Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins | 811 - Shellfish | 1103 - Other microbiology (includes protozoology, phycology, etc.) | 50% |
| 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins | 811 - Shellfish | 1150 - Toxicology | 50% |
The goal of this Area of Expertise proposal is to enhance analysis capacity of the Salish Sea Research Center at Northwest Indian College to monitor for Paralytic Shellfish Toxins in the Salish Sea, Washington, USA. The Coast Salish People have been fishing in the Salish Sea since time immemorial, and depend on the shellfisheries for subsistence, ceremonial, and commercial purposes. Harmful algae and biotoxin events are common along the West coast of the US, and are a serious, persistent threat to shellfisheries. Through two previous NIFA awards Northwest Indian College and Lummi Nation have worked together to monitor for biotoxins in Bellingham and Lummi Bays, and have identified the most concerning Salish Sea marine toxin, Paralytic Shellfish Toxins, as needing additional monitoring. To accomplish our goal, we have identfied the University of California Santa Cruz as an 1862 Land Grant partner for this project, bringing extensive experience in biotoxin analysis and monitoring. The additional analysis capacity this proposal would provide (1) enhanced Paralytic Shellfish Toxin analysis at Northwest Indian College, (2) comparison of passive sampling monitoring tools for Paralytic Shellfish Toxins, and (3) field testing of passive samplers in Bellingham and Lummi Bay. This additional monitoring capacity may allow timely determination for biotoxin events in Bellingham and Lummi Bay, and has been identified as a priority for Lummi Nation because of the swift consequences to shellfish harvests that harmful algae events produce. This project would support at least nine undergraduate Native American students at Northwest Indian College through research internships.
The goal of this proposed project is to expand research capacity at the Salish Sea Research Center (SSRC) through a project identified as a high priority by our tribal partners. Previous NIFA funded projects have allowed the SSRC to enact high resolution biotoxin monitoring in the Salish Sea, and our tribal partners' Traditional Ecological Knowledge (TEK) has established the current and ongoing threat of Paralytic Shellfish Poisoning in these waters. Validation of Solid Phase Adsorption Toxin Tracking (SPATT) used to monitor Paralytic Shellfish Toxins (PSTs) in the Salish Sea will increase the research capacity of the SSRC, establish partnerships for our students with Lummi Natural Resources (LNR) and University of California Santa Cruz (UCSC), and provide valuable data for our tribal partners' shellfisheries management.
SPATT construction and extraction. Currently we use DIAION HP20 resin (Sigma-Aldrich) for our SPATT samplers in the Salish Sea, which has been shown to be an established resin for PSTs adsorption in the Eastern Pacific (Lane et al. 2010; Peacock et al. 2018). For extraction, SPATT bags are rinsed briefly with deionized water, and resin is extracted out of bag without vortexing. The first 10 mL of 50% MeOH extraction is used for ELISA and would be for LC/MS/MS analysis (Lane et al. 2010; Kudela 2011). We have documented PST adsorption onto HP20 SPATT in the Salish Sea (Fig. 2), though there is discrepancy in the literature as to HP20 efficacy for PST adsorption (Hattenrath-Lehmann et al. 2018). Therefore we will investigate different extraction methods (Lane et al. 2010; Boundy et al. 2015; Kudela 2017; Hattenrath-Lehmann et al. 2018), and compare SP700 and OASIS HLB alongside the HP20 resin for the following experiments: comparing for extraction, adsorption efficiency, and environmental deployment in the Salish Sea. Both SP700 and HLB indicate the adsorption of polar compounds and HLB has been widely used for both hydro- and lipophilic toxins (Sayfritz et al. 2008; Lane et al. 2010; MacKenzie 2010; Kudela 2017; Thomas et al. 2017; Roué et al. 2018).ELISA PST measurements. PSTs will be analyzed following MaxSignal ® Saxitoxin (PSP) ELISA Test Kit (BIOO Scientific, USA) and read on Molecular Devices SPECTRA MAX 340 PC plate reader (Peacock et al. 2018). Detection limit is 3 ng g-1 for mussel tissue as saxitoxin equivalents, with cross-reactivity from 4-100% for various derivatives. Detection limits are similar for SPATT but will be determined at analysis to identify MDL.Adsorption and extraction efficiency experiments. Laboratory trials will determine adsorption and extraction efficiency of PSTs (Lane et al. 2010; Kudela 2011). SPATT bags will be incubated in PST-fortified 0.2 µm filtered water from Lummi Shellfish Hatchery, stored in the dark at 4 °C. The initial target concentration will be 100 ng/L for each of the PST analogues available from the NRC, which currently includes 14 analogues. Salinity and pH will be measured prior to incubations, controlled for temperature, agitated constantly, and on a 12h: 12h light: dark schedule. Sample water will be