Effects of Feral Pigs (Sus scrufa) on Fecal Contamination in Manoa Watershed Dashiell Dunkell M.S. Thesis Proposal Advisors: Dr. Carl Evensen, Dr. Greg Bruland Department of Natural Resource and Environmental Management University of Hawaii at Manoa Fall 2008 Abstract: Fecal contamination of water bodies is a concern to resource managers because of human and ecological health risks. In Hawaii, feral pigs have invaded many ecosystems, and their effect on water quality and microbial contamination is not well known. Feral pigs can disrupt soil layers, increase erosion, alter nutrient processes, and adversely effect native plants and animals. Feral pigs may also harbor and transmit many infectious waterborne pathogens dangerous to humans. In Hawaii, fencing and eradication of feral pigs has been performed in sensitive terrestrial ecosystems, especially on the islands of Hawaii and Maui, but watershed and water quality effects of feral pigs have rarely been researched. This project proposes to (1) determine if feral pig exclusion influences fecal indicator (Enterococcus sp.) bacteria abundance in runoff; (2) quantify spatial and temporal variation in enterococci abundance in runoff and streams; (3) investigate the correlations between enterococci and other water quality parameters such as total suspended solids (TSS) and dissolved oxygen (DO). The approach will involve runoff water samples from eight paired fenced/unfenced runoff plots, and stream samples from multiple sites, throughout the Manoa watershed on Oahu. Runoff samples will be taken monthly from July 2008 through March 2009, after significant storm events. Stream samples will be taken during both wet and dry periods throughout the project. Samples will be tested for enterococci, nutrients, and common water quality indicators such as TSS, DO, temperature and pH. Soils samples from runoff plots will be tested for antecedent soil moisture. Percent ground cover and canopy cover estimations for each plot will also be recorded. Statistical analysis of enterococci abundance will allow us to test the hypotheses: (1) that fecal contamination of runoff is increased by the presence of feral pigs; (2) enterococci will be correlated with total suspended solids and nutrients in runoff and stream samples; and (3) enterococci abundance in runoff and stream samples will be highest in fall and winter because of high rainfall levels, pig breeding patterns, and increased fruit production. This study will result in better information for the management of Hawaiian watersheds and ecosystems. 1 Introduction: Feral pigs (Sus scrofa) are found in a diverse range of habitats on all continents except Antarctica, as well as many oceanic islands. Early Polynesian settlers first introduced pigs to the Hawaiian Islands as an important food source (Katahira et al, 1993). Later Captain Cook brought European pigs during his first voyage to Hawaii. Many other introductions followed, and pigs became feral and dispersed throughout all the major Islands. Now the only main Hawaiian Islands free of feral pigs are Lanai and Kaho’olawe (Noguiera et al, 2007). The pigs are considered pests that can be dangerous to native biota. Feral pigs can also harbor and spread many potential human pathogens. Fecal contamination caused by pigs has rarely been studied in the Pacific Islands, and never in Manoa. This study will aid natural resource managers, helping them identify the effects of feral pigs, and informing them on the effectiveness of fencing as a tool for increasing water quality. This information will also allow landowners to protect the health of their riparian areas and could benefit swimmers or other recreational users of Hawaiian streams and rivers. The presence of pigs in the Manoa watershed is a divisive issue in the surrounding community, and among the many stakeholders who use the area. The pigs are hunted for their meat, and so a sustainable population is seen as a positive to some local people. However, many scientists, environmentalists, and local residents are concerned about the damage pigs cause to the native vegetation and riparian areas. There are also concerns about the disturbance and danger of hunting the pigs and the use of hunting dogs. The possible threat to human health is an important concern and the many recreational users of the Manoa Valley should be informed of the risks. There is anecdotal evidence that pig populations are increasing, and that pigs are interacting with humans more often. Pig threats to human health may also be increasing and the need to study this issue is urgent. The Manoa Valley watershed is the ideal place for this study for many reasons. Foremost, the University of Hawaii at Manoa is situated such that evaluation and analysis of samples will be fast and straightforward. Likewise, the timely sampling of streams and runoff plots will be expedited when significant rain events do occur. This study site is also benefited by the fact that feral pigs are the only large non-human mammal that occurs in the study area, unlike many of the other Pacific Islands where deer (Axis axis, Odocoileus hemionus) and goats (Capra aegagrus) can