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Twenty-five essential research questions to inform the protection and restoration of freshwater biodiversity

Freshwater biodiversity is declining at an unprecedented rate. Freshwater conservationists and environmental managers have enough evidence to demonstrate that action must not be delayed but have insufficient evidence to identify those actions that will be most effective in reversing the current trend. Here, the focus is on identifying essential research topics that, if addressed, will contribute directly to restoring freshwater biodiversity through supporting ‘bending the curve’ actions (i.e. those actions leading to the recovery of freshwater biodiversity, not simply deceleration of the current downward trend). The global freshwater research and management community was asked to identify unanswered research questions that could address knowledge gaps and barriers associated with ‘bending the curve’ actions. The resulting list was refined into six themes and 25 questions. Although context-dependent and potentially limited in global reach, six overarching themes were identified: (i) learning from successes and failures; (ii) improving current practices; (iii) balancing resource needs; (iv) rethinking built environments; (v) reforming policy and investments; and (vi) enabling transformative change. Bold, efficient, science-based actions are necessary to reverse biodiversity loss. We believe that conservation actions will be most effective when supported by sound evidence, and that research and action must complement one another. These questions are intended to guide global freshwater researchers and conservation practitioners, identify key projects and signal research needs to funders and governments. Our questions can act as springboards for multidisciplinary and multisectoral collaborations that will improve the management and restoration of freshwater biodiversity.

Aquatic Conservation: Marine and Freshwater Ecosys

Genetic diversity of immature Kemp's ridley (Lepidochelys kempii) sea turtles from the northern Gulf of Mexico

The Kemp’s ridley ( Lepidochelys kempii ) is the world’s most endangered sea turtle species. Predominately nesting at only one beach in Mexico, this species declined to an estimated 300 females in the mid-1980s. Conservation efforts in the United States and Mexico, including a head start programme in southern Texas in which hatchlings were reared in captivity for several months before being released into the wild, resulted in the recovery of this species. Although genetic data have previously been used to assess the success of the head start programme and dispersal of individual adults, data on immature turtles sampled at foraging areas and adult females sampled at the main nesting beach in Mexico are lacking. Genetic characterization of immature individuals is important for understanding recruitment, survival, and population demography, while genetic data on individuals from Mexico are essential for understanding dispersal and overall genetic diversity in this species. To address these gaps, mitochondrial DNA data were collected from 106 immature individuals sampled at four different foraging sites in the northern Gulf of Mexico and from 18 nesting females at the primary nesting beach in Mexico. Two previously unknown mitochondrial DNA haplotypes were discovered among the immature individuals. Except for these two new haplotypes, the genetic diversity of immature individuals in the northern Gulf of Mexico closely corresponds to that of adults sampled in Mexico, which suggests that much of the diversity within the nesting population can be found among immature animals dispersing to foraging grounds, including locations in the northern Gulf of Mexico. Continued monitoring of the genetic variation of different life stages of this species across its distribution range will help assess the success of conservation programmes by ensuring the maintenance of genetic diversity and representation of this diversity across the species’ distribution range.

Aquatic Conservation: Marine and Freshwater Ecosys

Assessment of recovery potential for the American horseshoe crab (Limulus polyphemus): An application of the IUCN green status process

According to an International Union for Conservation of Nature (IUCN) Red List assessment (RLA), the American horseshoe crab ( Limulus polyphemus ), an iconic coastal species, is at risk of extirpation in some regions within its range where small and vulnerable populations occur. However, the RLA does not consider future status beyond viability and does not attempt to identify the conservation necessary to effectively mitigate threats and recover the species to full ecological functionality. To aid in conservation planning for vulnerable species, the IUCN developed the Green Status of Species assessment (GSA) process to complement the RLA. This paper describes the application of the GSA process to assess the recovery potential of the American horseshoe crab. First, specific Limulus populations within spatial units for conservation were delineated, and their statuses were defined based on viability and ecological functionality. Then conservation actions were identified that would promote recovery and affect their near- and long-term population status under different conservation scenarios. Horseshoe crab conservation has relied on, and will continue to depend on, effective harvest regulation. However, as currently conceived, conservation is not expected to mitigate habitat loss at the scale required to restore range-wide ecological functionality, primarily because habitat loss is widespread and affected by climate change. Thus, the GSA results, while indicating that there is potential for near-term recovery gains, reveal that long-term recovery is in doubt owing to expected loss of habitat. To conserve critical habitats for spawning and early life stages and achieve ecological functionality, it is imperative to identify and develop conservation plans at appropriate spatial scales. Unfortunately, such plans do not currently exist and need to be established. The GSA Green Score can then serve as a metric for monitoring recovery and gauging the effectiveness of conservation implementation.