regularly tested for toxin concentrations, and adsorption will be analyzed by LC/MS/MS, as well as dissolved organic carbon (DOC), which can impact adsorption in seawater (Kudela 2017). To determine SPATT desorption characteristics we use the extraction protocol described in the previous section to determine optimal recovery volumes (Kudela 2011).Resin saturation values. To convert SPATT from qualitative to quantitative measurements, resin saturation determination is necessary. For this, we will use a Freundlich isotherm equation to determine the equilibrium adsorption isotherms of PSTs and seawater (Limousin et al. 2007; Li et al. 2011; Zhao et al. 2013; Salonen et al. 2017; Kudela 2017). This would determine the kinetics constants for the Salish Sea water and the three resins we would test (HP20, SP700, and OASIS HLB). A "good" resin should produce a linear curve, and a resin that exhibits similar kinetics as a function of temperature, salinity, pH, etc. is desirable.Method adaptation for PSTs extracted from SPATT using an Agilent 6460 LC/MS/MS at NWIC. Boundy et al. (2015) has established a method to determine PSTs in marine tissue samples using a Waters Xevo TQ-S MS/MS coupled to a Waters Acquity UPLC i-class flow thru. This instrument is complementary to the Agilent 6460 LC/MS/MS instrument at NWIC. This method has reduced issues with using LC/MS for PSTs, including sample matrix problems, presence of salts, and co-extractives (Dell'Aversano et al. 2005; Sayfritz et al. 2008; Boundy et al. 2015). Briefly, our SPATT samples would be extracted after completion of laboratory experiments for best extraction protocols and use an additional optimized graphitized carbon solid phase extraction (SPE) clean-up (ENVI-Carb cartridge; Sigma-Aldrich; Boundy et al. 2015). SPE will be performed on a vacuum manifold and LC/MS/MS analysis will use a HILIC column (Acquity UPLC BEH Amide). Methods will be optimized for LC/MS/MS analysis on our instrument using NRC standards and MRM (Boundy et al. 2015).Comparison of SPATT resins and PST toxin analysis. At our sampling sites we will hang HP20, SP700, and HLB SPATT resins weekly, as well as mussels at Lummi Bay, to determine the best environmental SPATT for the Salish Sea. These samples will be analyzed for PSTs using both the above adapted method for the LC/MS/MS and our current ELISA method.
Target Audience
The target audience for this project are the tribal undergraduate students at Northwest Indian College who are excited to participate in natural resources management, oceanography, or aquaculture. We also hope to reach Pacific Northwest Tribal shellfishers, Lummi Natural Resource employees, and state and federal shellfish managers. This project was discussed with 10 undergraduate Indigenous research interns, and with our Lummi Natural Resources partners. Additionally, a graduate student at our land-grant partner school (UC Santa Cruz) worked with undergraduates at Northwest Indian College. We routinely have informal conversations with commercial, recreational, and subsistence Native and non-Native shellfish harvesters while we are collecting samples. We also have a K-12 module that we use with the local 3rd grade Lummi Nation School classrooms where we discuss red tide, harmful algae, and safe shellfish harvesting. A presentation on this topic was also given to a Bellingham School District 1st grade class at Geneva Elementary school. We also participated in outreach with a local preschool program by allowing the students to be introduced to intertidal animals.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided training for four student interns in summer of 2023, and four student interns in academic year internships in biotoxins analyses, laboratory preparation, participation in field studies, monitoring strategies, and engagement with Research 1 schools, graduate students, and Lummi Natural Resources scientists. One intern completed a post-baccalaureate internship and then was hired as a research technician in the Salish Sea Research Center. This technician is working directly on completing the dual SPATT deployment for PSTs. The PI has provided the work that is being done at multiple research conferences, and has presented the data to partners and collaborators.
Dissemination Streams
We have multiple meetings with Lummi Natural Resources, routine meetings between the PD/Co-D lab, as well as local and natural conferences. We also used the results from this project to develop native curriculum that we share with 3rd and 5th grade students at Lummi Nation School and Bellingham School District.
Next Reporting Steps
By next reporting period we anticipate that we will have processed most of the dual SPATT experiments, and have developed up a data report for the laboratory kinetics experiments at UCSC. We will continue to report the data to our tribal, state, and scientific partners, and the PD/Co-D will have additional meetings to discuss the project. We hope to write the data up into a peer-reviewed manuscript, with a target date of Fall 2024.