occupy the same habitat as pigs. Manoa Valley is diverse in terrain, relief, and rainfall; which should lead to varied results in Enterococci presence. The outlet of the Manoa watershed, Manoa Stream, feeds into the Ala Wai Canal, an urban estuary which borders Waikiki. The Ala Wai Canal outlets just west of Waikiki Beach, one of the most heavily-used and most well-known beaches in the state of Hawaii. Increased pollution entering the Ala Wai should be of great concern to business leaders and politicians because of Hawaii’s dependence on tourism, not to mention to local surfers and beachgoers. Fecal contamination of water bodies is of great concern and is regulated by the EPA to ensure safety. The great diversity of pathogenic microorganisms transmitted by contaminated water and the difficulty and cost of directly measuring all microbial pathogens in environmental samples leads to the use of indicators, organisms that may indicate the presence of sewage and fecal contamination, for monitoring and regulation of recreational and drinking waters (Wade et al, 2006), (Gersberg et al, 2006). Indicator 2 organisms commonly inhabit the intestinal tract of warm-blooded animals. They are found in fecal material at high concentrations and are easier to measure in the environment than are pathogens. Although indicator organisms do not cause illness under normal conditions, they represent a measure of fecal contamination. The previous installation of runoff plots in the study area has provided a unique opportunity for study. The paired plots will allow for the gathering of data from areas both affected and unaffected by pigs, and will allow for different statistical methods of analysis then samples from unpaired plots. Feral pigs have been shown to have negative effects on soil properties as well as ecosystem functions and biodiversity (Hone, 2002), but no one has ever investigated any link to fecal contamination in runoff to pigs in Manoa. In Hawaii, the streams and ocean are highly prized for recreation, and tourists visit from all over the world to enjoy the abundant natural resources. Any possible threat to public health or environmental quality needs to be explored and researched. Literature Review: Invasive Species- Introduction of invasive species is known to be of great concern all over the world, but especially for island ecosystems (Donlan and Wilcox, 2008). Invasive species introduction is one of the main reasons Hawaii is home to 31% of the species on the U.S. endangered species list (Allison and Miller, 2000). Hundreds of exotic species have been introduced to Hawaii, yet feral pigs are considered one of the worst (Nogueira, 2007). Indigenous forests on oceanic islands such as New Zealand and the Hawaiian Islands have evolved in isolation from major landmasses and in the absence of mammalian herbivores. As a result, indigenous flora in such areas exhibit a high degree of endemism and are vulnerable to damage from mammalian herbivory (Sweetapple and Nugent, 2004). Biological invasions are a global phenomenon that can accelerate disturbance regimes and facilitate colonization by other nonnative species (Cushman et al, 2004). A study of a California grassland found that feral pigs promoted the colonization of exotic species (Cushman et al, 2004). Introduced ungulates also have the potential to change the rate and trajectory of recovery of patches of forest that have been damaged by natural and human-induced disturbances (Wilson et al, 2006). In the presence of ungulates, vegetation will often reestablish on these patches more slowly and with a different species composition than in the absence of ungulates. Impacts of exotic mammals on plant regeneration and other processes are fairly well known, however watershed level effects of feral pigs have not been studied much in the Pacific islands, and peer-reviewed research is scarce. In Hawaiian rain forests, an unharvested pig population is potentially capable of doubling every 4 months (Katahira et al, 1993). Except for malnutrition, disease, or cold weather during farrowing at high elevations, there are no known natural factors limiting pig populations. Pig Behaviors- Pigs consume and trample understory plants, disperse plant propagules, degrade native bird habitat by disturbing the understory and influencing forest succession, produce breeding sites for mosquitoes, and disrupt nutrient cycling (Katahira et al, 1993), (Nogueira, 2007). Pigs are omnivorous and have even been shown to threaten endangered shorebirds through predation of eggs (Donlan et al. 2007). Pig 3 rooting and other foraging activities are known to have adverse effects on soil compaction, erosion, ground cover, and water infiltration. These activities can also decrease biodiversity and bulk density. Pig rooting was found to be more frequent in upper elevations and drainage lines in watersheds in Australia (Hone, 2002). In Volcanoes National Park, Hawaii, a study found rooting behavior caused 70% of fresh and intermediate disturbance during pig activity transects (Katahira et al, 1993). Pig activity in the 5 x 10m plots ranged from 0.1-3.6%, with a mean of 0.7%. Estimated pig density