Aquatic Conservation: Marine and Freshwater Ecosys

Abundance of five sympatric stream dwelling mussels varies with physical habitat

Freshwater mussel species regularly co-occur in streams forming assemblages, but the extent of shared versus unique instream habitat features that contribute to their distribution and abundance is poorly understood. In Massachusetts, a rare species, Alasmidonta varicosa , is often found with four other species: Alasmidonta undulata , Strophitus undulatus , Margaritifera margaritifera , and Elliptio complanata , yet variation in species composition within assemblages raises questions of potential species-specific habitat associations. Identifying species-level habitat information is critical at a spatial scale that malacologists can use to identify translocation or restoration areas. This study investigated whether species abundance varied by mesohabitat type (riffle, run, dam pool, scour pool), instream habitat characteristics, and within-reach location (centre versus edge). From 2016 to 2019, freshwater mussel surveys were conducted in nine streams across Massachusetts and associated habitat information was collected. Species abundances were similar across mesohabitat types. Elliptio complanata was the exception, whereby higher abundances occurred in runs and dammed pools than in riffles. Unique species relationships with habitat existed for M. margaritifera with macroalgae and emergent vegetation, and A. varicosa with heterogeneous substrate. Flow transitions, such as depositional areas that create heterogeneous substrates, may provide habitats for A. varicosa . Most mussel species were distributed with higher abundance in the river centre than the edge; E. complanata was the only species with a higher abundance at the river edge. Locations with high abundance varied based on unique relationships with pebble heterogeneity ( A. varicosa ), depth ( A. undulata ), large wood ( A. undulata ), and canopy closure ( E. complanata ). Including physical characteristics in a holistic assessment of habitat that incorporates fish and landscape attributes may further an understanding of river reaches that best support translocated and propagated freshwater mussels.

Aquatic Conservation: Marine and Freshwater Ecosys

Evaluation of extinction risk for stream fishes within an urban riverscape using population viability analysis

1. The Santa Ana River in the Los Angeles region of California demonstrates common habitat degradation symptoms that are characteristic of the urban stream syndrome. These impacts have altered the Santa Ana River community structure, with few species as impacted as the native Santa Ana sucker (sucker; Pantosteus santaanae ). 2. Consequently, a recovery plan developed for sucker identified the need for a population viability analysis (PVA) to assess sucker extirpation risk. However, PVAs can be data-intensive and are subject to several sources of bias when standardized protocols are absent. 3. More than 20 years of sucker and arroyo chub (chub; Gila orcuttii ) surveys using different methods were compiled to build an integrated hierarchical multi-population PVA to estimate trends in abundance and extirpation probability of these native fishes from the Santa Ana River. 4. PVA modelling indicated similar patterns in sucker and chub abundance along the Santa Ana River, with the highest abundance of both species in the upper regions of the river during the early 2000s and downstream in recent years (2018–2022). Extirpation risk was estimated to be greatest near wastewater treatment facilities, where native fish abundance estimates have been zero since 2018. Extirpation risk was lower downstream of the wastewater treatment facilities for both species, although extinction risk was higher for sucker than chub throughout the river. 5. As the model evolves and more data are collected, the PVA could be used to assess the effects of various management actions, such as non-native predator removals and native fish re-introductions, on sucker and chub persistence.