Target Audience
Our target audience is tribal undergraduate students at Northwest Indian College who are interested in natural resources management, oceanography, or aquaculture, We also hope to reach Pacific Northwest Tribal shellfishers, Lummi Natural Resources, and state and federal shellfish managers. This project was discussed with six undergraduate Indigenous research interns, and with Lummi Natural Resources partners. Additionally a graduate student on this project worked with undergraduates at Northwest Indian College and participated in the summer internship activities. We routinely converse with commercial, recreational, and subsistence Native and non- Native shellfishers conducting water quality or shellfish harvesting. We have a K-12 module that we use, with zoom at the beginning of the year, and in person as the year progressed for ourwork in local Indigenous 3rd grade classrooms when we discuss red tide, harmful algal blooms, and safe shellfish harvesting. We also participated in outreach with a local preschool program by allowing the students to be introduced to intertidal animals.
Changes / Problems
Nothing Reported
Training & Professional Development
This project has provided training for three student interns in summer of 2022, and three student interns (one intern from the summer continued) in academic year internships in biotoxins analyses, laboratory preparation, participation in field studies, monitoring strategies, and engagement with Research 1 schools, graduate students, and Lummi Natural Resources scientists. Training for three recently hired (one full time, two part time) researchers has been done in field and laboratory practices. The PI has provided the work that is being done at multiple research conferences, and has presented the data to partners and collaborators. The peer-mentor graduate student from Co-I Kudela's lab was able to visit and engage with Indigenous researchers at the Salish Sea Research Center and work with undergraduate interns.
Dissemination Streams
Results have been disseminated through multiple meetings with Lummi Natural Resources, routine meetings between the PI/Co-I lab, and local and national conferences. This information is also provided as context in our native developed curricular that we share with 3rd and 5th grade students at Lummi Nation School. The PI participated in the monthly NIFA/USDA tribal meetings (zoom) and presented the work on SPATT and shellfish toxins related to this project. The PI has also presented this work to the Northwest Indian College board of trustees, and the science department at Northwest Indian College.
Next Reporting Steps
We will focus on completing our Goal 2 of our proposed project, which is the SPATT kinetics. We anticipate finishing up the determination of method extraction for the SPATT, and then processing the tandem SPATT that we have been collecting in the last year from Fairhaven Dock. We will work closely with our land-grant partner (UCSC) for the toxin kinetic experiments, and continue to process, analyze, and report the data to communities of interest as it comes in. We also anticipate presenting the data at conferences in the next year. <br><br>
<br>What was accomplished under these goals? The greatest impact this project has had to date is the on the Salish Sea Research Center's ability to expand testing for Paralytic Shellfish Toxins (PSTs) in the Salish Sea. We now have an established Liquid Chromatography Mass Spectrometry Mass Spectrometry (LC-MS/MS) method dedicated to identifying PSTs in the Salish Sea. Considering the continuing threat of PSTs to safe shellfish harvests, this is an important development. Goal 1: Produce a quantitative method for PST extraction from (SPATT) by liquid-chromatography QqQ mass spectrometry (LC-MS/MS) at the SSRC at NWIC. Objective 1: Use available literature to optimize the SSRC's LC-MS/MS and verify the PST method for SPATT. We a method by Bounty et al. (2015) and an application note (Agilent Technologies, 6460A LC-MS/MS; Turner and Toglyesi 2019) to develop and optimize our PST method. These methods, which are designed for shellfish, are working for the shellfish samples and particulate toxin samples we have collected as part of this project. Our Solid Phase Adsorption Toxin Tracking Samplers (SPATT) are extracted in methanol, while this method uses acetonitrile for extraction. We are completing experiments to determine if we lose toxin through extraction or analysis with different reagents. We have also deployed parallel SPATT at Fairhaven docks and are processing to determine if there is a loss of toxin in the environmental samples. Objective 2: Enhance NWIC's research capacity with new PST analyses. In 2022 BIOO Scientific stopped producing Enzyme-linked immunosorbent assay (ELISA) testing kits which we had been using since our monitoring program began. We have switched to the Eurofins Abraxis ELISA kits for PSP analysis and are doing a side-by-side comparison for continuity purposes. We continue to have concerns with the quality of ELISA for quantification of PSTs in SPATT and are excited that we are moving forward with the LC-MS/MS method. Goal 2: Investigate SPATT toxin kinetics for PSTs in the Salish Sea Objective 1: Complete uptake and depuration laboratory experiments on SPATT resin for toxin kinetics and produce an exponential decay fit. We have started these experiments are running them in tandem, using methanol for one