An Application Plan 

Our scoping and design phases included a workshop with marine mammal researchers and NOAA decision makers to build tools for this purpose. Using these tools and the literature7, we identified PCBs and dioxins as a high-interest multiple stressors scenario for NRDAs considering Houston area dolphins. Ideally, resource managers would have data about chemical exposures and individual doses, their resulting physiological and behavioral responses, the respective effects on health and individual survival, and what that means at the population level (Figure 1, blue arrows). With protected and cryptic species like dolphins, such comprehensive data is difficult, but during our workshop we identified key uncertainties that could be addressed to most benefit NRDA decision makers going forward (Figure 1, orange overlays).

We group the expected findings based on the four research questions proposed in the Research and Development plan:

We would analyze blubber samples from previous remote biopsy surveys and from a new remote biopsy survey to better understand average contaminant loads (both PCBs and dioxins) across the population, as well as individual doses compared to each animal’s ranging pattern (via sightings history) and health (via epigenetics). We would compare contaminant loads across sex, age, and reproductive life-history parameters of Houston area dolphins, as well as with other dolphin populations across the Southeast U.S. (SEUS)8-9. We would test the prediction that dolphins that spend more time near contaminated sites will have larger contaminant loads10.

With contaminant burdens in hand, we would then be able to evaluate PCB and dioxin exposure and doses to inform 1) cell culture exposures and 2) PCoMS modeling efforts11 and NRDA decision making. A key aspect of our co-production process would be to come together after all the potential samples have been collected and before any new analytical chemistry is performed, to ensure that we collectively agree on the best selection of samples to inform our research questions.

We would conduct laboratory exposures with cell lines to better understand the physiological impacts of dioxins alone, PCBs alone, and both chemicals together. By exposing cells to a range of chemical exposures in a controlled experimental set up, we would be able to define dose-response curves for each contaminant and for the combined exposure, as well as identify the specific thresholds at which cellular and molecular effects take place. Our results from the analytical chemistry laboratories would help us define the specific mixtures of PCB and dioxin congeners and their relative amounts, so that the exposures are as relevant as possible to the doses experienced by Houston area dolphins. We would work as a team to identify the specific HPT axis, immune system, and other physiological endpoints that would be most useful for interpretation of wild dolphin health assessment and strandings data, and therefore for marine mammal NRDA decision making. These endpoints would likely include free and bound thyroid hormone levels (e.g., triiodothyronine [T3], thyroxine [T4], and thyroid stimulating hormone [TSH]), selected mRNA transcript levels, and/or cell activity/signaling assays. We would focus on commercially-available human, primate, and/or rodent cell lines with reasonable HPT signaling pathway behavior, which would maximize the number of established kits, reagents, and assays available for the endpoints of interest.

With the three dose-response curves in hand, we would be able to identify whether the two contaminants have an interactive toxic effect on endpoints12-15. Multiple chemical compounds operating through the HPT pathway and molecular target/mechanism may be more likely to produce additive effects, whereas operating through the same pathway but different mechanisms may produce non-additive effects. We can use the dose-response curves to better interpret what the doses measured in our analytical chemistry efforts mean for the health of Houston area dolphins. This would inform how decision makers characterize impacts from specific contaminants in the context of the multiple stressor reality of the Houston area. We would stay flexible about adding additional endpoints, as possible, based on new information about how POPs impact the HPT axis, immune function, and other aspects of physiological health. In the future, if NOAA decides to pursue an injury assessment of Houston area dolphins, these data would be critical for any development of more case- or site-specific experiments with other cell lines, such as primary marine mammal cell lines from HPT tissues.

