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An assessment of aquatic organisms in surface water and groundwater of the Cumberland Valley karst of south
central Pennsylvania
Geo 546: Geo-environmental Research 1
Spring 2015
Student: Sarah R. Bartle1
Advisor: Dr. Thomas P. Feeney2
1
Department of Biology, Shippensburg University, 1871 Old Main Drive, Shippensburg, PA 17257
Department of Geography- Earth Science Shippensburg University, 1871 Old Main Drive, Shippensburg, PA 17257
2
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Table of Contents
1. Abstract………………………………………………………………………………………………………………………………………………………….P.3
2. Introduction……………………………………………………………………………………………………………………………………………………P.3
2.1 Overview……………………………………………………………………………………………………………………………………………..P.3
2.2 Purpose and Significance…………………………………………………………………………………………………………………..….P.4
3. Background and Literature Review………………………………………………………………………………………………………………….P.5
3.1 Aquatic subterranean fauna…………………………………………………………………………………………………………………P.5
3.2 Subterranean habitats………………………………………………………………………………………………………………………….P.7
3.3 Connectivity to surface sources……………………………………………………………………………………………………………P.8
3.4 Fauna as monitors and tracers……………………………………………………………………………………………………………P.10
3.4.1 Surface water………………………………………………………………………………………………………………………P.11
3.4.2 Groundwater……………………………………………………………………………………………………………………….P.12
3.4.3 Sampling methods……………………………………………………………………………………………………………….P.14
3.4.4 Limitations…………………………………………………………………………………………………………………………..P.15
3.5 Objectives…………………………………………………………………………………………………………………………………………..P.16
4. Study sites and previous research…………………………………………………………………………………………………………………P.17
4.1 Resurgences and possible sources……………………………………………………………………………………………………..P.17
4.2 Welsh Run Cave system……………………………………………………………………………………………………………………..P.19
5. Methods……………………………………………………………………………………………………………………………………………………….P.21
5.1 Data Collection……………………………………………………………………………………………………………………………………P.21
5.1.1 Biotic…………………………………………………………………………………………………………………………………..P.21
5.1.2 Abiotic…………………………………………………………………………………………………………………………………P.23
5.2 Analyses…………………………………………………………………………………………………………………………………………….P.24
6. Results and Discussion………………………………………………………………………………………………………………………………….P.24
6.1 Hydrologic Conditions…………………………………………………………………………………………………………………………P.24
6.2 Feasibility of aquatic organisms as basic tracers………………………………………………………………………………..P.27
7. Conclusions…………………………………………………………………………………………………………………………………………………..P.31
8. References……………………………………………………………………………………………………………………………………………………P.34
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Figures
Figure 1. Stygoxene, stygophile and stygobite……………………………………………………………………………………………………...P.7
Figure 2. Study sites…………………………………………………………………………………………………………………………………………….P.18
Figure 3. Welsh Run Cave system……………………………………………………………………………………………………………………….P.20
Figure 4. Welsh Run Cave stygoxenes…………………………………………………………………………………………………………………P.21
Figure 5. Passive flow sampling method……………………………………………………………………………………………………………..P.22
Figure 6. Sampling location in Welsh Run Cave…………………………………………………………………………………………………..P.22
Figure 7. Dykeman Spring flow model…………………………………………………………………………………………………………………P.25
Figure 8. Average organisms sampled at Welsh Run Cave and Green Spring………………………………………………………P.26
Figure 9. Organisms sampled at Big Spring………………………………………………………………………………………………………….P.27
Figure 10. Organism map for all sites………………………………………………………………………………………………………………….P.28
Figure 11. Green Spring stygobite isopod compared to surface water isopod…………………………………………………….P.31
Appendices
Appendix A. Fauna counts from Welsh Run Cave and Green Spring…………………………………………………………………..P.39
Appendix B. Photographic catalog of Welsh Run Cave and Green Spring organisms………………………………………….P.40
Appendix C. Organism map for Green Spring and potential contributors……………………………………………………………P.41
Appendix D. Organism map for the Welsh Run system……………………………………………………………………………………….P.42
Appendix E. Welsh Run system dye traces..…………………………………………………………………………………………………………P.43
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1.
Abstract
The life of subsurface systems is significantly understudied and largely unknown. Many subterranean systems
are comprised of diverse communities and recently these organisms have been used for monitoring groundwater quality
and for tracing sources of groundwater. By understanding the aquatic communities and possible transport of organisms
within subsurface systems, it may be possible to monitor groundwater quality and pinpoint contributing water sources
by using fauna as basic tracers. This study focuses on four groundwater systems including Dykeman Spring, Big Spring,
Green Spring, Welsh Run Cave and resurgence, and several surface streams in the carbonate region of the Cumberland
Valley in south central Pennsylvania. This study documents subsurface organisms, examines the feasibility of using
subterranean aquatic fauna as basic tracers, and explores whether abundances of fauna are affected by changes in
hydrologic conditions and various water quality parameters. Analysis showed that the presence of organisms varied
dependent on the hydrologic condition of the system. The diffuse system of Dykeman Spring contained no organisms,
the conduit systems of Welsh Run and Green Spring contained many organisms, and the intermediate system of Big
Spring contained very few organisms. The presence of organisms showed no relationship to the subtle fluctuations in
stage, pH, specific conductivity, temperature, or dissolved oxygen possibly due to the continuous baseflow conditions
during the study period. The reference system of Welsh Run revealed two distinct surface contributors to the Welsh
Run Cave shown by diatoms and algae. Green Spring contained diatoms that showed a definite surface water
contributor even during baseflow conditions. Three diatom types were found in the flow from the resurgence, but only
two were found in the flowing surface waters sampled. This suggests that two or more different sources contribute to
Green Spring, however, the definite sources still remain unknown.
2.
Introduction
2.1
Overview
Subterranean ecosystems are intriguing topics of study globally, yet remain significantly understudied.
Groundwater accounts for approximately 97 percent of the Earth’s freshwater (Gibert and Deharveng, 2002) and its life
is largely unknown. Subterranean aquatic ecosystems provide unique opportunities to study evolution, biodiversity,
and food webs of organisms that live in relatively contained and isolated systems. Subterranean systems share
characteristics such as the absence of light, little variation in temperature, limited food resources, high physical
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fragmentation (Gibert and Deharveng, 2002) and inter- and intraspecific competition (Culver, 1976) that result in
specialized organisms. Despite the extreme conditions associated with subterranean life, many subterranean aquatic
systems are inhabited by diverse communities largely comprised of crustacean fauna (Gibert and Deharveng, 2002;
Culver et al., 2000). Studies have focused on organism responses to the stresses of subterranean life including loss of
eyes and pigmentation, morphological changes to appendages, increased longevity, smaller clutch sizes, decreases in
metabolism, etc. (Culver, 1982). Recently, studies have focused on the use of subterranean aquatic fauna for monitoring
water quality by comparing the abundances and diversity of organisms before and after a pollution event (Malard et al.,
1996; Simon and Buikema, 1997; Krejca and Weckerly, 2007). Sampling subsurface aquatic systems may also give
information about the source(s) of water contributing without the use of dye injections from the transportation of
surface water organisms accidentally into groundwater systems (Rouch and Danielopol, 1997; Pipan and Culver, 2007;
Maurice and Bloomfield, 2012). By understanding the aquatic communities and possible transport of organisms within
groundwater systems, it may be possible to monitor groundwater quality and pinpoint plausible contributing water
sources by using fauna as basic tracers.
