Grantee Research Project Results
2007 Progress 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 Period Covered by this Report: January 1, 2007 through December 31,2007
Project Amount: $449,735
RFA: Ecology and Oceanography of Harmful Algal Blooms (2004) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Water
Objective:
We hypothesize that physiologically based toxicokinetic models mathematically analogous to the type developed in vertebrates can be adapted for marine invertebrates based on the known physiology of decapod crustaceans and bivalve mollusks. These models will be used to predict the uptake and disposition of the marine algal toxin domoic acid in Dungeness crabs, Pacific razor clams, and blue mussels. Validation of individual kinetic model predictions and trophic transfer of domoic acid will be achieved through a combination of focused laboratory experiments and the use of large-scale estuarine mesocosms containing razor clams and Dungeness crabs. Laboratory studies will determine the toxicokinetics of domoic acid in shellfish after intravascular injection and repetitive hemolymph removal. This technique will be used in conjunction with controlled laboratory feeding studies to develop a detailed data set on the uptake, tissue distribution and elimination of domoic acid in shellfish. In mesocosm studies, razor clams verified to contain domoic acid will be collected from contaminated Washington State coastal sites. The clams will be added to the mesocosm along with adult Dungeness crabs (previously unexposed to domoic acid), which will feed on the clams. Individual clams and crabs will be repetitively monitored for hemolymph concentrations during the study.
Work Status: Efforts during the past 12 months have been focused on probing the physiological mechanism(s) associated with domoic acid sequestration in the crab hepatopancreas and whether a common mechanism exists that can also account for the large interspecies variation in retention among bivalves. These studies follow-on from previous efforts which documented the unusual toxicokinetic behavior of domoic acid in crabs (summarized below). These studies are considered important in the final development of the crab physiologically-based toxicokinetic model for domoic acid. It is clear that a better understanding of the partitioning of domoic acid between the hepatopancreas and hemolymph is needed to allow accurate prediction of domoic acid toxicokinetics.
Approach:
We have successfully applied recent improvements in analytical detection of domoic acid to study the excretion of the toxin in shellfish after intravascular injection and repetitive hemolymph removal. This technique will be used in conjunction with controlled laboratory feeding studies to develop a detailed data set on the uptake, tissue distribution and elimination of domoic acid in shellfish. Physiologically based kinetic models will be developed and specifically parameterized for crabs and bivalves using a combination of recently published and experimentally determined values for the cardiovascular and gastrointestinal / digestive systems of shellfish. Validation of model predictions will initially be performed from indoor laboratory studies and then from the results of a large scale estuarine mesocosm containing razor clams and crabs. In the mesocosm study, 500 clams verified to contain domoic acid will be collected from contaminated Washington State coastal sites. The clams will be added to the mesocosm along with adult Dungeness crabs (previously unexposed to domoic acid), which will feed on the clams. Individual clams and crabs will be repetitively monitored for hemolymph concentrations during the study. Crabs will also be intravascularly injected with 15N-labeled domoic acid to allow simultaneous determination of elimination and uptake of domoic acid.
Progress Summary:
Recent findings from this project (described in more detail below) indicated that in both mussels and razor clams, domoic acid 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. These kinetic results suggested interspecies differences in domoic acid retention are more attributable to differences in the physiological mechanism of excretion as opposed to the specific binding of domoic acid (or lack thereof). Results from the crab studies are interesting in that they reveal a strikingly different pattern of disposition depending on whether the domoic acid dose is administered orally or by intravascular injection. When crabs are gavage dosed with domoic acid (mixed into clam meat), essentially the entire dose is absorbed from the stomach within 2 hours and deposited within the hepatopancreas. The domoic acid concentration in other tissues such as hemolymph, muscle, kidney, gonads and gills are more than 100x lower than in the hepatopancreas (see year 2 annual report). Thus, the hepatopancreas has an enormous capacity to retain domoic acid with the tissue: hemolymph concentration ratio being more than 1000 to 1. In contrast, when domoic acid is administered by direct intravascular injection, the majority of the dose is retained within the hemolymph compartment, and little extravascular distribution is observed (discussed in more detail below). These combined results suggest a diffusional barrier exists that prevents domoic acid exchange between the hepatopancreas and the hemolymph, which ultimately prevents distribution of domoic acid to edible tissues (e.g., muscle) after oral exposures. Based on these results, we developed a revised hypothesis to explain the unusual kinetic behavior of domoic acid in crabs and the interspecies differences among bivalves:
Domoic acid is a substrate for plasma membrane bound influx-efflux pumps (transporters), which can selectively pump domoic acid across large concentration gradients either into (influx) or out of (efflux) a cell. We hypothesized that upon oral dosing, domoic acid is selectively transported into hepatopancreas cells by a transporter located on the apical portion of the cell membrane that extends toward the sinus spaces forming the venous return of hemolymph, and retained due to the actions of transporters. When domoic acid is injected directly into the hemolymph, uptake into the hepatopancreas is decreased due to either a different class of transporter or one which works only on the basolateral membrane in an opposite manner on regions of the plasma membrane facing the arterial hemolymph. In bivalves, influx-efflux pumps may either facilitate elimination (efflux activity; mussels) or cause increased retention (influx activity; clams).