ranged from 0.8- 4.7 pigs/km2, with a mean of 0.8 pigs/km2. Pig activity and density in all 3 units combined had a statistically significant (R2 = 0.81, P = 0.01, n = 9) linear relationship. In Australia feral pig disturbance was found to be most common on flat slopes at high elevations, and least common on steep slopes at low elevations (Hone, 1995). The Manoa Valley has a high annual rainfall, and one study in Hawaii found an exponential rate of increase of feral pigs relating to antecedent rainfall (Caley, 1993). Several studies from Australia have also noted that pigs may especially thrive in subtropical or tropical rainforests. This appears to hold true with visual assessment and eyewitness reports of the high pig population living in Manoa. Reports in the local media have suggested that the feral pig interactions in urban areas is increasing as well, worrying homeowners and public health authorities (Nogueira et al, 2007). Foraging can also cause introduction of exotic species, especially Strawberry Guava which is invading many forests throughout the state. Feral pigs are known to spread strawberry guava (P. cattleianum) seeds through feces and rooting behavior causes disturbances that may enhance the spread of the exotic tree (Huenneke and Vitousek, 1990). Health Risks- The possible human health risks caused by feral pig activities and presence in a watershed has been documented and published in peer-reviewed journals. For example, studies of feral pigs in Australia have shown that the foraging and wallowing behavior of pigs can markedly increase the turbidity of water supplies, and more importantly, they can transmit and excrete a number of infectious waterborne organisms pathogenic to humans (Hampton et al. 2006). Specifically, populations of feral pigs may serve as an environmental reservoir of Cryptosporidium parvum oocysts and Giardia sp. cysts for source water (Atwill et al. 1997). Other important protozoan parasite pathogens, such as Balantidium, and Entamoeba, were detected from the feces of feral pigs caught in metropolitan drinking water catchments (Hampton et al 2006). All are potentially important waterborne human pathogens that pose a major threat to water quality. Bacteria: Indicator organisms (IOs) are commonly used to quantify fecal contamination of water bodies, and are an integral part of water management plans (Plummer and Long, 2007). Indictor organisms reside in the gastrointestinal tracts of humans and animals, and are used in the United States and throughout the world to assess the microbiological safety of drinking water, recreational waters, and shellfish waters. The U.S. Environmental Protection Agency recommends the use of Escherichia coli, a member of the fecal coliform group, as an IO for recreational waters in freshwater bodies and members of the genus Enterococcus (the enterococci) for both freshwater and 4 saltwater (Anderson et al. 2005). This study will use Enterococcus sp. bacteria to investigate fecal contamination by feral pigs in the Manoa watershed. Enterococci abundance is one of the three most common water quality tests in the United States (Noblea et al., 2003). From Santa Monica Bay, CA to the Great Lakes levels of indicator bacteria have been shown to correlate with incidence of illness reported by swimmers (Haile et al. 1999), (Wade, 2006). Though some research suggests that enterococci are free-living and reproduce in Hawaiian soils (Hardina and Fujioka, 1991), mesocosm experiments have found that enterococci do not multiply in subtropical waters and sediments (Anderson et al. 2005). Also recent, unpublished data from remote, very undisturbed locations on Oahu has found extremely low enterococci levels in surface waters (Ragosta, 2008). A study in Massachusetts showed the highest IO densities occurred during spring and summer, and the lowest densities during the fall and winter (Plummer and Long, 2007). However, this result was likely caused by the presence of housing complexes in the study area. Human contamination was greater when there was less dilution from rain and surface flow. A study in California (Cushman et al, 2004) found that pigs preferred riparian areas during the summer months. Objectives: 1 Determine if feral pigs have an adverse effect on runoff water quality and increase fecal indicator bacteria; 2 Investigate temporal and spatial differences in enterococci presence in Manoa streams; 3 Determine if correlation exists between other water quality factors and the presence of enterococci; Hypotheses: (1) Enterococci concentrations in runoff are increased by the presence of feral pigs; (2) Total suspended solids and nutrient levels in stream/runoff water will positively correlate with enterococci abundance; (3) Enterococci levels in runoff will be highest during fall and winter because they are bound to sediment and mobilized in wet season rain events. Approach: The project will require periodic water sampling at several different stream sites in the Manoa watershed, as well as the eight runoff plots/pig exclosures already installed by former NREM M.S. student, Chad Browning, under direction from Dr. Greg Bruland and Dr. Carl