2.2
Purpose and significance
In order to effectively manage groundwater systems it is important to understand both the hydrology of the
watershed, the status of chemical transport, and recent research suggests that it is equally important to understand the
composition of fauna within a system (Danielopol, 1981; Malard et al., 1996; Simon and Buikema, 1997; Fox et al., 2010).
A consistent supply of clean water in a subterranean system is crucial for the organisms that live there (Culver et al.,
2000), but also to individuals that reside downstream from resurgences. The quality of this water is important from all
surface water sources including conduit flow from sinks and swallets, which are major contributors of toxins (White et
al., 1995), or from more diffuse flow from infiltration via the soil or epikarst. Due to the sensitivities of some
subterranean organisms in certain systems including caves, aquifers, and wells, invertebrates can be used as
groundwater quality monitors. Additional research on poorly studied karst organism ecology will add to the rising field
of identifying and using aquatic subsurface organisms as biomonitors and possibly tracers (Malard et al., 1996; Simon
and Buikema, 1997; Pipan and Culver, 2007; Fox et al., 2010). The purpose of this study is two-fold: (1) the first purpose
is to find if aquatic invertebrates are affected by certain water quality parameters and hydrologic conditions. (2) A
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secondary purpose is to determine the feasibility of using subterranean aquatic fauna as basic tracers for connections to
surface waters. This study may help to understand flow contributors to three resurgences and one cave system in the
Cumberland Valley while documenting organisms that are present and noting their effects to changes in hydrology and
several other water quality parameters.
3.
Background and Literature Review
3.1
Aquatic Subterranean Fauna
Examining the types and determining the effects of hydrologic conditions and water quality on organisms
requires, at minimum, a basic knowledge of their distributions and evolutionary status. The abundances and
biodiversity of subterranean organisms worldwide are high for the amount of stress and specialization needed for
survival, but are comparatively much less than surface ecosystems. Records of cave organisms date back to the
seventeenth century from Croatia and Slovenia when eyeless white salamanders were found washed out of the regions
caves, and were at the time thought to have been the larvae of dragons (Culver et al., 2000). There are approximately
7,000 discovered subterranean species globally (Gibert and Deharveng, 2002). The most diverse subterranean
ecosystems include the Lezsource aquifer in France which contains 32 species (Malard and Gibert, 1997) and the
Dalmatian Cave in Croatia which contains 40 species of troglobites and 40 species of stygobites (Culver, and Sket, 2000).
Deharveng and Bedos suggest that, on average, approximately 20 to 25 subterranean species can be expected to be
present in a cave worldwide (Deharveng and Bedos, 2000). The country with the largest known numbers of
subterranean species is the 48 contiguous United States in which approximately 20 percent of the land area is comprised
of karst bedrock (White et al., 1995; Sket, 1999). There are several small countries, however, such as Slovenia and
Croatia that contain more species per unit of area (Sket, 1999). Surprisingly, compared to temperate regions such as the
Eastern United States, the tropical regions which have many subterranean aquatic systems contain very few species of
subterranean organisms despite its high level of biodiversity at the surface (Culver et al., 2000).
The southeastern United States is one of the most abundant and diverse regions for subterranean organisms
(Master et al., 1998). This karstic region contains diverse physical geography, humid climate, and old lithology which
have all aided in favorable conditions for organisms to reside and evolve over time (Culver et al., 2000). In 1998, Peck
described a total of 1,353 individual species in the United States with epikarst species included (Peck, 1998). In 2000,
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not including epikarst species, Culver described a total of 927 species and 46 subspecies for a total of 973 species from
caves in the 48 contiguous states. In reference to these species, 673 were troglobitic and 300 were stygobitic. The
stygobitic fauna of the United States were geographically concentrated and less than 17 percent of United States
counties in the 48 contiguous states contained even one stygobite or troglobite. The lack of biodiversity in various
counties is likely not a result of inadequate sampling, but is more related to the concentration of caves within the county
or surrounding counties. Culver suggests there is a strong relationship between the abundance of subterranean
organisms in an area and the number of caves in the area. For example, Jackson County, Alabama has almost twice as
many subterranean organisms (Culver et al., 2000) and more than three times as many caves than any other Alabama
county (Moss, 1998) which shows how density of organisms is determined by the density of caves.
There are still many understudied groups such as copepods, oligochaetes (Appendix B), and diplurans as well as
unexplored habitats such as epikarst that would likely change the species counts described by Culver (Culver et al.,
2000). Despite the lack of documentation, in most subterranean systems copepods are the most abundant aquatic
organisms with over 1,000 groundwater species worldwide. Copepods dominate species richness and have colonized
almost all types of habitats in groundwater including caves, aquifers, and epikarst on all continents where surveys for
copepods have taken place (Galassi, 2009). All obligate subsurface organisms have evolved specifically to their occupied
subsurface habitat resulting in many species.
Aquatic fauna of cave systems undergo various evolutionary stages to become truly stygobitic dependent upon
the hydrologic condition of the system. Culver (1976) describes the evolution of subsurface dwellers in four stages. The
evolution is depicted most accurately by studies conducted on cave amphipods and isopods in the Appalachian karst
region (Fleming, 1973; Holsinger, 1967; Steeves, 1969). The first stage is described with species retaining eyes and
pigment (Culver, 1976). These organisms may be described as stygoxenes or stygophiles which can freely transition
from subsurface to surface life or are accidental introductions to a subterranean system (Culver et al., 2000) (Figure 1).
These organisms are said to be directly connected to the surface water source from which they originated. The second
stage occurs when species become restricted from a direct surface connection. This occurs when water tables drop and
species become isolated in caves. In this stage, species display greatly reduced eyes and lack of pigmentation (Culver,
1976) and are described as stygobites which are obligate cave dwellers (Culver et al., 2000). The third stage of evolution
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occurs as organisms increase their range within the subterranean system. As species increase their ranges, regressive
evolution continues and organisms in the third stage are defined as having vestigial eyes and complete loss of
pigmentation (Culver, 1976; Holsinger, 1969; Steeves, 1969). The fourth stage of evolution occurs as an organisms
dispersal abilities decrease due to physical barriers or decrease in dispersal ability (Hack, 1969; Poulson and White,
1969). The decrease in dispersal ability results in speciation often confined to single karst systems (Holsinger, 1969) in
which species have no pigmentation or eyes (Culver, 1976) (Figure 1). By analyzing the evolutionary stage of
subterranean aquatic organisms it may be possible to determine the relative time a species has been subject to
subterranean life, but not an absolute time (Culver, 1976; Holsinger, 1967).