California

Surface detection probability of leatherback turtles at the Rio de La Plata and its Maritime Front: Implications for density estimates

The Río de la Plata and its Maritime Front (RLPMF) serve as a key, seasonal feeding area for leatherback sea turtles ( Dermochelys coriacea ) in the Southwestern Atlantic Ocean. While leatherback turtles can be frequently observed in these waters, there is little data on regional abundances and relative densities, which hinders threat assessments and the development of effective conservation strategies. Consequently, given that in this region parameters needed for in-water leatherback turtle density estimates have not been previously published, the objective of this study was to investigate the surface availability bias, g(0). This parameter requires region-specific considerations for factors like aggregation behaviour and environmental conditions. To address these gaps, our study assessed leatherback turtle aggregations in the RLPMF using satellite tag data and direct drone observations. Through satellite tracking, surfacing behaviour data of six leatherback turtles, g(0) was estimated to be 0.3183. This analysis represents the first of its kind in the region, providing an important contribution to sea turtle conservation studies. It enables substantial progress in characterising surface time, which is essential for understanding leatherback diving and surface behaviour in the region and for improving relative density estimates in the Southwestern Atlantic Ocean. Furthermore, this contribution represents a resource for planning more effective conservation strategies to mitigate threats and promote population recovery.

Río de la Plata and its Maritime Front

Quantifying the extent of river fragmentation by hydropower dams in the Sarapiquí River Basin, Costa Rica

Costa Rica has recently experienced a rapid proliferation of dams for hydropower on rivers draining its northern Caribbean slope. In the Sarapiquí River Basin, eight hydropower plants were built between 1990 and 1999 and more projects are either under construction or proposed. The majority of these dams are small (<15 m tall) and operate as water diversion projects. While the potential environmental effects of individual projects are evaluated prior to dam construction, there is a need for consideration of the basin-scale ecological consequences of hydropower development. This study was a first attempt to quantify the extent of river fragmentation by dams in the Sarapiquí River Basin. Using simple spatial analyses, the length of river upstream from dams and the length of de-watered reaches downstream from dams was measured. Results indicated that there are currently 306.8 km of river (9.4% of the network) upstream from eight existing dams in the Sarapiquí River Basin and 30.6 km of rivers (0.9% of the network) with significantly reduced flow downstream from dams. Rivers upstream from dams primarily drain two life zones: Premontane Rain Forest (107.9 km) and Lower Montane Rain Forest (168.2 km). Simple spatial analyses can be used as a predictive or planning tool for considering the effects of future dams in a basin-scale context. In the Sarapiquí River Basin, we recommend that future dam projects be constructed on already dammed rivers to minimize additional river fragmentation and to protect remaining riverine connectivity.

Aquatic Conservation: Marine and Freshwater Ecosys

Factors influencing densities of non-indigenous species in the ballast water of ships arriving at ports in Puget Sound, Washington, United States

Oceanographic characteristics and the presence of international shipping in Puget Sound, Washington, USA contribute to its vulnerability to non-indigenous species (NIS) invasions. To evaluate NIS arriving in ballast water, zooplankton was sampled in 380 ballast tanks of ships after they entered Puget Sound. Taxa were classified into a higher risk group of coastal organisms (including known NIS), and a lower risk group of largely oceanic species. Most ships reported conducting mid-ocean ballast water exchange (BWE). However, despite state regulations requiring BWE, and apparent compliance by ship operators, most sampled tanks from both transpacific and coastal routes had coastal zooplankton densities exceeding internationally proposed discharge standards. BWE efficiency models and controlled before-and-after BWE experiments indicate that BWE consistently removes most coastal zooplankton. However, this study found that although the empty-refill method of BWE significantly reduced coastal plankton compared with un-exchanged tanks, the flow-through method did not, and in either case remaining coastal plankton densities presented appreciable risks of introducing NIS. Densities of high risk taxa were consistently and significantly higher from US domestic trips dominated by tank ships carrying ballast water from California, and lower in samples from trans-Pacific trips dominated by container ships and bulk carriers with ballast from Asia. These findings are probably a result of the dense and diverse NIS assemblages present in California and other US west coast estuaries and the comparatively short transit times between them and Puget Sound. While it appears that BWE can effectively replace NIS with less risky ocean species, new reporting, verification, and operational procedures may be necessary to enhance BWE efficacy. In the long-term, the introduction of ballast water treatment technologies may be required to significantly reduce the discharge of risky organisms from commercial ships if BWE practices do not become more effective. Copyright ?? 2008 John Wiley & Sons, Ltd.

