., muscle) after oral exposures.
We also explored whether this diffusional barrier is unique for DA or exists for other organic acids with similar physicochemical properties. Intravascular dosing experiments were performed with the structurally related compound kainic acid and also with dichloroacetic acid (DCAA). These organic acids were injected IV into crabs in a manner identical to that of DA. The toxicokinetic analysis indicated both kainic acid and DCAA, were more rapidly excreted (t½ < 100 hrs), and have larger steady-state volumes of distribution (780 & 456 mL/kg), indicating extravascular distribution was occurring. These findings would suggest the unusual kinetic behavior of DA in crabs is specific to DA and not a fundamental kinetic property of crabs with respect to organic acids.
Follow-on studies – Integrating the findings from the bivalve and crabs studies suggest a specific physiological process is occurring that sequesters DA in specific tissues (hepatopancreas in crabs) or limits excretion from the animal. Our working hypothesis to explain this phenomenon is that some type of cell membrane bound, transporter protein such as an invertebrate homolog to the organic anion transporters (OATs) are regulating the transport of DA in shellfish. We would hypothesize that upon oral dosing, DA is selectively transported into hepatopancreas cells by a transporter located on the apical portion of the cell that extends toward the sinus spaces forming the venous return of hemolymph. When DA is injected directly into the hemolymph, uptake into the hepatopancreas is decreased or blocked due to transporter activity in a different cell type or one which operates only on the basolateral portion of the cell. To obtain experimental evidence in support of this hypothesis, we performed two types of experiments: in vitro studies using isolated hepatopancreas tissue that measured DA uptake and protein binding and in vivo studies with shellfish that were co-exposed to DA and known inhibitors of transporters (verapamil and cyclosporine A). Verapamil is a competitive inhibitor of p-glycoprotein (pgp) type transporters, which are believed to be similar to the multi-xenobiotic transporter described in bivalves. Cyclosporin A inhibits both pgp and OAT transporters. These in vivo experiments were designed to determine whether co-exposure altered the toxicokinetic behavior of DA in crabs, clams and mussels. The results from these studies indicated that verapamil treatment (but not cyclosporine A) increased the steady-state volume of distribution by 28% in crabs, which had the effect of lowering the hemolymph concentration of DA after IV injection (Figure 1). In mussels, verapamil treatment caused more complex changes in DA toxicokinetics, with a reduction in extravascular distribution but also increased clearance. In razor clams, verapamil treatment did not appear to alter DA elimination. In vitro studies with isolated hepatopancreas tissue provided additional evidence that DA is actively sequestered by the hepatopancreas. When 1 g of tissue was incubated 12 hrs with 45 µg of DA, 68% (30.6 µg) was absorbed by the tissue. Subsequent homogenization of the tissue followed by differential centrifugation to isolate the cytosol fraction indicated 79% of the absorbed DA was in the cytosol. Ultrafiltration of the cytosol through a 10,000 MW cut-off filter indicated that 85% of the DA passed through the filter. This latter result indicates DA is not appreciably bound to cytosolic proteins. These results are consistent with DA being actively transported into the hepatopancreas but not bound to any specific intracellular carrier protein(s).
Future direction:
Ongoing studies during FY09 are working with isolated hepatopancreas cells to characterize the rate of DA absorption by the cells and more specifically establish sensitivity towards inhibitors of transporter proteins. A more complete characterization of DA uptake by hepatopancreas cells will aid development of a physiologically based toxicokinetic model for DA in Dungeness crabs.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 6 publications | 3 publications in selected types | All 3 journal articles |
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Lefebvre KA, Noren DP, Schultz IR, Bogard SM, Wilson J, Eberhart BT. Uptake, tissue distribution and excretion of domoic acid after oral exposure in coho salmon (Oncorhynchus kisutch). Aquatic Toxicology 2007;81(3):266-274. |
R831703 (Final) |
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Schultz IR, Skillman A, Woodruff D. Domoic acid excretion in dungeness crabs, razor clams and mussels. Marine Environmental Research 2008;66(1):21-23. |
R831703 (2007) R831703 (Final) |
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Schultz IR, Skillman A, Sloan-Evans S, Woodruff D. Domoic acid toxicokinetics in Dungeness crabs: new insights into mechanisms that regulate bioaccumulation. Aquatic Toxicology 2013;140-141:77-88. |
R831703 (Final) |
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Supplemental Keywords:
Transporters, toxicokinetics, HPLC, sequestration, kainic acid , RFA, Scientific Discipline, Water, ECOSYSTEMS, Ecosystem Protection/Environmental Exposure & Risk, Aquatic Ecosystems, algal blooms, Environmental Monitoring, Ecological Risk Assessment, Ecology and Ecosystems, estuaries, pharmacokinetic models, trophic transfer of phycotoxins, algal bloom detection, algal toxins, trophic interactions, benthic algae, domoic acid producing diatomsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.