Figure 1: Stygoxene fish from Welsh Run Cave in Franklin County PA that can freely transition between surface and
subsurface aquatic habitat (A). Stygophile Meridon diatoms that require photosynthesis from the Welsh Run
Conococheague Spring that were passively introduced into the subsurface system and are at stage one (B). Many of
these organisms will succumb to subsurface life as would be the case with photosynthesizing organisms such as diatoms
and algae. Stygobite amphipods from Cleversburg Sink in Cumberland County PA that are obligate to subterranean life
and have reached stage four of evolution (C).
3.2
Subterranean habitats
The morphology and physiology of subterranean organisms show different adaptations dependent upon the
system and habitats they utilize. Adaptations include simplifications of body structure and reductions in the complexity
of their appendages (Galassi, 2009). In most cases, morphological adaptations in subterranean systems include the
enlargement of antennae and appendages displaying less complexity, and lack or reduction of pigmentation and eyes
with a general trend of non-constructive adaptations (Mejía-Ortíz and Hartnoll, 2006). The spatial distributions and
morphology of copepods in particular are related to habitat type including sediment size, availability of food, and past
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geologic and climate processes. These factors can result in a significant degree of endemism amongst organisms within
subterranean systems (Galassi, 2009). The organisms found in subterranean ecosystems are site specific often to a
habitat or resource that they utilize.
There are several basic subterranean habitat types that can be occupied by organisms. These habitats include
conduit cave streams (Culver, 1976), shallow cave lakes, aquatic epikarst, and the phreatic zone in which organisms live
in permanent groundwater bodies. The majority of cave streams studied within the Appalachian karst region consist of
riffles and pools (Culver, 1976). The most popular species for the study of subterranean habitats, including amphipods
and isopods, most often prefer riffles to pools (Culver, 1973) as riffle habitats have a high rate of food flow per unit of
cross-sectional area (Culver, 1976). The riffle habitat can be broken down into smaller microhabitats. For example,
amphipods and isopods utilize the undersides of rocks, which are considered microhabitats, within a riffle while avoiding
open areas of the riffle that are subject to higher velocity flow where they are more easily dislodged (Culver, 1976).
Cave streams are among the most popular types of habitats studied by biospeleologists.
A large percentage of subsurface fauna are found in the less commonly studied epikarst habitats, which are
often difficult to sample. Pipan and Culver suggest that epikarst habitats comprise about 20 percent of all aquatic fauna
in any given region. This research found that ceiling drips in caves could be used to sample for aquatic epikarst fauna
and that copepods were the most common organisms originating from the epikarst. Sampling over varying space and
time determined that continuous sampling for three to four months was needed to sample 90 percent of cave species
resulting from one continuous drip, that five continuous cave drips were needed to account for 90 percent of the species
present in a cave, and that five caves were necessary to sample 90 percent of epikarst species in a region (Pipan and
Culver, 2007). This study is an example of how sampling for subterranean fauna can be complex and site specific
dependent upon habitats and access to the systems.
3.3
Connectivity to surface sources
Subterranean system communities are almost all supported by allochthonous surface water or epikarst material.
Food sources in subterranean systems have a strong influence on the evolution and ecology of organisms that live there
(Culver, 1982). Conduit cave stream food sources are primarily dissolved organic matter (DOM) or particulate organic
matter from surface waters rather than from primary production of photosynthesis (Culver, 1985). Cave streams have a
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simple food web with detritus as the primary food source and trophic structures that can consist of collectors,
shredders, scrapers, and in some systems, predators (Simon et al., 2003). Subterranean systems show patterns of loss
in predators due to limited supply of food sources which results in a truncated food web (Gibert and Deharveng, 2002).
Troglobitic organisms can also be sustained by water that leaves residue of POM resulting from water level rise and fall
in the system (Culver et al., 2000). Dependent upon hydrologic input, different surface food sources are transported
into subterranean systems in different ways. Some subsurface systems are provided with food sources from large
openings such as a swallet or sink allowing transportation of coarse particulate organic matter (CPOM) which can sustain
larger or more diverse populations of organisms. Food sources can also enter a system by percolation which allows only
fine particulate organic matter (FPOM) and DOM into the system which may not sustain as large or diverse of
populations (Culver, 1982).
The limitations of food in subterranean systems has effects on the overall biodiversity of a system as well as a
bottom-up control food web in which the availability and abundance of organic material from surface systems supports
the survival of stygobitic organisms (Gibert and Deharveng, 2002; Schneider et al., 2011). In one study, mud from cave
pools was used in a laboratory setting to determine the extent to which the mud was utilized as a food source in a
subterranean habitat by a cave amphipod. The cave mud was found not to support sufficient development in the
laboratory setting. These results suggested that the life in the subterranean system was not supported solely by the
cave mud, but also from leached organic materials entering the systems via cave drips into the muddy cave pools. This
study showed that DOM and FPOM were important contributors to sustaining life in subsurface systems (Dickson, 1979).
Some systems, however, are completely isolated from a surface source of organic material and are solely
supported by chemoautotrophic production (Gibert and Deharveng, 2002). Although chemoautotrophy is an alternative
for systems that have no source of organic material, this source of production is not widespread. There are a limited
number of caves that contain the needed quantity or type of chemicals needed by chemoautotrophs (Sarbu et al., 1996;
Hose et al., 2000). Autochthonous production is so uncommon in subterranean systems, at least those that are known,
there are very few documentations of such systems. The Movile Cave system in Romania contains microorganisms that
display carbon fixation by sulfide oxidation. This system is said to be similar to deep sea vents which also have a
chemoautotrophic food source and significant biodiversity. In the Movile Cave system, 60 percent of the cave organisms
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are sustained by the chemoautotrophic energy base (Sarbu et al., 1996). Other autochthonous chemoautotrophic
systems in subterranean environments include Parker Cave system in Kentucky and the San Marcos Spring artesian wells
in Texas. In one study from the sulfidic Cueva del Azufre cave stream in Tabasco State, Mexico it was determined that
fish and aquatic invertebrates contained gut contents and stable isotope ratios of tissues (13C:12C and 15N:14N) that
indicated that the carbon and nitrogen in their system was derived from chemoautotrophic production within the cave.