While some data are available on how PCBs/dioxins impact dolphin health16, none are available for Houston area dolphins. Although we would have a small number of stranded cases with necropsy and pathology information about individual dolphin health, most of the biopsied dolphins would not have paired health data. However, we can analyze epigenetic patterns (e.g., DNA methylation levels at particular sites in the genome) from skin tissue to understand overall health of individual dolphins17. The epigenetic signatures of unhealthy individuals can appear older or younger than their actual age (e.g., smokers appear older than their actual age18). We will collect skin samples, extract DNA, and analyze epigenetic profiles to estimate chronological (age based on number of years since birth) and biological ages (age based on health, stress, and lived experience). We would test our prediction that if Houston area dolphins are exposed to dioxins and PCBs, there would be a discrepancy between their biological and chronological ages, due to adverse health from the toxic effects of the chemicals.

We have developed elastic net and random forest models with ~750 epigenetic dolphin samples19 so that at a population level, we could compare the Houston area dolphins to relatively healthy reference populations in the SEUS and/or populations facing a variety of other environmental stressors.

In the future, if NOAA decides to pursue an injury assessment of Houston area dolphins, our epigenetic and analytical chemistry data can then be used to target specific animals for health assessments, or to provide a broader context for how to interpret future health assessment information. Thus, decision makers could better link the endpoints from the cell studies, veterinary examinations of Houston area dolphins, and the bigger-picture epigenetic biological age/health of the individuals and population. Our epigenetic models are continually updated to incorporate additional information, such as evaluating epigenetic trends/markers based on specific health endpoints, so as new information becomes available, we would incorporate them in updated and/or new analyses to help decision makers.

Survival, at both the individual- and population-level, is a key factor in understanding PCoMS and assessing injury for NRDAs with marine mammals. We would conduct three surveys (in Years 1, 3, and 5) to inform spatial capture-mark-recapture (CMR) analysis, allowing us to estimate population 2-year and 5-year survival rates across the five-year project period. We would leverage existing data from past surveys1, 20-22 and compare the Houston-area rates to other populations across the SEUS23. We could also leverage our parallel work on another project, the Veterinary Expert System for Outcome Predictions (VESOP), to generate and compare health endpoints and VESOP survival predictions from the epigenetics analyses with the survival estimates from the CMR analysis. We expect that the Houston area dolphin population would have lower survival rates compared to populations from less contaminated areas.

Together, research on these four priority questions would facilitate future Houston-area NRDAs by providing decision-makers with context for understanding health data vs exposure data in the local dolphins, including contaminant burdens, survival rates, overall health, and dose-response relationships for singular and combined PCB/dioxin exposures. Addressing these uncertainties would enable NRDA decision-makers to better evaluate jurisdiction under the preassessment phase and develop targeted studies to answer site-specific concerns as part of the injury assessment and restoration planning activities. Importantly, our studies would be meant to help decision makers by addressing current priority data gaps to facilitate future NRDAs but they would not be a substitute for NRDAs. In other words, our proposed work would not be designed as a standalone study, but as one important part of a co-produced, broader set of research and decision-making about dolphins and the numerous National Priorities List Superfund sites undergoing cleanup in the Houston area.

Resource managers have been unable to identify ways to fund the proposed work. Thus, our co-production lead, natural resource managers, and research team would all work closely (via dedicated in-person meetings and conference calls) to evaluate next steps at each critical milestone during our project. Such milestones would include planning, analysis, and interpretation phases for:

• each field effort (CMR surveys and the remote biopsy survey), 

• the cell study, 

• the epigenetics sample selection and analysis, 

• the analytical chemistry sample selection and analysis, and 

• the PCoMS, epigenetic, and CMR modeling efforts. 

This adaptive management process would focus on how our ongoing efforts are helping to address the uncertainties and relationships within our conceptual model (Figure 1).

NOAA resource managers have identified several sites in the Houston area that involve NRDA-related evaluations, preassessments, injury assessments, and restoration planning over the next six to ten years, and anticipate more sites/incidents in the future. The process and criteria for determining if a NRDA is warranted are complex. A specific timeline for the process depends on an unpredictable combination of actions by and discussions with other stakeholders and natural resource Trustees. Having conceptual models developed, methods ready, and data available to reduce uncertainty on cumulative effects from multiple contaminants for dolphins would be a huge asset for informed decision making. NOAA will also be evaluating restoration activities that are scheduled in the Houston area within the next six to ten years. All of our proposed activities and the products we would develop would strive to incorporate iterative data collection, analysis, and application with stakeholders.