Aquatic Conservation: Marine and Freshwater Ecosys

Characterizing mauka-to-makai connections for aquatic ecosystem conservation on Maui, Hawaiʻi

Mauka-to-makai (mountain to sea in the Hawaiian language) hydrologic connectivity – commonly referred to as ridge-to-reef – directly affects biogeochemical processes and socioecological functions across terrestrial, freshwater, and marine systems. The supply of freshwater to estuarine and nearshore environments in a ridge-to-reef system supports the food, water, and habitats utilized by marine fauna . In addition, the ecosystem services derived from this land-to-sea connectivity support social and cultural practices (hereafter referred to as socio-cultural) including fishing, aquaculture, wetland agriculture, religious ceremonies, and recreational activities. To effectively guide island resource management, a better understanding of the linkages from ridge-to-reef across natural and social usages is critical, particularly in the context of climate change, with anticipated increasing temperature and shifting precipitation patterns. The objective of this study was to identify spatial linkages that promote multiple and diverse uses, following the ridge-to-reef concept, at an island-wide scale to identify regions of high conservation importance for aquatic resources. We selected the Island of Maui as a study representative of many Pacific islands. Diverse datasets, including agricultural lands within watersheds, wetland locations, presence of stream species, indicators of freshwater input from streams, coral cover, nearshore fish biomass, socio-cultural data such as fishpond locations, wetland taro cultivation, beach recreation use, and lastly the dynamically downscaled Coupled Model Intercomparison Project Phase (CMIP5) future climate projections scenarios (Representative Concentration Pathway (RCP) 4.5 & 8.5) were used to examine the spatial linkages through hydrological connectivity from land to the sea. Zonation spatial planning software was used to prioritize areas of high management and conservation value and to help inform aquatic resources management. The resulting prioritized areas included many minimally disturbed watersheds in east Maui and western nearshore and coastal zones that are adjacent to diverse coral reefs. These results are driven by the importance of fish biomass and coral reef distribution as well as traditional wetland taro cultivation and coastal access points for recreation. These results underline the importance of examining ridge-to-reef systems for aquatic resource management and including important social and cultural values in resource management upon planning adaptation strategies for climate change. Improving our understanding of diverse natural and socio-cultural influences on habitat conditions and their values in these areas provides an opportunity to strategically plan future management and conservation actions.

Hawaii

Aquatic bird disease and mortality as an indicator of changing ecosystem health

We analyzed data from pathologic investigations in the United States, collected by the USGS National Wildlife Health Center between 1971 and 2005, into aquatic bird mortality events. A total of 3619 mortality events was documented for aquatic birds, involving at least 633 708 dead birds from 158 species belonging to 23 families. Environmental causes accounted for the largest proportion of mortality events (1737 or 48%) and dead birds (437 258 or 69%); these numbers increased between 1971 and 2000, with biotoxin mortalities due to botulinum intoxication (Types C and E) being the leading cause of death. Infectious diseases were the second leading cause of mortality events (20%) and dead birds (20%), with both viral diseases, including duck plague ( Herpes virus ), paramyxovirus of cormorants ( Paramyxovirus PMV1) and West Nile virus ( Flavivirus ), and bacterial diseases, including avian cholera ( Pasteurella multocida ), chlamydiosis ( Chalmydia psittici ), and salmonellosis ( Salmonella sp.), contributing. Pelagic, coastal marine birds and species that use marine and freshwater habitats were impacted most frequently by environmental causes of death, with biotoxin exposure, primarily botulinum toxin, resulting in mortalities of both coastal and freshwater species. Pelagic birds were impacted most severely by emaciation and starvation, which may reflect increased anthropogenic pressure on the marine habitat from over-fishing, pollution, and other factors. Our study provides important information on broad trends in aquatic bird mortality and highlights how long-term wildlife disease studies can be used to identify anthropogenic threats to wildlife conservation and ecosystem health. In particular, mortality data for the past 30 yr suggest that biotoxins, viral, and bacterial diseases could have impacted >5 million aquatic birds.

