Our Students and Their Research

Sulfide is the product of sulfate reduction which occurs under anaerobic conditions, including in marine sediments that’s been weather deprived. Sulfide is highly toxic to organisms such as worms and seagrasses that may inhabit marine sediments. Sulfate reduction may be promoted by organic matter loading, which occurs, for example, below oyster aquaculture cages. Therefore, the accumulation of sulfide is a potential undesirable consequence of bottom cage oyster aquaculture. Oyster aquaculture is a major source of commerce along the East Coast and may positively affect the nutrients/ water quality that it surrounds. Sulfate is the second most abundant anion in seawater after chloride and the proximity of sulfide accumulation under oyster cages has caused a rise of concern for environmental monitoring. Denitrification and anaerobic ammonium oxidation (anammox) are biogeochemical processes that naturally remove reactive nitrogen (N) by converting nitrate or ammonium and nitrite to N2 gas, which is released from the water to the atmosphere. The purpose of this project is to test if electrochemical interventions help remove sulfide and promote denitrification. Two methods of electrochemical interventions were tested under oyster farming cages to evaluate which is the most effective in reducing the amount of sulfide that is accumulating within the sediment. This is still an ongoing process, but preliminary data shows that benthic microbial fuel cells can generate small amounts of low voltage current, suggesting it may be mitigating sulfide accumulation. Now the challenge is trying to see if these interventions can withstand longer periods of time to mitigate the sulfide accumulation.  

Rates of ecosystem metabolic properties, such as plankton community respiration, can be used as an assessment of the eutrophication state of a waterbody. However, given the additional labor involved in measuring biogeochemical rate processes, few monitoring programs regularly measure these properties and thus few long-term monitoring records of plankton metabolism exist. Here, we communicate the results of an analysis of nine years of biweekly plankton community respiration rates made as part of the monitoring program at the Chesapeake Biological Laboratory, situated in the lower Patuxent River estuary, a tributary of Chesapeake Bay. We found that particulate nutrients (carbon, phosphorous, and nitrogen) were the most highly correlated co-variates with respiration rate, and both statistical and kinetic models including variables for season, water temperature, and particulate nitrogen were able to explain 74% of the variability in respiration rate. We also found that both particulate nutrients and respiration rate were enhanced when tide height was higher compared to lower. Follow-up measurements of respiration rate over ten consecutive days and during high and low tide on three separate days also support the enhancement of respiration with high tide. It is possible that this could be due to the influence of the Chesapeake Bay by bringing particulate nutrients from the highly productive mid-bay. This analysis of a long-term record of metabolic rates has implications for how we interpret long-term records of measurements made at fixed locations in estuaries.

Eastern oyster (Crassostrea virginica) larvae exhibit several types of migratory and swimming behavior that has been well observed in nature and studied in laboratory settings, but less is known about larvae behavior within oyster hatcheries. Water conditions and larvae are assumed to be uniform in larval tanks, but this assumption doesn’t consider evolved behaviors. We used a Sequoia LISST-100X particle-size analyzer (LISST) to detect oyster larvae behavior within hatchery rearing tanks. The LISST was verified with a comparison to a hand count using 2-day-old larvae. Larvae profiles were taken from five mass larval tanks (MLTs) at the Horn Point Laboratory Oyster Hatchery (HPLOH). Additionally, a conductivity, temperature, and depth logger (CTD) was used to profile the water conditions of seven MLTs. Results were inconclusive for detecting larvae behavior because linear regression models showed that larval density was a function of depth for only two out of the five tanks. CTD data suggested that conditions within and among tanks were not homogeneous. Overall, the larvae profiles created using the LISST was a proof of concept for easily detecting oyster larvae, and results from this study point to new avenues of research to help hatchery managers improve larvae production.

Shoreline flow is challenging to measure and model due to its complexity and high potential for turbulence, requiring more datapoints to get satisfactory resolution. NanoDrifters are small low-cost drifters designed to be deployed in large numbers in shallow regions to characterize these complex flows. The technology and deployment techniques of these drifters were optimized over the course of three different deployments along the Choptank River: one off Todd’s Point, one in Horn Point Cove, and one at a living shoreline in Hurst Creek. The data acquired from these deployments showed that oyster castles reduced the flow of water and caused looping motion of flows which move along them, an effect which continues to a lesser extent post interaction. Flows were found to be faster at near maximum outflow than at slack tide in tidal inlets, and the mean speeds of these flows were much higher than their displacement as a result of the looping motion. The drifters were also found to be useful to create a rudimentary map of flows within a region. These results indicate that the drifters are useful for analyzing such motion, though they were found to experience noticeable slippage from wind and stokes drift. Future models of the NanoDrifter should incorporate a greater subsurface area to mitigate this effect.