extraction, and acetonitrile for the second (described above). Currently we are using spiked standards that have been purchased from the National Research Council (NRC) from Halifax, Canada. These experiments are ongoing. We have not begun the uptake/depuration laboratory experiments, but will focus the next reporting period on this objective (and objective 2, below). Objective 2: Compare SPATT resins for PST best adsorption and extraction, and (3) comparing SPATT resins for best adsorption for PSTs in the Salish Sea. We have collected tandem SPATT resins in the Salish Sea, but are not extracting them until we determine the best method (see above). We anticipate focusing our efforts on this goal during the next reporting period. Goal 3: Work closely with LNR to monitor for toxins in the Salish Sea. Objective 1: Support shellfish monitoring with passive toxin samplers. We work closely with our Lummi Nation partners, and share our biotoxin and microscopy analyses when PSTs or the causative agent (Alexandrium spp.) are present in the Salish Sea. We have three passive toxin samplers deployed weekly. Lummi Nation continues to send shellfish samples for toxin analyses to the state of Washington, and are using both datasets to manage their shellfish harvests. Objective 2: Train students and researchers on field collection, laboratory preparation, and analysis of biotoxins. This reporting period we have 6 students who have worked in the lab/field on this project. The students are tasked with collecting weekly monitoring sampling, extracting SPATT, and harvesting, shucking, and preparing shellfish. One student is working with our research technician on analyzing the SPATT using ELISA. Goal 4: Increase Indigenous scholars in STEM geosciences. Objective 1: Support at least nine NWIC students in paid internships for research. During summer 2022 we supported four student interns who participated in the field work, lab analysis, and meetings with shellfish and research scientists. Students learned how to prepare SPATT, place it in the field, collect it, process it in the lab, and have access to the data set. Some of the students participated in the LC-MS/MS analysis, and some of the students are participating in the ELISA analysis of the SPATT. Currently we have three students working during the academic year as interns. Objective 2: Collaborate with LNR researchers, R1 university resources with UCSC, and NWIC students. During summer 2022 we had a visiting graduate student researcher from UCSC. She participated as a peer mentor to the undergraduates and helped them with SPATT processing. The student is working on a different toxin, but is using the same type of SPATT. As such, the student was able to offer guidance and support for the undergraduate interns who worked during the summer. The peer mentor also offered support for undergraduates who were interested in applying for graduate school. We continue to work with marine research and shellfish biologists at Lummi Natural Resources, and our student interns were able to collaborate in field studies with LNR researchers. The PI/Co-I (Drs. Peacock and Kudela) held a PI meeting in person in October 2022, and continue to meet regularly on zoom for this project. <br><br><b>Publications</b><br>
Target Audience
Our target audience is tribal undergraduate students at Northwest Indian College who are interested in natural resources management, oceanography,or aquaculture, Pacific Northwest Tribal shellfishers, Lummi Natural Resources, and state and federal shellfish managers. This project was discussed with twelve undergraduate Indigenous research interns, and with Lummi Natural Resources partners. We routinely converse with commercial, recreational, and subsistence Native and non-Native shellfishers conducting water quality or shellfish harvesting. We have a K-12 module that we use (via zoom) for our work in local Indigenous 3rd grade classrooms when we discuss red tide, harmful algal blooms, and safe shellfish harvesting.
Changes / Problems
Some of the lab work has been delayed due to COVID-19 laboratory closures. We are working diligently to catch up on the lab-required objectives. We have only just recently had a student intern participate in this project, again, because of regulations regarding student participation in lab sampling due to COVID-19 closures. We anticipate these delays being mitigated over the next year.
Training & Professional Development
This project has provided training for three student interns, and three researchers in biotoxin analysis, laboratory preperation, participation in field studies and moitoring strategies, and engagement with research 1 schools and tribal managers. Training for the interns is in small groups as an intern cohort, and also through one-on-one mentoring. Addditional training has been provided for the recently hired research assistant (a former intern), and the research assistant has also provided guided training to the interns.
Dissemination Streams
We continue to partner with Lummi Natural Resources to disseminate project updates to our communities of interest. To date, as we work on method development, that is the researchers at Lummi Natural Resources, though we anticipate a wider audience once we have a working method. We also include information on Paralytic Shellfish Toxins and harmful algal blooms in our native developed curricula that we share with 3rd and 5th grade students at Lummi Nation Schools.