This was contrary to fishes in non-sulfidic cave streams which obtained carbon and nitrogen from photosynthetic
allochthonous sources or bat guano and detritus from surface runoff. In the Cueva del Azufre system, sulfur-oxidizing
bacteria are directly consumed by fish. This study is one of the first documentations of vertebrates being sustained by
carbon and nitrogen originating from in system chemoautotrophic production (Roach et al., 2011). These systems are
influenced very little by surface water contributors unlike the majority of subsurface systems which are supported by
allocthonous materials and are often influenced heavily by anthropogenic pollutants.
3.4
Groundwater fauna as monitors and tracers
Subterranean organisms have recently been used as groundwater quality monitors and even tracers for
identifying contributing sources. Pollution to groundwater and its organisms originates from surface sources. These
sources enter the system via percolation and conduit flow (Fox et al., 2010). Organic pollution to groundwater can harm
organisms by either direct toxicity or by disturbing their trophic interactions (Notenboom et al., 1994). Common sources
of groundwater pollution include agricultural runoff, sewage effluent, septic system leachate, sink hole filling leachate,
and legacy contaminants. These sources can contain high levels of nutrients, organic matter, and toxic waste (Kofoid,
1900; White et al., 1995; Simon and Buikema, 1997; Fox et al., 2010). Subsurface aquatic organisms can also be
vulnerable to groundwater drawdown. When the water table drops, aquatic subsurface habitats can disappear, or
dependent upon the system, can result in saltwater intrusion (Culver et al., 2000). Due to the direct connections to
surface systems most often associated with karst, invertebrates in groundwater systems have been used as biomonitors
for subterranean water quality (Malard et al., 1996; Simon and Buikema, 1997; Krejca and Weckerly, 2007). Using
groundwater organisms as biomonitors is a relatively new approach to the field of groundwater monitoring. Organisms
can be used as biomonitors by examining abundances of stygobites compared with those of stygophiles and stygoxenes
in a system, examining abundances of sensitive invertebrates compared to non-sensitive invertebrates, and examining
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densities of organisms that may thrive under polluted conditions (Malard et al., 1996; Simon and Buikema, 1997).
Organisms may also be used as basic tracers by comparing the fauna of different subsurface or surface waters to the
fauna in the system of interest. This method of tracing, however, is complicated in that mechanisms which determine
the distribution of organisms in a karst system are not well understood due to complex hydrologic processes of surface
and subsurface connections and difficulty of sampling (Pipan and Culver, 2007; Maurice and Bloomfield, 2012).
Determining the similarities and differences between surface fauna and different groundwater systems may help to
determine approximate sources of contributing waters (Rouch and Danielopol, 1997).
3.4.1
Surface water
Surface waters that contribute to subsurface systems are far less problematic to analyze in terms of water
quality and connections than groundwater. Connections between surface waters are visible from flowpaths within the
topography or observations, while groundwater flowpaths are largely unknown. Water quality in relation to organisms
is examined in surface streams using several variations of Indexes of Biotic Integrity (IBI’s), while subsurface system
organisms can often be difficult to quantify or even identify. Surface stream IBI’s are statistical tools used to classify the
severity of anthropogenic pollution by examining the health of surface stream communities by evaluating physical
characteristics of habitat, substrate, flow patterns, channel stability, and water chemistry as well as biosurveys of
invertebrates and/or fish (Davis and Simon, 1995; Novotny, 2004; PADEP, 2012). Various IBI’s have been developed for
different types of surface streams, such as limestone versus non-limestone (PADEP, 2012; Botts, 2014), that have
different compositions of biota to allow accurate assessments of the health of populations. There are currently three
types of streams in Pennsylvania that have a unique method to determine the biotic integrity of the system including
true limestone spring streams (PADEP, 2009), low gradient pool streams, and wadeable riffle-run streams (PADEP, 2012).
A healthy surface stream is expected to support balanced populations of organisms (Karr and Dudley, 1981; Davis and
Simon, 1995; Davies and Jackson, 2006), which can be very different from site specific subsurface systems. Organism
composition within subterranean systems, unlike surface streams, are expected to be different at each site (Culver,
1976), and it is therefore difficult to determine universal biological indicators of pollution in subsurface systems.
Biological indicators of wadeable riffle-run surface streams in Pennsylvania often include species of mayflies, stoneflies,
and caddisflies which signify a healthy system. Anthropogenic pollution sources are likely if any of these species are
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absent from such a surface system. In some cases, high abundances of organisms such as Simuliidae or Chironomidae
may indicate polluted waters. Many streams differ in biological composition based on physiochemical, climatological,
and geological factors in which the same IBI’s cannot be used. For example, invertebrate communities within limestone
streams are different from those in freestone streams in that they usually display low diversity and high abundances
which are a result of a relatively constant thermal and flow regime. Limestone streams in Pennsylvania displayed four
taxa that accounted for approximately 75 percent of total organisms collected when developing the limestone stream
IBI including isopods, amphipods, mayflies and Chironomidae (PADEP, 2012; Botts, 2006). Surface water organisms,
however, are often very different than those in site specific subterranean systems and thus biotic integrity indices
cannot be adopted by subsurface systems.
3.4.2
Groundwater
The majority of true subterranean aquatic organisms are more sensitive to pollution than surface organisms. The
partial reason for the high sensitivity to contamination in obligate subsurface fauna is due to their extended longevity
resulting in the bioaccumulation of harmful compounds in their systems (Dickson et al., 1976). Pollution of groundwater
from agriculture and development can also result in the invasion and colonization of organisms that utilize polluted
surface water sources, including coliform bacteria and oligochaete worms, which can outcompete obligate organisms
(Holsinger, 1966; Stein et al., 2010). Various studies are using the presence of certain types of organisms to determine
extent and source of pollution. Danielopol (1981) used the morphology and abundances of subterranean ostracods
compared to the morphology and abundances of surface water ostracods as an indicator for wells that were
contaminated by surface water sources (Danielopol, 1981). A study in Virginia examined the density of stygobitic
crustaceans present in cave pools polluted by septic system effluent from minimal to high pollution levels. All polluted
pools contained high conductivity levels, nutrient concentrations, and fecal coliforms while highly polluted pools in
addition displayed decreased levels of dissolved oxygen. The density of isopods was highest in low and moderately
polluted pools (maximum of 74.6 individuals per meter squared), and absent from highly polluted pools. Amphipods
were completely absent from all levels of polluted pools. This study determined that isopods can utilize septic effluent
for food, but overall the effluent was detrimental to the system (Simon and Buikema, 1997).
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In some cases, a single compound alone may not largely affect populations or communities, but harmful
synergistic relationships can exist among toxic compounds. These relationships, however, can be difficult and expensive
to determine and monitor. In Missouri, several caves were examined for pollution in areas with heavy agricultural land
use to explain the extirpation of the Grotto Cave Sculpin from various systems. This study suggests that one pollution
compound could not explain the extirpation of the species, but rather the combination of several pollution compounds
acted synergistically resulting in detrimental effects on both aquatic vertebrates and invertebrates (Fox et al., 2010).