The eastern oyster, Crassostrea virginica, is a prevalent foundation and commercial fishery species along the eastern coast of the United States (Wilberg 2011). Recently, restoration efforts have been made in order to restore oysters in the Chesapeake Bay, successfully increasing oyster populations in the wild and allowing for growth in commercial fisheries instead of overharvesting of wild oysters (Gray 2022). However, with increased rainfall in the spring months causing decreased salinity followed by the increased summer temperatures, oysters populations are showing mass mortality (e.g. McCarty et al 2020). This experiment tests the utility of hormesis or ‘stress priming’— the increase in stress tolerance following initial exposure to a sublethal stress—for C. virginica through a low salinity stress treatment on juvenile spat (approximately 1 year old). In order to determine the success of stress priming, a secondary stress condition was applied to oysters given varying days of primary stress. Oysters were measured for heat shock protein (HSP) expression and basic phenotypic traits (growth and survival). Throughout both the primary and secondary stresses, gill tissue—the main immune organ of oysters—was sampled from oysters for RNA and relative expression of Hsp70 and Hsp90 was measured via qPCR (quantitative Polymerase Chain Reaction). Beta actin was used as a housekeeping gene while the stress family of heat shock proteins, specifically Hsp70 and Hsp90, were quantified and the change between Beta actin and each HSP was calculated via the 2^-ΔΔCt method of analysis. Oysters showed very little changes in height and weight, but showed distinct differences in mortality with 2 days primary stress (2dPS) group showing the best stress priming. 8 days primary stress (8dPS) oysters showed anti-stress priming qualities via increased mortality and Hsp70 expression; 14 days primary stress (14dPS) showed some stress priming via reduction of Hsp70 expression but still had elevated mortality. 

The Susquehanna Flats, a fresh water site located near the mouth of the Susquehanna River, supports the greatest and most diverse population of submersed aquatic vegetation (SAV) in the Chesapeake Bay. The SAV bed traps suspended solids and removes nutrients before they approach downstream reaches of the Bay. A filamentous benthic cyanobacteria (Lyngbya) was first observed in the early 2000s and continues to reappear on a seasonal basis. The cyanobacteria grows on benthic and plant surfaces and as nutrients are depleted throughout the summer, it moves up the water column and begins fixing atmospheric nitrogen, eventually floating on the surface in dense mats. This research investigated potential negative interactions between Lyngbya and SAV by studying the effect of various nutrients on plant growth and nitrogen fixation rates as well as aerobic and anaerobic microbial dynamics within Lyngbya filaments and sediment through measuring oxygen and methane fluxes. Bioassays were conducted in triplicate with nutrient additions of 10 µM NO₃^-, 5 µM PO₄^3-, 10 µM NH₄⁺, and NO₃^- + PO₄^3- (N+P). Sub-samples from the bioassays were taken to measure the nitrogen fixation rates of the Lyngbya in the respective treatments using the acetylene reduction assay. Lyngbya that had sediments rinsed off (clean) and Lyngbya with co-occurring sediment left undisturbed (dirty) were compared for nitrogen fixation rates and nutrient exchange. Methanogenesis rates were analyzed for the sediment collected at Sea Grant Site 23. The bioassays demonstrated increased growth in July compared to June, with strong stimulation from the N+P treatment. Nitrogen fixation rates peaked with the addition of PO₄^3- in June with higher nitrogen fixation rates associated with Lyngbya that had sediment trapped in its matrix. Methanogenesis rates appeared to be higher in July in anaerobic conditions, correlating with decreased daily net oxygen production for Lyngbya samples with sediment. We hypothesize that when nutrients are high, the cyanobacteria can focus their energy towards growing and as nutrients become depleted, phosphorus in particular is drawn out of the sediment. Sediment nutrient uptake causes the Lyngbya to blanket the bottom substrate, changing the environment from aerobic to anaerobic. This research may contribute to Chesapeake Bay nutrient models of the SAV bed at the Susquehanna Flats.  