Next Reporting Steps
During the next reporting period our focus is on completing goal 2 of our proposed objective, which is to investigate SPATT toxin kinetics in the Salish Sea. We anticipate completing the uptake and depuration laboratory experiments and comparing resins in both the Salish Sea and also in the lab. We will begin hanging tandem (multiple) resins at each field collection site in January 2022. We also anticipate finishing the quality assurances on our method development and having a standard operating procedure for PST analysis on NWIC's LC/MS in the next year. As we hope COVID restrictions on the reservation and at the lab lift, we anticipate being allowed to include more student interns in this project, either in the field, or for laboratory analysis. We anticipate having a cohort of interns for our summer season in 2022. We will work closely with our land-grant partner (UCSC) for the toxin kinetic experiments, and continue to process, analyze, and report the data to communities of interest as it comes in. We also anticipate presenting the data at conferences as travel becomes more available in the next year. <br><br>
<br>What was accomplished under these goals? The main impact this project has had is on the Salish Sea Research Center's ability to expand testing for Paralytic Shellfish Toxins (PSTs) in the Salish Sea, through supplies and dedicated field collection using SPATT resin at our 3 marine collection sites. We have successfully completed the first method development necessary for the new method on the LC/MS, and anticipate having an established method to quantifiy PSTs in by the next reporting period. We have also had successful engagemnt with tribal partners, and our research 1 (UCSC) school. Lastly, this project has had an impact on our student interns, through providing field and lab analysis training on biotoxin collection and preperation for analysis. Goal 1:Produce a quantitative method for PST extraction from (SPATT) by liquid-chromatography QqQ mass spectrometry (LC-MS/MS) at the SSRC at NWIC. Objective 1:Use available literature to optimize the SSRC's LC-MS/MS and verify the PST method for SPATT. We are developing an LC/MS/MS method based on Bounty et al. (2015), and an application note for our Agilent instrument for PSTs. For verification purposes, both labs (NWIC and UCSC) are analyzing tandem samples to verify the method. We have run a small test batch of samples, and are using NWIC's instrument to optimize PSTs that are being analyzed. Objective 2:Enhance NWIC's research capacity with new PST analyses. Currently, NWIC is continuing to test for PSTs using the ELISA PSP saxitoxin kit (BIOO Scientific). As this method can be less than accurate for quantification purposes, we are focusing on making the LC/MS/MS method a viable alternative, and are making progress on this. We are still analyzing for both, until we are confident in the LC/MS/MS methodology. Goal 2:Investigate SPATT toxin kinetics for PSTs in the Salish Sea Objective 1:Complete uptake and depuration laboratory experiments on SPATT resin for toxin kinetics and produce an exponential decay fit. Objective 2:Compare SPATT resins for PST best adsorption and extraction, and comparing SPATT resins for best adsorption for PSTs in the Salish Sea. This Goal has not yet been completed, but we anticipate based on our timeline of PST analysis that laboratory kinetic experiments will be started in the winter 2022. Goal 3:Work closely with LNR to monitor for toxins in the Salish Sea. Objective 1:Support shellfish monitoring with passive toxin samplers. We continue to work closely with our Lummi Nation partners, and routinely share our biotoxin and microscopy analyses when PSTs or the causative agent (Alexandriumspp.) are present in the Salish Sea. We have three passive toxin samplers deployed weekly, at Gooseberry Point (Lummi Nation), Fairhaven Docks (Bellingham), and 2 nm offshore at the Se'lhaem Buoy (Bellingham Bay). Lummi Nation continues to send shellfish samples for toxin analyses to the state of Washington, and are using both datasets to manage their shellfish opening and closing events. Objective 2:Train students and researchers on field collection, laboratory preparation, and analysis of biotoxins. On student, hired in 2019 has transitioned into a fulltime research assistant role. This researcher is a gradate of NWIC's Native Environmental Science program and now processes and preps the biotoxin samples from field collections. She is trained on all biotoxin procedures. Two other NWIC interns have just started (November 2021) in the lab when personnel COVID restrictions were lifted and are being trained on field collection protocols. Goal 4: Increase Indigenous scholars in STEM geosciences. Objective 1: Support at least nine NWIC students in paid internships for research. Currently we have one student intern in a paid research internship on this project. This student is doing most of the field collection for the biotoxin program. We anticipate more student interns as COVID restrictions lift in the near future. Objective 2: Collaborate with LNR researchers, R1 university resources with UCSC, and NWIC students. We have routine meetings with LNR researchers, and the student interns are involved. We also work with LNR shellfish and water quality researchers to do frequent shellfish and water quality monitoring studies on or near Lummi Reservation. LNR researchers also routinely collect shellfish for us to test for paralytic shellfish poisoning in the lab. We have had some zoom meetings with researchers (namely graduate students) at UCSC, and anticipate more active support on working with interns in summer of 2022. <br><br><b>Publications</b><br>