Evensen. The eight sites for runoff plots are located throughout the Manoa watershed, with three of the plots near stream sampling areas. Each site consists of two paired 5 x 10 m plots, one surrounded by a fenced pig exclosure and one unfenced. The exclosures are constructed of 14 gauge utility fencing approximately 5 x 10 m in area and 0.91 m tall, with barbed wire around the bottom to prevent pig disturbance. Within each individual 5 x 10 m plot is a 4.2 x 1.2 m runoff plot that channelizes 5 any runoff into a buried receptacle. Runoff plots are oriented to run from higher elevation to lower elevation, with slopes ranging from 5-27%. Plots are sheltered from addition overland flow from outside the plot by 15 cm tall plastic pieces buried approximately 7.5 cm deep. The plastic pieces form a water tight barrier completely surrounding the runoff plot. Gathering of runoff is accomplished by the collection end of the runoff plot, a triangular metal collector that funnels all runoff to a 10 cm by 5 cm opening. Attached to the opening of the runoff collector by a metal feed tray is an 18.9 L plastic bucket. A hole cut in the side of the bucket allows the feed tray to deposit runoff directly into the bucket, while a watertight lid ensures that no direct rainfall will enter the bucket. Each month a dry period will be targeted to initiate runoff collection and activate each site. Buckets will be emptied and wiped clean and throughfall gauges emptied. Ground and midlevel vegetation cover will be estimated and soil samples taken. Three randomly selected 5mm soil cores will be taken from inside each plot, within the fence or marked areas, but outside of the runoff plot. These cores will then be analyzed in the lab for antecedent soil moisture. Total runoff amount will be estimated by measuring runoff depth in the bucket, then using a regression formula to give volume. After measuring depth, runoff collected in the bucket will be thoroughly mixed, and then water samples will be taken. Disposable sterile gloves will be worn during all water sampling procedures. One sample from each bucket will be taken in a sterile single-use container for Enterococci testing, and one sample from each will be taken in a clean, acid-washed bottle for use in determining total dissolved solids, nutrients and other water quality indicators. Runoff samples will be taken monthly throughout the year, after significant rainfall events. Stream samples will be taken in the midpoint, middle depth of the stream. Stream samples will be taken after the same rainfall events as runoff samples, and also several times during the study period after significant dry spells. Disposable sterile gloves will be worn at all times during stream sampling procedure. Stream sampling sites will be located in multiple spots throughout the Manoa and Palolo Valleys, located as close as possible to the runoff plots. Stream sampling sites will be located above Lyon Arboretum, below Manoa Falls, in Palolo stream, and other areas in the upper Manoa Valley. The samples lower in the watershed will still be above areas of homes, so sewage system contamination will be avoided. 6 Figure 1. GIS map of Manoa Watershed slope with streams, trails, and the eight runoff plot sites. All water samples will be tested for enterococci, nutrients, pH, EC, total suspended solids and other common water quality indicators. The enterococci test will be performed using an IDEXX instrument (IDEXX Laboratories, Inc., Westbrook, Maine). This process is the only commercial microbiological test included in the AWWA’s Standard Methods for Examination of Water and Wastewater, 20th Edition. The nutrient tests (NO3-, NH4+, total N, and total P) will be performed by the Agricultural Diagnostic Services Committee (ADSC) laboratory at University of Hawaii at Manoa. Water quality tests will be performed in the field or in the laboratory using an YSI 556 Multiprobe device (YSI, Yellow Springs, OH), in accordance with Standard Methods. These will include dissolved oxygen (DO), pH, electrical conductivity (EC), turbidity, and temperature. 7 September Enterococci (2nd Run) 4000.0 3000.0 2000.0 Fenced CFU 1000.0 Unfenced 0.0 LY MC Site MF PP Fenced RT WR Figure 2. September Enterococci abundance results. The location of stream sampling sites will allow for differences to be seen in relief, dominant vegetation, and rainfall. The upper part of the watershed is characterized by steep terrain, higher rainfall rates, and is dominated in places by strawberry guava and bamboo. The lower part of the watershed has flatter terrain and lower rainfall. The quarterly sampling of all sites will allow for any seasonal/temporal differences in fecal contamination to be seen. Runoff plots are situated in areas with varying slope, rainfall, and vegetation cover. This will permit us to analyze differences in enterococci abundance based on those parameters. The wet period samples of streams and runoff plots will be taken when the USGS online real-time rain gauge at Kanewai Field: (http://waterdata.usgs.gov/hi/nwis/uv/?site_no=211747157485601&PARAmeter_cd=00045) Figure 3. USGS Kanewai Field real-time rain gauge, 8/28/08. 