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New Hampshire Department of Environmental Services

Regional Advancement of Emerging Methods to Detect PSP Toxin

Contract Title: Contract with Resource Access International for Shellfish Tissue Testing
Grantee: Resource Access International
Award Period: October 19, 2012-December 31, 2012

Original Proposal

This project was a joint Maine and New Hampshire effort to develop side-by-side comparison of paralytic shellfish poisoning (PSP) toxin analyses from the mouse bioassay (MBA) test with results from high pressure liquid chromatography (HPLC), with the goal of producing sufficient data to determine if HPLC can/should become an approved analytical method in the National Shellfish Sanitation Program (NSSP). Approval of the HPLC method would provide managers with another laboratory analytical tool that will likely be faster, more precise, and possibly more cost-effective than the MBA method. The Maine Department of Marine Resources (MEDMR) performed mouse bioassay (MBA) tests on samples from the 2009 PSP season and saved the unused shellfish homogenate for testing at the NHDES lab. The preserved homogenate was planned to be used by the New Hampshire Department of Health and Human Services (NHDHHS) lab in the project entitled "Regional Application of an Alternative HPLC Lab Method to Detect PSP Toxin." Project end date was June 30, 2011.

Project Summary

The New Hampshire Division of Public Health Services Public Health Laboratories (NH DPHS PHL) Water Analysis Lab, formerly the NHDES Environmental Services Laboratory, completed a study to develop the Post-Column Oxidation-High Performance Liquid Chromatography (POCX-HPLC)/Fluorescence method for the analysis of paralytic shellfish toxins and to compare that method to the standard mouse bioassay (MBA). Although the original intent of this project was to use the "Lawrence HPLC" method, Maine Department of Marine Resources (MEDMR) and NHDES decided to focus the project on the HPLC method using rapid post-column oxidation and fluorescence detection. This HPLC method is less cumbersome in the laboratory and was endorsed by the Interstate Shellfish Sanitation Conference in October 2009. This project was scheduled to end on June 30, 2011, but NMFS approved a number of changes to this project in March 2012. These changes were largely needed due to a major reorganization of the laboratories at NHDES and the NH Department of Health and Human Services (NHDHHS) as part of the June 2011 state budget.

The HPLC analyses for this project were performed using a Hatachi EZChrome Elite HPLC-Post Column Oxidation/Fluorescence Detection System. Due to two different incidents that caused sample freezers to fail and samples to thaw, only blue mussel homogenate from the 2009 NHDES and 2010 MEDMR red tide seasons was used in this HPLC analysis.

The NH DPHS PHL Lab found that sample preparation and analysis using the POCX-HPLC/Fluorescence method were straightforward and could be completed by a laboratory scientist experienced in common lab techniques and HPLC training. The HPLC results compared favorably with the MBA results for this study. The costs associated with the HPLC method appear comparable to the MBA method; however, equipment maintenance and replacement, especially due to the potential harshness of the shellfish extracts, could make the method cost-prohibitive. The lab recommended that more work be done to understand and evaluate the life of the analytical column under real-time production conditions. This could be cost-prohibitive for a low-volume lab, such as the NHDHHS lab, which only processes 60-65 samples per year. The lab was not able to assess the robustness of the HPLC method under a real-time production environment. Lab staff believe that having the necessary reagents, the fresh mobile phases in particular, ready at all times could pose a challenge, especially if samples come to the lab with little advance notice. The timeliness of generating laboratory results under real-time production conditions might also be a constraint, because preparing extracts for HPLC analysis requires an hour of lab time per sample as well as the time for analysis and data review. The Lab concluded that more laboratory work needs to be completed before New Hampshire decides to adopt the PCOX-HPLC method in its Shellfish Program.

In collaboration with Maine and the Interstate Shellfish Sanitation Conference (ISSC), NHDES added another paralytic shellfish poisoning (PSP) toxin detection method to this project. ISSC was interested in evaluating the utility of a PSP analytical procedure developed by Abraxis LLC of Pennsylvania. The method was being used in a pilot program as a screening tool to allow commercial shellfish harvesting in federally closed waters in the Gulf of Maine. The protocol instituted the use of Abraxis onboard and dockside testing methods in order to verify that surf clam harvesting locations in closed waters had PSP toxin levels below 80g/100g. NHDES understood that the Abraxis method could be a useful and potentially less expensive test for state PSP monitoring programs, but an evaluation of how the Abraxis test compared to the mouse bioassay tests was needed.

In this project, MEDMR, NHDES, and the ISSC analyzed archived shellfish tissue samples from Maine and New Hampshire waters for which mouse bioassay results had already been generated. The Abraxis enzyme-linked immunosorbent assay (ELISA) test method was scheduled to be considered by the ISSC for approval at the October 2011 conference. The comparative data generated by this project were presented at the conference, and the shipboard method using rapid extraction was accepted into the National Shellfish Sanitation Program (NSSP) for use with the Dockside Testing Protocol.