To initially test this hypothesis, we first characterized the toxicokinetics of domoic acid after intra-vascular injection in crabs, mussels and clams. For these studies, domoic acid was dissolved in a crab or molluscan saline. Crabs were injected by inserting a dose-filled syringe tipped with a 25G needle through the arthroidal membrane and into the pereiopod artery of the 4th 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. Subsequent to these studies, kinetic studies were performed in crabs and bivalves that had been co-treated with known inhibitors of efflux pumps, verapamil and cyclosporine A (crabs only). Verapamil is a well-established competitive inhibitor of p-glycoprotein (pgp) type pumps. Cyclosporin A inhibits both pgp and OAT-1 (organic anion transporters) pumps. If these classes / types of transporters are involved in regulating domoic acid disposition in marine invertebrates, inhibition of transporter activity should alter the toxicokinetics of domoic acid after intravascular (IV) and oral administration. For most experiments, domoic acid was analyzed using HPLC with UV or fluorimetric detection. In select studies, ELISA methods were also used to analyze domoic acid.
A summary of current results is presented in Figures 1 and 2 and Table 1. In crabs injected with domoic acid, the hemolymph concentration changes little during the initial 24-36 hrs after injection and then declines in a log-linear manner. This kinetic behavior was observed at both the 1 mg/Kg and 0.1 mg/kg domoic acid doses (Figure 1 top). The volume of distribution (Vss parameter in Table 1) of domoic acid in crabs is quite small and approximately 150 - 282 mL/kg, depending on dose (Table 1). This value is similar to or less than, the total volume of hemolymph in crabs, suggesting very little of the injected domoic acid distributes outside the hemolymph compartment. These results are surprising given the established ability of the hepatopancreas to retain domoic acid after oral dosing (see year two annual report). We expected a much larger apparent volume of distribution for the injected domoic acid due to uptake into the hepatopancreas. Instead, most of the injected DA is retained in the hemolymph compartment providing the experimental basis for suggesting a diffusional barrier exists that prevents entry of domoic into the hepatopancreas after intra-vascular dosing. In contrast to the findings from the crab studies, the volume of distribution of domoic acid in bivalves is quite large, varying between 4746 – 5425 mL/kg in mussels and clams (Table 1, based on data shown in Figure 1 bottom and Figure 2 top). This would indicate extensive extravascular distribution of domoic acid occurs in both species. Consistent with past studies of domoic acid retention in bivalves, pronounced differential clearance was also observed between mussels and clams. In mussels, DA declines in a log-linear manner with a elimination half-life of 55 hrs (Table 1). In clams, DA levels initially declined and then appeared to stop or even increase slightly, which prevents estimation of clearance and elimination half-life (Figure 1 bottom). These results and corresponding kinetic analysis provide the basis for proposing that differential retention of domoic acid in mussels and clams is attributable to differential elimination rather than binding to a high affinity binding protein.