Reef-building corals are geochemical archives that allow reconstruction of temperature in marine environments over seasonal to centennial time scales. The strontium (Sr) to calcium (Ca) ratio of the coral skeleton is inversely related to the sea surface temperature (SST) in which the coral grows. Theoretically, seawater Sr/Ca variability can affect the accuracy of coral Sr/Ca paleothermometry, but most oceanographers assume Sr/Ca is near constant and seawater Sr/Ca measurements are often not made. In this study, I used seawater Sr/Ca and Siderastrea siderea and Diploria strigosa coral Sr/Ca data to investigate the impact of seawater Sr/Ca fluctuations in coral reef environments on coral Sr/Ca-based paleotemperature studies. The coral Sr/Ca and SST calibrations generally demonstrated an inverse linear regression, as expected from many prior calibrations. However, the regression slopes were not significantly different from zero when the coral Sr/Ca data were corrected for seawater Sr/Ca. Future work should use higher precision seawater analytical methods to avoid adding noise when correcting the coral Sr/Ca data.  

Corals are slowly disappearing, disrupting ocean benthic ecosystems that they create. Restoration efforts seek to revive dead or diseased coral reefs by out planting young coral in these areas. However, active coral restoration largely relies on the use of high greenhouse gas materials such as concrete. The goal of our study is to create and identify a low greenhouse gas concrete alternative to be used in coral restoration that maximizes the settlement of Porites astreoides coral larvae and minimizes greenhouse gas emissions. I conducted an exploratory settlement study of P. astreoides larvae on seven cement tile types, observing and quantifying the number of larvae that settled and metamorphosed on the tiles after 48 hours. While the average larval settlement rate differed among tile types, a Kruskal-Wallis ANOVA revealed that these differences were not significant (p=0.23). The lower greenhouse gas alternative concretes were not significantly better or worse than the existing infrastructure for the settlement of P. astreoides larvae. Based on the materials tested, I propose that the biocement made with Vitacal, or a similar CaCO₃ mixture, should be used in reef restoration initiatives. 

Although many studies have examined Crassostrea virginica, also known as the Eastern oyster, and its relationship to the nitrogen cycle, few have studied nitrogen cycling in aquaculture settings through on-bottom aquaculture. As aquaculture is on the rise in the United States (Ray et al. 2021), a better understanding of these impacts may allow for the promotion of nitrogen crediting systems regarding the oysters’ ability to reduce eutrophication (Newell 1988). We hypothesized that Eastern oysters in on-bottom cages in high-energy farms have limited effects on nitrogen cycling, due to high flow rates dispersing biodeposits. Through sediment-water fluxes, porewater collections, and surface sediment samples, we quantified the impacts of oyster aquaculture off the coast of Solomons Island, MD.

Although no sizable difference between the percent carbon and percent nitrogen levels in the sediment was detected, sediment-water fluxes and porewater samples did show dramatic changes. Heightened levels of oxygen, ammonium, and nitrite fluxes suggested an amplification of biodeposits under the cages, and porewater data showed sulfide and ammonium levels were consistently higher in the farm. Furthermore, these data show ammonium and sulfide levels increasing with depth, which peaked in the summer.

Overall, this study suggests that high-flow oyster aquaculture farms concentrate
biodeposits, leading to elevated nitrogen and phosphorous concentrations, increased respiration and ammonium fluxes, and higher sulfide accumulation. This study suggests a decrease in nitrification and an increase in denitrification under cages, allowing for both the enhancement of nitrogen loss and recycling on the farm. These conclusions did not support our hypothesis that high energy conditions will limit a farm’s effect on nitrogen cycling. Future studies including larger sample sizes and tracking the impacts of cages versus bags should also be investigated, as these tests may continue to show support for implementing oyster aquaculture in nitrogen crediting systems.