We will would make our final report available to all relevant stakeholders in the NOAA DAARP and OPR offices, local Houston area (e.g., the Gulf Coast Protection District), and other Federal, State, and non-government offices—similar to how we have disseminated our findings from the DWH NRDA to natural resource Trustees and decision makers. We would also hold an End-of-Project virtual workshop dedicated to the transfer and application of our findings with the same stakeholders, focusing on both what uncertainties our project addressed and any priority data gaps that remain. Our team would also present our findings at scientific conferences and submit the co-produced work to peer-reviewed journals. Stakeholders and natural resource managers would not have to wait until the end of the project to see our progress, as we include a series of conference calls and an in-person meeting for the project team and stakeholders to discuss adaptive management and transfer/application of our findings (see our milestones Gantt chart).

Our project would also include: 

1) at least three paid internships for underrepresented minority undergraduate students to gain access to field research opportunities and to broaden and strengthen our research team; 

2) two training workshops for local Houston area volunteers to assist with the field surveys and develop field biology/research skills; 

3) an annual research assistantship/apprenticeship position for students interested in pursuing a graduate degree in marine mammal science; and 

4) two post-doctoral fellows to run analyses and interpretation of the cell line study and the spatial CMR survival and PCoMS modeling efforts. 

We would hope these opportunities and experiences will enrich the environmental toxicology and marine mammal conservation fields that underpin effective, science-based decision making for Gulf protected species and NRDAs.

Most of our co-investigator team was part of our working group that scoped and designed this project together in Galveston, TX. We would continue to consult closely with the working group from that effort, as needed. Between our project team and working group members, we have a broad network in the marine mammal, NRDA, and toxicology fields, and we were deeply integrated into the marine mammal assessments for the DWH NRDA. Our project would benefit because the co-production lead, management bodies, and local stakeholders most involved with our management decision have been and would continue to be co-investigators throughout the project.

We have both 1) a group of institutions and agencies that cover the various aspects of decision making, research, local stakeholders, and community engagement related to our management decision, and 2) individuals who throughout their careers have worked on research, resource management, and outreach/engagement within co-production environments. Our fields of NRDA science and protected resource management have a rich history in this approach to addressing challenges. Our potential team is built on successful working relationships from past projects and would continue to implement the co-production lessons we have learned from those experiences. 

Dr. Michel Gielazyn is a natural resource manager and co-investigator representing the NOAA ARD team making decisions about NRDAs in the Houston area and beyond. She has >19 years working for NOAA to evaluate contamination of natural resources and identify ways to make the public whole for the injuries. 

Dr. Teresa Rowles is a natural resource manager and co-investigator representing the NOAA OPR team making decisions about restoration planning, NEPA evaluations, and wildlife management actions in the Houston area and all U.S. waters. She is a veterinarian with >30 years of health and conservation experience, and has served as NOAA’s MMHSRP coordinator for >25 years. 

Both Drs. Gielazyn and Rowles were integral in the scoping and design phases of the project and will ensure that all our activities are framed in co-producing scientific support for Houston area NRDAs and marine mammal decision making.

Our lead investigator, Dr. Ryan Takeshita, has >15 years of experience with toxicology and marine mammals, including with boundary-spanning teams on injury/restoration evaluations for NRDAs. During DWH, he served as a technical consultant and project manager that worked closely with NOAA resource managers and researchers to coordinate and report on toxicology research portfolios and marine mammal assessments. Dr. Takeshita led the scoping and design phases of this project and will oversee the project team’s activities and collaborative efforts, including NMMF’s support of field activities and data analysis, as well as contribute to technical analysis/interpretation and the drafting, editing, and finalization of all deliverables.