Marine Ecology Progress Series

One hundred pressing questions on the future of global fish migration science, conservation, and policy

Migration is a widespread but highly diverse component of many animal life histories. Fish migrate throughout the world's oceans, within lakes and rivers, and between the two realms, transporting matter, energy, and other species (e.g., microbes) across boundaries. Migration is therefore a process responsible for myriad ecosystem services. Many human populations depend on the presence of predictable migrations of fish for their subsistence and livelihoods. Although much research has focused on fish migration, many questions remain in our rapidly changing world. We assembled a diverse team of fundamental and applied scientists who study fish migrations in marine and freshwater environments to identify pressing unanswered questions. Our exercise revealed questions within themes related to understanding the migrating individual's internal state, navigational mechanisms, locomotor capabilities, external drivers of migration, the threats confronting migratory fish including climate change, and the role of migration. In addition, we identified key requirements for aquatic animal management, restoration, policy, and governance. Lessons revealed included the difficulties in generalizing among species and populations, and in understanding the levels of connectivity facilitated by migrating fishes. We conclude by identifying priority research needed for assuring a sustainable future for migratory fishes.

Frontiers in Ecology and Evolution

The rapid return of marine-derived nutrients to a freshwater food web following dam removal

Dam removal is increasingly being recognized as a viable river restoration action. Although the main beneficiaries of restored connectivity are often migratory fish populations, little is known regarding recovery of other parts of the freshwater food web, particularly terrestrial components. We measured stable isotopes in key components to the freshwater food web: salmon, freshwater macroinvertebrates and a river specialist bird, American dipper ( Cinclus mexicanus ), before and after removal of the Elwha Dam, WA, USA. Less than a year after dam removal, salmon returned to the system and released marine-derived nutrients (MDN). In that same year we documented an increase in stable-nitrogen and carbon isotope ratios in American dippers. These results indicate that MDN from anadromous fish, an important nutrient subsidy that crosses the aquatic&ndash;terrestrial boundary, can return rapidly to food webs after dams are removed which is an important component of ecosystem recovery.

Washington

Drought in the U.S. Caribbean: Impacts to freshwater ecosystems

Healthy and functioning freshwater ecosystems are needed for successful conservation and management of native fish and invertebrate species, and the services they provide to human communities, across the U.S. Caribbean. Yet streams, rivers, and reservoirs are vulnerable to the effects of extreme weather events, urbanization, energy and water development, and other environmental and human-caused disturbances (Neal et al., 2009). One major management concern is the impact of prolonged drought on freshwater ecosystems. Drought impacts streamflow, dissolved oxygen content, water quality, stream connectivity, available habitat, and other important freshwater habitat characteristics necessary for sustaining fish and invertebrate populations (Covich et al., 2006). These changes can impact species interactions, abundance, life history events, and the presence of native and non-native species (Larsen, 2000; Covich et al., 2006; Ramírez et al., 2018). Drought impacts aquatic ecosystems and species both in the short-term and long-term, depending on the severity and duration of the event (e.g. Covich et al., 2006 ). In Puerto Rico, all native freshwater fish, shrimp, and snail species spend part of their lives in estuarine and marine ecosystems and depend on being able to move between these habitats to survive, so maintaining connectivity is key (e.g., Engman et al., 2017 ). Freshwater ecosystems also provide recreational, cultural, and ecological value to humans ( Kwak et al., 2007 ; Neal et al., 2009 ). For example, some communities in Puerto Rico engage in artisanal shrimp and freshwater crab fishing ( Neal et al., 2009 ). Artisanal fishing for postlarvae gobioids, known colloquially as “cetí” also occurs at the river mouths of large drainages and has strong cultural significance in parts of Puerto Rico, such as Arecibo (Kwak et al., 2016). The U.S. Virgin Islands (USVI) is particularly sensitive to drought, because almost all streams are ephemeral and typically only flow after rainfall. These intermittent channels, known locally as “ghuts”, run down the surface of steep slopes, rather than through the ground, and are important sources of freshwater. Natural springs are often located in ghuts and can form pools of freshwater that serve as habitat for wetland and migratory birds, freshwater shrimp and fish, and amphibians ( Nemeth and Platenburg, 2007 ; Gardner, 2008 ).