The Eastern Oyster (Crassostrea virginica) is an organism of significant ecological and economic importance. Oyster farming is demonstrably beneficial not only to the economy, but also – in many scenarios – to the coastal environment. However, as oyster farming efforts increase, there is the potential for some negative impacts to the environment. Aquaculture methods generally consist of areas of caged oysters where oyster biodeposits, a combination of oyster feces and pseudofeces, can accumulate on the seafloor. When oyster biodeposits are not fully dispersed, they can increase the concentration of sulfide in the underlying sediment by stimulating sulfate reduction. Excessive amounts of sulfide can poison benthic organisms, causing decreases in vegetation, secondary production, and biodiversity. Thus, we aim to investigate methods that can be used to counter sulfide excess. Two such methods have been suggested - benthic microbial fuel cells (BMFC) and electrochemical snorkels (ES). This study serves as a pilot study for future research which will measure the ability of BMFC and ES to remove sulfide from sediment, so this study focuses on the design and construction of BMFC/ES regarding their ability to test sulfide concentrations. Some materials tested include, container material, electrode material and placement, wire material and insulation, and wire connectivity to the electrode. For the sake of future sulfide concentration measurements, I determined that the most effective BMFC/ES would be constructed in an acrylic plastic tubing, with electrodes designed from carbon fiber rather than carbon felt, electrodes and the conductive rod could be housed in a specially designed 3D printed platform, and titanium wires insulated with polyolefin shrink tubing and sewn in a circular pattern.

Changes in the biogeochemical processes found in the marine sediments of the Chesapeake Bay’s Patuxent River, have been currently influenced by sulfate intrusion due to sea-level rise. In our investigation of these biogeochemical processes we collected core samples along a salinity gradient in the river at 4 stations. Using one of the replicate core samples, characterization of the core was conducted by analyzing for trace metals iron (Fe) and manganese (Mn²⁺), porosity, methane in pore water, major ions, and sulfide (H₂S). The first 10 cm of the second core sample from each station was then used to create four slurry mixtures. From the homogenized sediment, triplicate sediment samples were taken for each treatment type for a total of 36 incubations; a potassium hydroxide (KOH) kill control, a sulfate enriched treatment and the native sediment at in situ sulfate concentrations. Station 1.5 showed the greatest concentrations of CH₄ (ppm) for all treatment types. The highest concentration of CH₄ was seen in the in situ overlying water treatment at 5270 ppm. High SO₄^2- treatment showed final CH₄ concentrations at 216 ppm and basified treatment showed average cumulative CH₄ at 52 ppm. Overall this study looks to further expand research needed to understand sulfate mediated anaerobic oxidation of methane (AOM), which is performed between methanotrophic archaea and sulfate-reducing bacteria, and how it affected by external factors such as sea-level rise due to sulfate intrusion (Segarra et al., 2013).

Atlantic menhaden are a migratory, schooling, forage fish species that serves as a crucial source of food for a variety of predators in coastal ecosystems and supports the largest fishery by biomass on the East Coast of the United States. Recent research indicates that a large portion of the Atlantic menhaden stock overwinter in offshore waters across the entire East Coast of the United States instead of migrating south to warmer waters off the coast of North Carolina and below as scientists had previously thought. However, the environmental drivers of this partial migration behavior are poorly understood, and little is known about Atlantic menhaden’s pelagic habitat preferences or how this species may respond to a changing climate. To investigate how depth, water conditions, latitude, and a storm event affect both the spatial presence and absence of Atlantic menhaden schools and the size distribution of these schools, I analyzed hydrographic data and Atlantic menhaden school location and size data that was collected during a winter 2022 Atlantic menhaden acoustic survey conducted off the coast of New Jersey. A generalized additive model created to explore the relationship between hydrographic conditions and Atlantic menhaden school size suggests that Atlantic menhaden school size increases with increasing depth greater than 17 meters. This insight could help improve the design of future Atlantic menhaden surveys, sustainably manage the offshore winter bait fishery for Atlantic menhaden, and improve our understanding of how oceanographic conditions affect Atlantic menhaden spatial distribution along greater portions of the East Coast.