Dr. Lori Schwacke is our co-production lead and the Director of Scientific Programs at the Marine Mammal Commission (MMC). She has >20 years of experience with biostatistics, epidemiology, and population health, both with NMMF and NOAA. These prior positions, in combination with her new role at the MMC, provide her with perspective and relationships that will be key to overseeing and evaluating effective co-production among our team. She led dolphin field studies and injury quantification for the DWH NRDA, and served as an expert for the NASEM Committee for Assessment of the Cumulative Effects of Anthropogenic Stressors on Marine Mammals. She will also assist with data analysis and modeling efforts.

Our team also consists of marine mammal and toxicology researchers that are especially well-suited to addressing our specific research questions in the Houston area. Ms. Kristi Fazioli (Environmental Institute of Houston; EIH) and Dr. Vanessa Mintzer (Galveston Bay Foundation; GBF) lead the Galveston Bay Dolphin Research Program, studying the Houston area dolphins since 2014. Ms. Heidi Whitehead leads the Texas Marine Mammal Stranding Network (TMMSN) and their variety of rescue, rehabilitation, research, and education efforts. These three groups will be critical to our planning and execution of our proposed field work efforts in the Houston area, as well as providing important local insight for the interpretation of the findings for the natural resource managers. Mr. Erol Ronje will serve as an unpaid collaborator to help with the Houston area finbase catalog. Ms. Fazioli will also coordinate the analytical chemistry analysis of dioxins. 

Dr. Irvin Schultz, the Environmental Chemistry Program Manager at NOAA NWFSC, will coordinate the analytical chemistry analysis of PCBs, and will provide important expertise on our interpretation of PCB/dioxin endocrine disruption. 

Drs. Aaron Roberts and Amie Lund (University of North Texas) will lead the proposed cell-line laboratory exposure experiments and interpretation. Dr. Roberts is an environmental toxicologist with broad expertise in designing laboratory exposures to inform wildlife risk assessments, including marine mammal studies; Dr. Lund studies the molecular/cellular mechanistic pathways by which environmental pollutants impact physiological systems. 

Dr. Ashley Barratclough is a conservation medicine veterinarian with NMMF and will lead our epigenetic evaluation of Houston area dolphin health. 

Finally, we anticipate collaborating with statisticians (such as Dr. Len Thomas at the University of St Andrews) to lead the modelling efforts for survival and integrating our results into a PCoMS framework, using the products developed during our scoping/design phases.

Our project team has worked closely with each other across a variety of NRDA and natural resource decisions, including co-producing research plans and assessment reports, and we appreciate how a better understanding of multiple stressors could benefit the decision-making process for marine mammal assessment, conservation, and restoration. Most of our team was also part of the Working Group during the scoping and design phases of the project. Although marine mammals aren’t typically associated with any group of “resource users”, we believe that our project team represents the breadth of stakeholders involved in conserving and managing our U.S. marine mammal populations, including government (NOAA and MMC), local conservation groups conducting education and outreach with the community (EIH, GBF, and TMMSN), and broader non-profit/academic groups focused on conducting research to inform responsible conservation management decisions (NMMF, UNT, and St Andrews). 

Here, we include a list of "End Users" (and their current affiliations) from our scoping and design phases of the project. We anticipate that these individuals would remain important stakeholders in the application of our proposed work.

In total, we estimate that the work proposed in the Research and Development Plan could be conducted with a budget of approximately $2 million. This includes funds for a multi-institutional, multi-disciplinary team of scientists and resource managers, including funds for NOAA partners to travel to team meetings and for data management services. Application would then mostly fall upon NOAA OR&R and PRD to identify appropriate funding for NRDA and resource management decision-making.

If we are unable to secure funding through the NOAA RESTORE FFO-2023 funding opportunity, then we will revise this Application Plan later in 2023 to update any new information and to identify additional potential sources of funding.

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