Puerto Rico, U.S. Virgin Islands

A conceptual framework and methods for studying the connectivity of fishes

Connectivity is a multifaceted concept that has important implications for the management and conservation of marine and freshwater fishes. We developed a conceptual framework that encompasses multiple, interrelated categories of connectedness, including landscape (e.g., structural, functional) connectivity and ecological (e.g., trophic, genetic, demographic) connectivity, that together shape the flow of organisms, energy and information across ecosystems. We also synthesised six key methods that can be used to study connectivity of fishes: (1) telemetry, including satellite, acoustic, radio and passive integrated transponders (PIT), (2) mark-recapture, (3) environmental tracers, including stable isotopes and otolith-microchemistry, (4) genetics, (5) community structure analysis and (6) emerging technologies and tools (e.g., remote sensing and artificial intelligence). For each method, we describe the categories of connectivity it can assess and provide real-world examples where they have been effectively used. We also identify limitations of each method. This article highlights the diverse and evolving toolbox of methods used to assess fish connectivity, underscoring the need for continued collaboration, innovation and integration of new approaches to refine our understanding and address remaining challenges in this critical area of aquatic ecology and fisheries management.

Fish and Fisheries

Fish-assemblage variation between geologically defined regions and across a longitudinal gradient in the Monkey River Basin, Belize

Linkages between geology and fish assemblages have been inferred in many regions throughout the world, but no studies have yet investigated whether fish assemblages differ across geologies in Mesoamerica. The goals of our study were to: 1) compare physicochemical conditions and fish-assemblage structure across 2 geologic types in headwaters of the Monkey River Basin, Belize, and 2) describe basin-scale patterns in fish community composition and structure for the benefit of conservation efforts. We censused headwater-pool fishes by direct observation, and assessed habitat size, structure, and water chemistry to compare habitat and fish richness, diversity, evenness, and density between streams in the variably metamorphosed sedimentary geologic type typical of 80% of Belize’s Maya Mountains (the Santa Rosa Group), and an anomalous extrusive geologic formation in the same area (the Bladen Volcanic Member). We also collected species-presence data from 20 sites throughout the basin for analyses of compositional patterns from the headwaters to the top of the estuary. Thirty-nine fish species in 21 families were observed. Poeciliids were numerically dominant, making up 39% of individuals captured, followed by characins (25%), and cichlids (20%). Cichlidae was the most species-rich family (7 spp.), followed by Poeciliidae (6 spp.). Habitat size and water chemistry differed strongly between geologic types, but habitat diversity did not. Major fish-assemblage differences also were not obvious between geologies, despite a marked difference in the presence of the aquatic macrophyte, Marathrum oxycarpum (Podostemaceae), which covered 37% of the stream bottom in high-nutrient streams draining the Santa Rosa Group, and did not occur in the low-P streams draining the Bladen Volcanic Member. Correlation analyses suggested that distance from the sea and amount of cover within pools are important to fish-assemblage structure, but that differing abiotic factors may influence assemblage structure within each geologic type. The fauna showed weak compositional zonation into 3 groups (headwaters, coastal plain, and nearshore). Nearly 20% of the fish species collected have migratory life cycles (including Joturus pichardi , Agonostomus monticola , and Gobiomorus dormitor ) that use freshwater and marine habitats. Some of these species probably rely on a natural flow regime and longitudinal connectivity for reproduction and dispersal of young, and natural flow regime and longitudinal connectivity are important factors for maintenance of functional linkages between the uplands and the coast in the ridge-to-reef corridor where the Monkey River is located. Therefore, we suggest that the viability of migratory fish populations may be a good biological indicator of upland-to-estuary connectivity important both to fishes and coastal ecosystem function. We recommend follow-up studies to substantiate the relative strengths of relationships between community structure and abiotic factors in contrasting geologies and to examine potential bottom−up responses of stream biota to the higher nutrient levels that were observed in stream waters draining the Santa Rosa Group geologic type.