Additional research on various system effects from different types and concentrations of pollutants may yield certain
ecological criterion that may be used to assess water quality (Stein et al., 2010).
Subterranean fauna composition may also be influenced by other nearby ecosystems especially during
hydrologic events. Several studies have focused on the composition of fauna in the hyporheic zone to monitor
groundwater communities as many shallow subsurface waters are influenced by such systems. The quantity and quality
of recharge from these areas can significantly impact the success and survival of strategist stygobitic fauna (Schmidt and
Hahn, 2012; lepure et al., 2013). Hyporheic communities after flood events have been shown to change in composition
resulting in the decrease or depletion of stygobitic fauna and the colonization of tolerant species such as oligochaetes
and cyclopoids. Stygobite demise after such an event is likely due to their adaptation and dependence on the
consistency of the groundwater environment which suggests that hyporheic communities are sensitive to large
disturbances, such as a flood event (Hancock, 2006), and may be influencing the groundwater community composition
of a nearby system. Unnatural events also affect the composition of hyporheic communities. Contaminated hyporheic
zones are not able to support true stygobitic crustacean communities, whereas pristine hyporheic zones show a
dominance of stygobitic crustaceans. Hyporheic communities that are subject to pollution are often comprised of more
tolerant organisms and areas with higher levels of contamination are often largely comprised of species of cyclopoida
and ostracoda (lepure et al., 2013). Further studies concerning hyporheic zones and the possible contribution to
subterranean systems may consider the investigation of comparing organisms in surface water hyporheic zones to
nearby karst groundwater communities.
Organisms may be used as basic tracers for determining an approximate source of water by comparing
subterranean fauna to other systems. A study from Europe determined that the number of species of crustaceans,
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including cyclopoids, harpacticoids, and ostracods, found in subterranean habitats was similar to the number of species
found in nearby surface waters (Rouch and Danielopol, 1997). Microbial interactions with stygobitic fauna have been
found to indicate possible sources of pollution from specific types of agricultural land use such as irrigated versus nonirrigated (Korbel et al., 2013). In epikarst flow, Pipan and Culver (2007) used 27 copepod species sampled in cave drips
from Slovenia and West Virginia to determine isolated distributions of certain copepod species. The localized
populations of some species resulting from ancestral colonization of copepods showed distinct species being present at
single locations in the epikarst. This study suggested that these copepods and other organisms like them may be used to
trace various flow paths through the epikarst based on their distributions originating from an ancestral surface
population at a single location without the need of chemical tracers (Pipan and Culver, 2007). The concept of using
organisms as tracers, however, is complicated in that mechanisms for stygobite species distributions are not fully
understood because of complex processes within isolated systems that are often difficult to sample and study beyond
one narrow access point (Pipan and Culver, 2007; Maurice and Bloomfield, 2012). Detailed studies of the hydrogeology
of an area can often help to determine possible mechanisms for organism distributions and connectivity of ground water
and surface water. The use of DNA analysis on various organisms may in the future yield valuable information about the
connectivity between aquifers, hydraulic conductivity, and surface water contributors (Maurice and Bloomfield, 2012).
3.4.3 Sampling Methods
Sampling for aquatic subterranean invertebrates is not an exact science and the sampling method used is most
often site specific dependent upon the hydrology and the accessibility of the system. Methods for sampling subsurface
organisms include plankton nets, kick nets, pumps, bailers, trapping, and stalactite drip traps (Pipan and Culver, 2007;
Hose and Lategan, 2012), although these trapping techniques may be modified or new variations of traps can be made
to suit the location where trapping. Plankton nets can be used to catch organisms in larger pools and kick nets may be
used to sample invertebrates within the riffle areas of cave streams. Where groundwater is not accessible via a cave,
bailers and pumps may be used to extract invertebrates. Electric pumps are the most widely used method for sampling
groundwater organisms. Baited traps are used to attract invertebrate species in accessible cave waters (Hose and
Lategan, 2012). Epikarst species are often sampled by stalactite drip traps where invertebrates dispersing through cave
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drips are captured by a funnel net with a container at the end of the funnel (Pipan and Culver, 2007). A single method or
combination of these methods may be used to sample for aquatic invertebrates within a system.
3.4.4
Limitations
Methods for invertebrate monitoring in groundwater systems are site specific dependent upon the biology and
the hydrogeology of the system (Malard et al, 1996). Subterranean systems, even if close in proximity, can have varying
and distinct biodiversity as organisms evolve differently according to habitat when isolated (Culver, 1976). The
evolution of cave fauna can be related to that of islands. Cave systems are erratic and in many cases extremely isolated
which results in the evolution of many different species and subspecies. The characteristics of a system are very
different in terms of quantity and quality of surface flow input, habitat morphology, habitat size, and competing species
which results in the evolution of differing morphological characteristics and survival among fauna. In some cases when
direct flow is cut off, obligate cave species can disperse to other subterranean systems through drips and seep pools
(Holsinger, 1975; Pipan and Culver, 2007) allowing new species to colonize a system and increase competition with
species already present in a system. The difficulty of using species in subsurface systems for monitoring water quality is
that the fauna inhabiting the system may not be an indicator of anthropogenic pollution, but may have been the only
fauna that had access to colonize the system and were successful in developing characteristics needed for survival in
high stress subsurface environments. Culver (1976) states that even if you study one subterranean community, which
can be a model system in itself, you have not understood all systems as each one does not model the same thing
(Culver, 1976). Organisms that inhabit a system are most often not known to have existed with or without pollution.
Therefore, effects of pollution on communities cannot be examined except during a pollution event. This, however, may
be impossible to accomplish in a system that has already been affected by frequent or continuous pollution and has
been colonized by tolerant organisms. In this case, various water quality parameters should be evaluated to quantify
the degree and type of pollution to note what organisms have been able to colonize a system under the polluted
conditions.
Subterranean systems must each be monitored individually to develop standards that cannot be adopted by
other systems as all parameters in subterranean systems are site specific. Due to the site specific variations of
organisms in groundwater systems, it is difficult to develop a standard monitoring system such as the use of biotic
P a g e | 16
indices in surface waters. Surface stream IBI’s cannot be adopted by subsurface systems as organisms common to
groundwater are not even identified as indicators in surface waters such as species of copepods and rotifers. The biotic
indicators of surface streams, which are largely insects, are most often not even present in subterranean systems. Biotic
indices in the future may be developed for groundwater systems, although this may be difficult as a biotic index would
need to be created for each system as organisms within subsurface systems are most often isolated and site specific.