8 records a 0.1 inch rainfall event or higher, or if the Manoa Stream gauge (http://waterdata.usgs.gov/nwis/uv?16240500) registers a sizable increase in flow due to a single storm event. Samples could also be taken after a rainfall event in Manoa of more than two inches in any 24 hours. The dry period samples will be taken only from the stream sites and only when there has been no significant rainfall event over seven days. If there is no optimum dry period, the samples will be taken when the USGS stream gauge registers less than the 88 year average. Two samples from each site will be taken back to the laboratory, one for the IDEXX machine and one for nutrient tests. Collected data will be managed with the use of Microsoft Excel spreadsheets. Analysis of the data will be performed using standard univariate and multivariate statistics, including the use of ANOVA or other methods. The paired runoff plots will allow for a matched pairs ANOVA test, analyzing the average contamination for each plot over the year long study period. Plotting each site over time will also allow for differences in fenced/unfenced runoff to be seen. As I take more statistics classes, different and more rigorous analysis of the data will be performed. Expected Outcomes: I hypothesize that the unfenced runoff plots will show higher levels of enterococci than will occur in the fenced plots. By the end of the study, the pig exclosures will have been in place for over a year, which should allow for differences to become evident. Runoff plots are in areas known to be frequented by pigs, and hopefully pig disturbance will occur inside unfenced plots during the study period. Review of literature on feral pigs found pig disturbance more often on flat slopes in higher elevations (Katahira et al, 1993). All the runoff plots are located low-moderate slopes and many are relatively high elevation in the watershed, which should increase likelihood of pig disturbance. ANOVA analysis of fenced vs. unfenced plots will allow for determination of relationship between enterococci levels and pig exclusion. Pig disturbance and excretion should cause increased fecal contamination that will manifest in the water samples. If this does prove to be true, it would likely add to calls for the management or removal of pigs from the ecosystem. I also expect results to show a link between periods of high rainfall and increased enterococci in streams. Increased runoff should cause enterococci, from feral pig activities in the watershed and from bacteria living in the soil, to be transported into stream waters. I predict that enterococci will correlate with turbidity, TSS, and nutrient levels in water. Bacteria are known aggregate with soil and other particles in water, and higher nutrients could be a result of fecal contamination. I posit that runoff and stream water contamination of enterococci will be highest during fall and winter. Winter because that is the period of highest rainfall, and theoretically, highest runoff. Fall is when rainfall is fairly high and strawberry guava trees are fruiting. One study found that when strawberry guava is fruiting it makes up a majority of pigs' diets (Noguiera et al, 2007). The guava trees occur throughout the watershed, often near the riparian zone (per author's obs.), and near or in many of the runoff plots. If enterococci in stream samples are found to be highest in the summer months then perhaps this highlights an unforeseen pig behavior that directly contaminates surface waters, and is a consequence of less dilution during low flows. If enterococci in 9 runoff samples are found to be highest in spring or summer this could be a result also be a result of unpredicted pig behaviors. This project will shed light on unanswered questions related to the management of natural resources such as: how does fecal contamination move through the watershed? The results should show rainfall events and pig foraging activities are responsible for fecal contamination. If enterococci counts in stream samples are higher after dry periods than wet periods, this would suggest that fecal contamination is not brought into the surface waters through runoff. This would highlight the need for keeping pigs out of riparian areas, so that they do not directly contaminate surface waters. Another pertinent question is: what are other factors that may influence the extent of fecal contamination? I believe the study will illustrate that areas with less runoff will have less fecal contamination. If this result is confirmed than the case could be made to try and remove pigs from the upper watershed and areas with higher runoff rates. If fecal contamination is found to be correlated with turbidity, then erosion control projects could help mitigate pig activities. If nutrient load is correlated with fecal contamination then steps could be taken to prevent fertilizer leaching from Lyon Arboretum or other sources. The data generated from this research will be available for resource and land managers throughout the Pacific Islands to help justify feral pig removal or management plans. 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