Journal of the North American Benthological Societ

Use of Numerical Models to Simulate Transport of Sewage-Derived Nitrate in a Coastal Aquifer, Central and Western Cape Cod, Massachusetts

The unconsolidated glacial sediments underlying Cape Cod, Massachusetts compose a regional aquifer system that is used both as a source of drinking water and as a disposal site for wastewater; in addition, the discharge of clean ground water from the aquifer system is needed for the maintenance of freshwater and marine ecosystems throughout the region. Because these uses of the aquifer conflict with one another in many areas of the Cape, local and regional planners have begun to develop sustainable wastewater plans that will facilitate the disposal of wastewater while protecting water supplies and improving the health of aquatic ecosystems. To assist local and regional planners in these efforts, the U.S. Geological Survey conducted a 2-year investigation to (1) assist local and regional planners in the evaluation of potential wastewater scenarios, (2) use results and interpretation from these analyses to develop hydrologic concepts transferable throughout the region, and (3) establish and test methods that would be of use in future evaluations. Wastewater-disposal scenarios need to be evaluated in the context of the regional ground-water-flow system. For a given rate of disposal, wastewater from sites at or near a regional ground-water divide is transported in a wider arc of flow directions, flows deeper in the system, and contaminates a larger part of the aquifer than does wastewater discharged from sites farther from the divide. Also, traveltimes of wastewater from sites near a ground-water divide to receptors are longer (as much as several hundred years) than traveltimes from sites farther from the divide. Thus, wastewater disposal at or near a divide will affect a larger part of the aquifer and likely contribute wastewater to more receptors than wastewater disposal farther from a divide; however, longer traveltimes could allow for more attenuation of wastewater-derived nitrate from those sites. Ground-water-flow models and particle tracking can be used to identify advective-transport patterns downgradient from wastewater-disposal sites and estimate traveltimes; however, these tools cannot predict the distribution of mass or concentrations of wastewater constituents, such as nitrate, in the aquifer. Flow-based particle-tracking analyses can be used to estimate mass-loading rates and time-varying concentrations at wells and ecological receptors by the accounting of mass-weighted particles discharging into the receptor of interest. This method requires no additional development beyond the flow model; however, post-modeling analyses are required. In addition, the method is based on the assumption that no mass is lost during transport, an assumption that likely is not valid in many systems. Solute-transport models simulate the subsurface transport of nitrate through the aquifer and predict the distribution of the mass of a solute in the aquifer at different transport times. This method does require additional model development beyond the flow model, but can predict timevarying concentrations at receptors. Estimates of mass-loading rates require minimal post-modeling analyses. Time-varying concentrations and mass-loading rates calculated for wells in eastern Barnstable by the two methods generally were in reasonable agreement. Inherent in the flow-based particle-tracking method is the assumption that mass is conserved along a given flow line and that there is no spreading of mass in the aquifer. Although the solute-transport models also incorporate a system-wide conservation of mass, these models allow for a spreading of mass in the aquifer, and mass is not conserved along a given flow line. As a result, estimates of concentrations and mass loading rates generally were higher in particle-tracking analyses than in solute-transport simulations. Results from the two types of simulations agreed best for wells that receive large amounts of wastewater with short traveltimes (less than 10 years) because insufficient transport

Scientific Investigations Report