Shoreline erosion is rapidly becoming a more prevalent issue. Factors contributing to this erosion include sea level rise, a product of the increasing rate of climate change. This has many negative impacts, both economic and environmental, as it causes property loss and degradation of nearshore water quality. Living shorelines are becoming a popular option to combat this erosion, as they are ecosystem friendly and provide a better alternative to traditional hard erosion structures. However, research into the variability of sediment and vegetation dynamics over space and time has lagged behind installation. There is a lack of knowledge about these shorelines, especially pertaining to their smaller scale spatial variability and temporal variability. This study aims to determine if the usual approach of taking 3 sediment cores along 3 transects perpendicular to the shoreline is sufficient to capture the small scale spatial variability, and to better identify temporal trends in sediment and vegetation characteristics of living shorelines. To test smaller scale variability, 2 new transects were established between the usual 3 transects at sites sampled over the last few years; in addition, 2 new sediment cores were taken between the standard 3 cores. A total of 25 cores were collected and then analyzed for grain size and organic matter content, which was then compared to 3 years of previously collected data to address temporal variability. Previously collected accretion rates were compared to determine the shoreline’s relative success in trapping enough sediment to keep pace with sea-level rise. Overall it was determined that the standard collection method is sufficient to address the small scale spatial variability, and that a longer time series (>3 years) is needed to understand temporal trends. More research on living shorelines and their temporal trends is crucial for understanding their potential sustainability over time.

To assess the ability of oyster aquaculture sites in the Chesapeake Bay to remove nitrogen via coupled nitrification-denitrification, a better understanding of the production and dispersal of oyster biodeposits is necessary. To track the movement of biodeposits at aquaculture sites, environmental DNA (eDNA) analysis of oyster DNA was used to track biodeposit particles. The research had three steps: measure the ability of eDNA to amplify DNA from oyster biodeposits, temporally measure the persistence of oyster DNA in the Chesapeake Bay environment, and, depending on the persistence of eDNA, to measure the spatial distribution of oyster biodeposits at each site. DNA amplification of the oyster genome was proved to be possible. However, the temporal and spatial analyses of these factors revealed that the sediment dynamics under and around aquaculture sites are more complex than anticipated. In the future, a study like this would require a multidisciplinary approach, and further measures should be taken to reduce heterogeneity and inhibition.

Acartia tonsa is a copepod species that lives in freshwater and saltwater areas of the Chesapeake Bay and is categorized as a cryptic species complex. Both freshwater (F) and saltwater (S) A. tonsa look nearly identical but there could be some differences that can characterize the F and S A. tonsa as individual species. The F and S A. tonsa cannot reproduce together in the lab, which implies that they cannot cross breed in the wild. This study investigated the morphometrics of females and the egg hatching rates of the F and S A. tonsa to find those differences, with hand drawn illustrations to visualize A. tonsa basic anatomy and understand what external anatomy characteristics would be measured for the morphometrics. For about four weeks, the F culture had contracted parasitic ciliates and couldn’t be used in this experiment, so only data from the S culture has been gathered and analyzed. The S culture underwent two hatching experiment trials and were photographed for morphometric analysis. The second trial showed that the S culture had an egg hatching rate of 0.27 eggs female^-1 day^-1, a hatching success of 0.53 (53%), and a clutch size of 18 eggs per egg-laying female. Previous studies have found the egg hatching rate and hatching success to be higher for the S culture than the F culture (Plough et al. 2018; Pierson et al. 2005). In addition, the general size for female prosome length is 0.9 mm for the S culture and 0.8 mm for the F culture (Plough et al. 2018). Supposedly salinity is a factor as to how the A. tonsa has separated itself in the Chesapeake.

The Chesapeake Bay plume is thought to be an injector of energy into the shallow coastal waters of the Atlantic Ocean. Identifying how the Plume affects and influences benthic invertebrates is important for understanding how connectivity across terrestrial-to-marine environments affects the biomass, biodiversity, and abundance of benthic macrofauna (e.g., polychaete, oligochaetes, bivalves). The hypotheses for this study were that there would be a peak in species biodiversity at benthic samples collected where there was intermediate plume influence, and that biomass and abundance is higher in sites closer to the mouth of the plume and lower in sites farthest from the bay. Macrofauna recovered from sites were enumerated, aggregated, and weighed by taxonomic groups. It was found that some factors of the plume influence biomass, abundance, and diversity of species.