The extinction of obligate and specialized subsurface organisms can also result from, in addition to pollution,
competition with other organisms, population death rates exceeding birth rates, and changes to the hydrologic cycle
(Culver, 1976). The consistency of the hydrologic cycle is critical for subsurface aquatic fauna as it provides clean water,
and most importantly, a consistent supply of food from allochthonous sources. Many caves in the Appalachian region
have direct sources of flow from swallets or sinks. Conduit systems can have strong flows and species that can
withstand the higher velocities of flow may be more successful than others (White and Stellmack, 1965). This suggests
that it may be difficult to determine why certain species inhabit a system as there are many different variables that
affect subsurface organisms including consistency of flow, substrate, degree of connectivity to the surface, and changes
to surface land use.
Determining connections to surface sources by using invertebrates as basic tracers may also be problematic.
The mechanisms for species distributions in karst systems are difficult to determine due to largely unknown complex
hydrologic processes that are often not feasible to sample beyond one access point (Pipan and Culver, 2007; Maurice
and Bloomfield, 2012). Identifying similarities and differences between surface and subsurface specimens may also not
be directly obvious, and identifying organisms to species level is not feasible for this research as specimens would need
to be sent to a taxonomist for identification. This study is among few to use subterranean organisms as tracers. Despite
its challenges, this study will give information about the composition of organisms inhabiting various systems, and
regardless of outcome, is being studied to understand the feasibility of using organisms as basic tracers.
3.5
Objectives
This study will monitor invertebrates and their relationships to water quality, changes in hydrology, and possible
surface water sources at three resurgences including Green Spring, Big Spring, and Dykeman Spring in Cumberland
County and the Welsh Run Cave System in Franklin County. The first objective is to determine if invertebrates are
P a g e | 17
affected by certain water quality parameters and hydrology of the system by measuring invertebrate abundances and
types as well as several water chemistry and hydrologic parameters to find if there is a relationship between the
organisms and the abiotic variables. A second objective is to sample organisms in several surface sources that are
plausible contributors to the resurgences and compare their types and morphology to the organisms sampled from the
resurgences to determine the feasibility of using subsurface invertebrates as basic tracers. The Welsh Run Cave System
will be used as a reference system for using invertebrates as basic tracers as there is access to a known contributing
source to the Welsh Run Cave via a swallet of the Welsh Run Stream, the Welsh Run Cave Stream, and the
Conococheague Creek resurgence where water from the Cave is draining.
4.
Study Sites and Previous Research
4.1
Resurgences and Possible Sources
This study examined a total of three resurgences including Green Spring, Big Spring, and Dykeman Spring,
several surface streams including Burd Run, Branch Creek, Middle Spring Creek, and ‘No Name’ Stream, and one cave
system which includes the Welsh Run Stream, Welsh Run Cave, Welsh Run Conococheague Spring, and Welsh Run Pond
Swallet (Figure 2). Green Spring in Newville, Cumberland County Pennsylvania supplies water to the Green Spring Trout
Hatchery and is of particular interest in this study. Previous data collection in the fall of 2014 showed high abundances
of several invertebrate species emerging from the flow of the resurgence. This resurgence is also known to dramatically
fluctuate in terms of water quality shown by a massive fish kill in early March of 2014. During this event, a total of
approximately 52,000 fingerling and 12,000 adult trout were killed. The dissolved oxygen (DO) levels at the time of the
fish kill were approximately 2 mg/L when, according to the Department of Environmental Protection (DEP), cold water
trout species need a minimum DO concentration of 7 mg/L to survive. The DEP suggests that there were two possible
sources for the contamination including fertilizer application on surrounding fields that was not within regulations, or
effluents released from the Shippensburg Wastewater Treatment plant around the same time as the fish kill. The DEP,
however, found no evidence to support or deny either potential source as a contributor. The DEP is not confident that a
source will be identified as the karst geology of the region results in subterranean streams that can transport
contaminated water for miles (Miller, 2014). Hydrology of Green Spring is a conduit flow system shown by flashy
hydrographs with abrupt rising and falling limbs. Surface sources of water contributing to the resurgence have been
P a g e | 18
speculated, but not confirmed after several attempts were made to trace water using dyes (Lasako, 2009). Surface
streams sampled in this study as possible contributors to Green Spring include Burd Run, Branch Creek, and Middle
Spring Creek below the Shippensburg wastewater treatment plant as well as a small stream flowing past Green Spring.
Figure 2: All sites sampled for organisms including groundwater and surface water sources in Cumberland and Franklin
Counties Pennsylvania.
The Dykeman Spring and Big Spring systems are also located in Cumberland County. Dykeman Spring is a diffuse
flow system (Lindsey, 2004), while Big Spring is an intermediate system between conduit and diffuse flow (Feeney and
Lasako, 2009). Water from Dykeman Spring originates from the colluvial wedge at the base of South Mountain resulting
in diffuse flow. In the past, when this resurgence supplied the borough of Shippensburg, treatment needs were minimal
P a g e | 19
suggesting few water impurities or contamination issues. The complete picture of flow contributors to Big Spring is not
known. In a previous study, Sulpho Rhodamine Blue (SRB) dye was released into a sinkhole collapse in a failed retention
basin 8.9 km west of Big Spring, and Uranine dye was released into a losing portion of the Yellow Breeches Creek 5.2 km
south of Big Spring. The SRB dye was evident in both of Big Spring’s resurgences 3.5 days after release following a
suspected flowpath parallel to the geologic strike. The west spring displayed a clear SRB breakthrough peak and the east
spring displayed a less distinct peak. The differences in breakthrough peaks of SRB showed differences in hydrology
between the west spring, which displayed more conduit flow characteristics, and the east spring, which is the focus of
this study, that displayed more diffuse flow characteristics. The uranine dye was not evident in both west and east Big
Spring, but was detected in the springs of the Huntsdale state fish hatchery one month later 9.5 km away to the east
(Hurd pers. comm., 2015). Sodium naphthionate was also released into a swallet located along Ott Road to the
southwest, but was not detected in Big Spring. The three resurgence sites in this study are ideal as there is differing
hydrology from diffuse flow at Dykeman Spring to conduit flow at Green Spring, and an intermediate between diffuse
and conduit flow at Big Spring. Potential surface sources for invertebrates were not sampled for the Dykeman and Big
Spring sites and the reason is explained in the results.