NHDES recognized that the Abraxis method could be a useful and potentially less expensive test than the MBA method for state PSP monitoring programs, but an evaluation of how the Abraxis test compared to the mouse bioassay tests for blue mussels in New Hampshire waters was needed. The NHDES Shellfish Program collected blue mussel samples at inshore and offshore locations from April 2011-September 2011. Those samples were analyzed using the standard mouse bioassay method by NHDHHS within 24 hours of sample collection. Any remaining homogenate from the samples was frozen and stored at the NHDHHS lab. NHDES staff and cooperating organizations analyzed the archived 2011 shellfish tissue samples using Abraxis ELISA kits.

When the Abraxis ELISA results were compared to the MBA results for the 2011 NH homogenate testing of PSP toxins, there was no consistent, linear relationship between the two methods. In one case, the MBA result was higher than the FDA quarantine limit of 80 g/100g, while the Abraxis result was less than the quarantine limit. In four cases, the Abraxis result was higher than the FDA quarantine limit, while the MBA result was lower than the quarantine limit. However, Abraxis scores on the lower end of that tests detection limit range (<20 µg/100g) compared well to MBA scores on the lower end of that test’s detection limit (<44 µg/100g). Overall, NHDES determined that more comparisons of these two methods was needed in order to provide managers with the necessary information to make an informed decision concerning the use of Abraxis ELISA shipboard testing kits as an alternative to the MBA method for PSP toxin testing.

Due to the mild intensity of red tide blooms over the previous few years, NHDES partnered with Resource Access International LLC to continue Abraxis testing of blue mussels during the 2012 red tide season. The NHDES Shellfish Program collected 47 blue mussel samples at inshore and offshore locations from April 2012-September 2012. Those samples were analyzed using the standard mouse bioassay method by NHDHHS within 24 hours of sample collection. Any remaining homogenate from the samples was frozen and subsequently delivered to Resource Access International. Resource Access International analyzed the samples using Abraxis ELISA kits and provided NHDES with recommendations for Abraxis testing as well as comparisons of results with the MBA method.

Resource Access International examined the potential effect of holding time for extracted samples on toxin scores. They conducted an informal study on several samples using extracts held for four days at -20 degrees and compared them to fresh extractions from the same homogenates, prepared on the day of the testing. Although the sample size was too small to make any statistical determinations, the data suggest that running "old" extracts through the Abraxis kit will result in slightly higher final toxicity scores. They recommended that the user extract and prepare only those samples which will be analyzed that day and leave any additional samples in homogenate form in the freezer until there is time to both extract and analyze the samples on the same day.

Resource Access International recommended that the user have additional Abraxis "Accessory Packs" on hand, beyond the exact number needed for testing purpose, in case any of the pre-filled vials are broken or lost some of the pre-measured diluent volume. The Abraxis ELISA kit is sensitive to operator error and variability, which can be observed at the end of the test in the calculated R2 and %CV values. They recommended that additional testing supplies be allocated above and beyond the exact number needed for samples, in order to accommodate a "practice run" for the operator to become more familiar with the testing method. Resource Access International also provided recommendations for maximum plate load. They state that because the final step in the assay must be completed within 15 minutes in order to obtain accurate results, the operator should only screen up to 10 discreet samples per run. Even though the Abraxis kit is technically capable of running as many as 26 discreet samples per plate, this would only yield 20 possible samples per plate. However, Resource Access International states that the results will be more accurate, which will make up for the additional costs associated with budgeting only 20 samples per kit.

Resource Access International compared the Abraxis ELISA results with the MBA results for blue mussel homogenate from the 2012 red tide season. Although they found no consistent, linear relationship between the two methods, they did observe an overall pattern in the sample results that could be used to reduce the number of MBA tests performed over the course of a red tide season. They state that a resource manager could potentially consider using the Abraxis kit to screen early season results and forgo the use of the MBA until Abraxis results approach 35 g/100g or higher. By following this recommendation, the manager could save on resource allocation for MBA testing, while still keeping a comfortable margin for protecting public health. Resource Access International did not recommend using the Abraxis kit during an active, ongoing PSP bloom in lieu of the MBA. They stated that additional collection of data is necessary before a resource manager commits to a complete transition to Abraxis testing as a replacement for the MBA method.

The US Food and Drug Administration (FDA) has also done considerable comparison in developing the Shipboard Screening/Dockside Testing Protocol for harvesting clams in previously closed federal waters. The FDA study of clams in federal waters and the NH study of blue mussels in NH state waters cannot be directly compared for a number of reasons. The FDA study was specific to surf clams and quahogs in a particular area of Georges Bank, while the NH study involved mussels from the New Hampshire coast. Toxicity profiles can be very specific for different species and regions. That is one of the reasons why the federal action to open some federal waters under the dockside testing program is so restrictive to a specific region, and a specific species. One should not expect results for New Hampshire mussels to always compare well with Georges Bank surfclams/quahogs. Furthermore, the FDA study involved hundreds of comparisons, while the NH study involved a total of 85 comparisons.

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