The results from the toxicokinetic studies of domoic acid in crabs and mussels co-treated with verapamil or cyclosporine are included in Table 1 and Figure 2. Based on preliminary analysis of this data, it appears that verapamil treatment did in fact alter the toxicokinetics of domoic acid in both species. The total clearance was increased in animals treated with verapamil, which caused a corresponding decrease in the plasma elimination half-life for domoic acid. In crabs, the area-under the curve during the initial 48 hrs after injection was decreased by more than 30% compared to controls (see AUC0→48 hr values in Table 1). Verapamil treatment also appeared to have a similar effect on mussels with respect to domoic acid elimination (Figure 2 and Table 1). Combined, these initial results are exciting as they are consistent with our proposed hypothesis to explain the disposition of domoic acid in crabs and bivalves.
Difficulties Encountered / Remedial Actions: The primary difficulty encountered this past year was associated with the use of ELISA assays for the low level detection of domoic acid in crab hemolymph. There appears to be an interfering substance(s) in crab hemolymph not present in bivalve hemolymph or even vertebrate blood plasma. To eliminate this interference, it was necessary to dilute crab hemolymph an extra 10-fold. Attempts to eliminate the interference using solid-phase extraction or selected denaturing steps (heat, strong acid) were unsuccessful.
Planned activity for Project Year 4: Activities planned for the remainder of this project are centered on completing development of the physiologically based toxicokinetic model for domoic acid in crabs and pursuing additional studies designed to test the hypothesis that membrane transporter proteins regulate domoic acid uptake into the crab hepatopancreas and are involved in the interspecies differences in retention among bivalves.
Changes of key personnel: None.
Expenditures: As of March 31, 2008, total expenditures for the project were $418,433, which corresponds to 93% of the total project funds.
Table 1. Summary of toxicokinetic parameters in Dungeness crabs, Mediterranean mussels, and razor clams after injection of domoic acid with or without co-administration of verapamil or cyclosporine A.
Treatment |
AUC0→48 hr |
AUC0→∞ |
Vss |
Clb |
t½, β |
(mg/ml h) |
(mg/ml h) |
(ml/kg) |
(ml/h/kg) |
(h) |
|
Control (0.1 mg/kg) |
38.26 |
214.2 |
153 |
0.9 |
114 |
Control (1 mg/kg) |
245.1 |
639.5 |
282 |
1.50 |
221 |
w/ Cyclosporin A |
216 |
570.2 |
187 |
1.67 |
76 |
w/ Verapamil |
168.1 |
449.4 |
226 |
2.22 |
72.3 |
Mussels |
6.8 |
14.1 |
4746 |
71 |
55 |
w/ Verapamil |
3.3 |
3.7 |
5425 |
268 |
18 |
Razor Clams |
n.d. |
n.d. |
5785 |
? <0.1 |
? >500 |
Note. The AUC0→∞, Vss, Clb and t½, β were calculated from the individual profiles (n=5, mean values shown on graph) using clearance-volume compartmental methods. n.d. = not determined.
Figure 1: Elimination of domoic acid from hemolymph after intra-vascular injection in D. crabs (top), razor clams (bottom). Each data point is the mean ± SD (n=3-6). Dose is indicated on graphs. Solid line is the predicted values using a two compartment clearance-volume model. Parameter estimates from this fit are listed in Table 1.
Figure 2. Effect of verapamil (20 μM or 20 μm/kg) or cyclosporin A (10 μm/kg) exposure on domoic acid elimination after intra-vascular injection (1 mg/Kg) in mussels (M. galloprovinciallis) or Dungeness crabs. Top: Mussel results; Solid line is the predicted values using a two compartment clearance-volume model. Bottom: Crab results. Observed data are plotted for each treatment group for comparison. Toxicokinetic parameters for this data is shown on Table 1. Note that mussels were exposed to verapamil by immersion in a 1 L solution beginning 12 hrs prior to domoic acid injection. Crabs were given injections of verapamil 2 hrs before domoic acid administration. Each data point is the mean ± SD (n=3-6).
Expected Results:
The validated models of domoic acid kinetics in shellfish will provide researchers and risk assessors a useful tool for exploration of mechanisms controlling selective retention of domoic acid by certain shellfish and allow more accurate predictions of depuration times to below permissible limits. When used in conjunction with forecasting models of Pseudo-Nitzschia blooms, the predicted levels of domoic acid in shellfish can be better estimated along with the potential economic consequences of recreational and commercial shellfish closures.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 6 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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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) |
Exit Exit Exit |
Supplemental Keywords:
exposure assessment, toxicokinetics, modeling, trophic transfer,, 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.