4.2
Welsh Run Cave System
The Welsh Run Cave system in Franklin County Pennsylvania is ideal for studying invertebrates as tracers as a
direct connection has been determined to an accessible cave stream and a resurgence. This study sampled a total of
three sites at the Welsh Run Cave system including the contributing source of the Welsh Run Stream, the cave stream,
and the resurgence of the cave stream. A qualitative study of the Welsh Run Cave system from 2011 using two dye
traces determined that there are likely several sources of groundwater flow contributing to a single Conococheague
resurgence via adjacent isolated paths (Hurd et al., 2011). Results from a trace using SRB dye released into the Welsh
Run Stream swallet showed that the flow from the swallet was a certain contributor to the water in the cave and a
partial contributor to flow from the Conococheague resurgence. The Tinopal optical brightener released into the sink
did not fluoresce in the cave, however, both the Tinopal and the SRB dye from the swallet were present in the samples
taken from the single resurgence in the Conococheague Creek (Hurd et al., 2011) (Figure 3). This suggests a complex
conduit groundwater system in this area as both dyes exited the groundwater system at the same location, but took
P a g e | 20
different routes to meet at the resurgence. A quantitative study from 2013 also determined that a large quantity of
discharge, but not all, in the Welsh Run Cave is from a sinking portion of the Welsh Run Stream. This research showed
that more than half of the flow in the cave is from unknown sources while the other portion is from the Welsh Run
Stream swallet (Bartle and Feeney unpubl data, 2013). This leaves the possibility for organisms to be present in the cave
that are of an unknown surface water origin. A certain connection with a surface source is evident by the presence of
stygophile fish in the cave stream. In the winter, there are often large schools of fish present in the cave (Figure 4). This
suggests a large resurgence in the Conococheague Creek where fish move uspstream into the cave, or a large swallet
opening where fish can migrate downstream from Welsh Run into the Cave as fish have been observed in both surface
sources. The diverse community and hydrology of this system makes it an ideal location to study transport of organisms
from a surface source and their responses to potential contamination.
Figure 3: Location of the Welsh Run Cave system in Franklin County, Pennsylvania and the dye trace at the Welsh Run
Stream swallet (red) using Sulpho-Rhodamine Blue (SRB) has directly linked flow from Welsh Run (thin blue line) through
the Welsh Run Cave (green) to the resurgence (yellow) in the West Branch Conococheague Creek (WBCC) (thick blue
line) with an approximate flow path (white dashed line).
P a g e | 21
Figure 4: School of fish, largely bluegills, in the Welsh Run Cave Stream (Left), and school of a larger, unknown fish
species utilizing flow through a fracture (Right).
The Welsh Run Cave system is surrounded largely by agricultural land use, and invertebrate communities in the
cave and contributing Welsh Run Stream may be influenced by agricultural contaminants. Research from 2014
examined nitrate concentrations in the Welsh Run Stream source and Conococheague Creek resurgence. This study
determined that nitrate contamination from January through April during various flow conditions did not exceed the
EPA recommended level of 10 mg/L maximum contamination load (MCL). Fluctuations in concentration levels inversely
followed fluctuations in flow levels which suggested that during higher flow, nitrate load is decreased thus decreasing
concentration. By examining other water quality parameters in the current study, effects of invertebrate abundances
may be linked to surrounding land use contamination.
5.
Methods
5.1
Data Collection
5.1.1
Biotic
This study captured invertebrates within flow by use of a passive sampling method. For resurgences, an 80
micron plankton net was placed within the flow, recessed in the bedrock (Figure 5). The net was placed as far into the
bedrock opening of the resurgence as possible to eliminate the possibility for contamination with organisms in the
stream not transported from the groundwater (Figure 5). Passive sampling for invertebrates within the flow took place
for 20 minutes unless otherwise specified. After 20 minutes, the plankton net was removed from the flow and water
collected from the plankton net was stored in a sealed container. Dykeman Spring was problematic to sample using the
passive flow method as flow was not strong enough to force water and the organisms through the plankton net. For this
P a g e | 22
location, a modification of the plankton net was made to capture the maximum amount of flow possible by increasing
the surface area of the plankton net opening to maximize velocity, thus forcing organisms into the net. The cave stream
was sampled by placing the plankton net in the narrowest part of the channel where velocity was greatest to filter the
most water possible at one point (Figure 6).
Figure 5: Sampling method using passive flow of water transporting organisms and filtering them through an 80 micron
plankton net. Any organism larger than 80 microns is captured in the plankton net (A). Plankton net recessed in the
bedrock within the conduit flow of Green Spring (B).
Figure 6: Area of channel in the Welsh Run Cave at the narrowest point where the plankton net was place to sample
passive transport of organisms (A) with scale of the passage shown by a yardstick (B).
Surface streams were sampled to determine types of organisms present and compare them to those present in
the sampled subsurface systems to find the feasibility of using aquatic fauna as basic tracers from surface sources. For
this sampling, the plankton net was placed in a riffle area of the surface stream during low flow conditions. Passive
sampling within the flow of the surface streams took place for 20 minutes, the net removed, and the water sample from
the net stored for examination. All samples from surface waters and groundwater were stored in a cool, dark location
P a g e | 23
and examined within six hours of collection to ensure the examination of live organisms. The plankton nets were
cleaned, rinsed, and dried between each use to avoid cross-contamination of micro-invertebrates between sites.
Water samples from the plankton net were examined under a dissecting microscope at low power in a small
petri dish. Water was pipetted from the sample into the petri dish in small intervals to ensure a lower density of
organisms per dish and a more accurate count of small invertebrates. The dish was separated into four quadrants so
that one entire quadrant was visible in the field of view. All invertebrates were individually counted in each quadrant
and recorded to the lowest taxa possible as identification to species would require sending the organisms to a
taxonomist and was not feasible for this study. Samples of organisms from each site were photographed through the
ocular lens of a compound microscope using the four or ten time magnification powers. Scales were made for each
image by measuring the field of view on the microscope based on the magnification.
5.1.2
Abiotic
At each sampling for invertebrates in the resurgences and the cave, several water chemistry parameters were
measured to find if invertebrates are affected by any changes to these parameters. Temperature, although expected to
remain constant at each site, was measured to note any change as this may be an indicator for a nearby surface source.
Temperature change is also noted in surface systems to affect reproductive activity in species of invertebrates,
especially copepods (Wua et al., 2013; Liu et al., 2014), and thus a change in temperature may result in a change to
invertebrate abundances. Specific conductivity (SpC) was also measured as SpC has been noted to affect certain species
of invertebrates (Bos et al., 1996). Although expected to remain stable, the pH of systems was measured to note
changes in chemistry that may be indicative of a nearby surface contributor. Changes of pH have also been noted to
affect composition of invertebrate fauna in surface streams (Fitzer et al., 2012), and if such changes in pH occur in the
subsurface system the same effects may occur. The DO of each groundwater site was measured to determine, if
fluctuations occur, the effects on invertebrate communities (Roman et al., 1993; Keister et al., 2000). Stage was also
recorded during the time of sampling at each site to find if abundances of invertebrates are affected by changes in flow.