In the early 2000s, at the mouth of the Susquehanna River in the freshwater regions of the Chesapeake Bay, a submersed aquatic vegetation (SAV) bed experienced a rapid resurgence, after complete disappearance due to sediment loading from Hurricane Agnes in 1972. Since then, a filamentous nitrogen-fixing cyanobacterium called Lyngbya carpets the bottom in the spring and floats to the surface in dense mats during the summer. As benthic nitrogen-fixing cyanobacteria, Lyngbya produces nitrogen compounds that may alter the nutrient dynamics in the submersed aquatic vegetation bed. This study aims to investigate environmental factors influencing nitrogen fixation rates by Lyngbya wollei and benthic sediment nitrogen fixation from the Susquehanna Flats with different seasonal proximities to the sediment in June and July along a N-S and E-W transect moving away from the mouth of the river. Bioassays with nutrient addition triplicate treatments of 10μm NO₃, 5 μm PO₄,,10μM NH₄ and 10μM NO₃ in combination with 5 μm PO₄ (N + P) were done. A suite of water quality parameters were measured at all study sites, and sediment and Lyngbya experiments took place under various conditions. Nitrogen fixation was measured using the acetylene reduction assay on a Shimadzu gas chromatograph. The June bioassay showed there was a strong positive response to the PO₄ treatment while NO₃ suppressed nitrogen fixation. We then found there were higher nitrogen fixation rates with Lyngbya growing on sediment than without and even higher rates with the addition of clay and sediment. In addition to this, Lyngbya biomass appears to increase with distance from the mouth of the Susquehanna River. Our findings lead us to hypothesize that earlier in the season the Lyngbya likely uptakes inorganic N (NO₃ and NH₄) from the sediment, so it doesn’t have to perform the energetically demanding process of nitrogen fixation. Once nitrogen is depleted it has a competitive advantage over non-diazotrophic organisms, by switching to nitrogen fixation. Phosphorus can then become the limiting nutrient which it can also access from the sediment for nitrogen fixation. After being removed from contact with the sediment the Lyngbya appears to acclimate to lower nutrient conditions in the water and switches to fixing non-limiting atmospheric nitrogen dissolved in the water. This study has implications for Chesapeake Bay nitrogen budgets and modeling the nutrient dynamics in this valuable SAV bed.

Climate change and eutrophication has resulted in water temperatures and dissolved oxygen levels deviating from their natural levels. In the marine environment, a stressor can be described as a variable that, as a result of human activity, exceeds its natural level in variation. Predicting fish population responses to exposure to multiple stressors is a challenge because the extensive needed is rarely available.  We use an agent-based modeling to explore changes in population level variables of Atlantic Croaker (Micropogonias undulatus) as a response to single and multiple stressors. We performed a 2x2 factorial simulation experiment using temperature at 2 levels (cool and warm) and dissolved oxygen at 2 levels (normoxia and hypoxia). Using the four simulations, we analyzed the responses of two population level variables: spawning stock biomass (SSB) and average weight of age 5 individuals. Our results showed that the effect of warming and hypoxia on SSB was not well estimated with the additive model but was well estimated with the multiplicative model. In contrast to SSB, the average weight of age 5 individuals was well predicted by both the additive and multiplicative models. Our analysis using an agent-based model of croaker showed that in some cases one can accurately predict the population responses to warming and hypoxia from single stressor effects. The generality of these results needs to be determined.

Marine snow plays an important role in the nutrient cycling and transport of particle associated bacteria to the dark deep of the ocean. Previous studies seemed to suggest that microbial abundance was lower in the bathypelagic zone compared to the epipelagic zone. However, the relationship between microbial abundance and particle sizes across the water column is unknown. Samples were obtained from the AT42-09 cruise aboard the R/V Atlantis to the East Pacific Rise in Spring 2019 then conserved in the freezer at -80°C prior to processing. These samples were prepared on black polycarbonate filter discs with DAPI stain then placed on microscope slides with a cover-slip coated in immersion oil. They were viewed under the Zeiss Axiophot Epifluorescence Microscope. Data analysis of obtained data from observations through a microscopy was carried out on Excel. The results were surprising as these data showed that the microbial abundance decreased until ~300 m where it started to increase. Other studies contrasted with a decrease in the microbial abundance across the water column (Bergauer et al. 2018). Across particle sizes, microbial abundance was expected to be at the highest on smaller particle sizes (Kostadinov et al. 2009). However, the data indicated otherwise with an inconsistency in the microbial abundance across particle sizes. To account for possible contributing factors to inconsistencies in data, negative controls were used to assess the possibility of bacteria contamination and most microbial abundance from samples had higher abundance versus the negative controls’ abundance which suggests the numbers were most likely real. These data did not arrive at a firm conclusion which may suggest some challenges from the methodology to be addressed.