The stage for Green Spring and Welsh Run Cave was recorded from pressure transducers installed at each site for other
groundwater studies. The stage for Big Spring was downloaded from the USGS website as this organization monitors to
flow of Big Spring. The stage at Dykeman Spring was recorded from a staff gage directly downstream of the resurgence.
P a g e | 24
5.2
Analyses
Organism abundances and diversity will be compared to the graphed results of hydrology and water
characteristics including type of flow in the system such as diffuse, conduit, and intermediate conditions, stage, specific
conductivity (SpC), temperature, pH, and dissolved oxygen (DO) to determine if there is any relationship between
abundances and/or diversity with any of these factors. Statistical analyses with this data is not feasible as it is not
possible to collect enough samples for there to be significant power to draw conclusions as identifying and counting
organisms is extremely time consuming and tedious. For using organisms as basic tracers, organism type and
morphology will also be compared between the groundwater system and surface waters to determine the likelihood of
pinpointing definite or at least possible contributors to the groundwater.
6.
Results and Discussion
6.1
Hydrologic Conditions
The abundances and diversity of organisms varied with different hydrologic conditions in terms of a diffuse,
conduit, or an intermediate system with both diffuse and conduit contributors. The diffuse system of Dykeman Spring
contained no organisms on all sampling dates. Due to the lack of organisms the stage, pH, temperature, specific
conductivity (SpC), and dissolved oxygen (DO) could not be used to determine a relationship with the presence and
abundance of organisms. The lack of organisms in this system is expected to be a result of colluvial mantle filtration
(Figure 7). One hypothesis is that as small streams and runoff are lost to the colluvial mantle at the base of South
Mountain, the pore space of the weathered bedrock through which the water infiltrates is unsuitable for sustaining the
populations and transport of subterranean fauna into the theoretical conduit flow under the colluvium which flows to
Dykeman Spring. A second hypothesis is that the pore space of the weathered bedrock of the colluvial mantle and the
underlying fracture system do not easily allow for established organisms to be caught in flow, as runoff slowly infiltrates
through the colluvium and fractures, and transported to the conduit system at greater depths.
P a g e | 25
Figure 7: Theoretical mechanism of flow to Dykeman Spring from the colluvial mantle and suspected underlying conduit
at the base of South Mountain. Arrows depict hypothesized flow direction (modified from Lindsey, 2004).
The conduit systems of Green Spring and Welsh Run Cave contained high abundances and diversity of organisms
on all sampling dates (Appendix A). The organisms in both of these systems consisted mainly of crustacean fauna
(Appendix B) including larval copepods known as nauplius, adult cyclopoid and adult harpacticoid copepods, daphnia,
and ostracods (Figure 8). Freshwater oligochaete worms were the next most abundant organism followed by various
species of rotifers in both systems (Appendix B) (Figure 8). The presence of various types of algae and diatoms varied
likely dependent on surface conditions favorable for blooms (Appendix B). Both algae and diatoms found in any of the
subterranean systems are indicative of a surface water contributor as both types of organisms rely on photosynthesis for
food production. The stage, pH, temperature, SpC, and DO showed no clear relationship with the presence and
abundance of organisms in either system. Long term monitoring over seasons and varying hydrologic conditions should
be done to determine the relationships of flow and other water characteristics including water quality to the presence of
organisms. This study largely took place at baseflow conditions with no significant precipitation events and therefore
may not be an adequate representation of how abundances of organisms may respond to different flow conditions. The
abundances of organisms in these systems are expected to be a result of passive introduction of the organisms via losing
surface waters or from colonization of subterranean organisms that are washed out of the systems.
P a g e | 26
Figure 8: Average number of organisms captured in Welsh Run Cave and Green Spring. The total number of sampling
days for Welsh Run Cave was four; the total number for Green Spring was eight. The brown bars represent crustacean
fauna, and the blue bars show all other fauna types.
The intermediate system of Big Spring contained a total of very few organisms across all sampling dates (Figure
9). Due to the small amount of organisms captured at this site, the stage, pH, temperature, SpC, and DO measurements
could not be used to determine a relationship with the presence and abundance of organisms. As the flow from the
intermediate system of Big Spring originates from a diffuse source at baseflow conditions and surface conduit sources
likely contribute during higher flow conditions, the lack of organisms in this system is expected to be a result of an
inconsistent conduit contributor. The same condition with no organisms due to colluvial mantle filtration, as evident in
Dykeman Spring, is also hypothesized to be the case in the Big Spring system. The inconsistent conduit contributor(s) is
presumed to not facilitate the colonization of organisms as these organisms would not be able to colonize an
intermittent surface contributor with partial dry conditions. The Plecoptera and Tardigrada found in the flow from Big
P a g e | 27
Spring are hypothesized to have come from narrow crevices above the spring covered in moss where these organisms
may also reside. For this reason and the scant amount of organisms sampled from the system, the Big Spring site was
excluded from the tracer analysis.
Figure 9: Organisms captured at Big Spring on four sampling dates.
6.2
Feasibility of using subterranean aquatic fauna as basic tracers
As organisms were not present in Big Spring and Dykeman Spring, these systems were not used for the analysis
of using fauna as basic tracers. Green Spring and the Welsh Run Cave system contained many and diverse organisms
which made these systems ideal for attempting the use of fauna as tracers. The Welsh Run Cave and associated
resurgence from the cave were used as a reference system for using fauna as tracers due to the close proximity and
access to a known surface water source (Figure 10).
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Figure 10: Organisms found at all sampling sites on all sampling dates.
The Welsh Run reference system for using aquatic organisms as basic tracers resulted in several interesting
finds. The Welsh Run Cave contained many obligate surface water organisms including various types of algae, diatoms,
and midge larvae as well as many organisms of unknown origin (Appendix D). An organism with an unknown origin in
this study means that it was unknown whether the specimen was a surface water organism washed into the
groundwater system, or if the organism was truly stygobitic. The known surface water contributor of the Welsh Run
Stream contained Pediastrum, Spirogyra, and Closterium algae (Appendix B). The suspected contributor of the Welsh
Run Pond Swallet contained no identified algae, but contained thousands of Meridon diatom colonies. There also were
thousands of what were named rotifer Type I sampled from the pond water that flowed into the Welsh Run Pond
Swallet. Surprisingly, very few of these rotifers were found in the Cave and resurgence which may suggest, if this water
is in fact contributing to the Cave and resurgence, that these organisms may not be reliable as tracers especially because
this is a relatively short system. There is a possibility that these organisms are very sensitive to transport into
P a g e | 29
groundwater and are easily destroyed during transport. This theory, however, would need to be confirmed with
experimentation using these rotifers.
Despite finding only a handful of rotifers, the Welsh Run Cave contained all types of algae identified in the Welsh
Run Stream and two small sections of Meridon diatom colonies, as were present from the pond swallet, possibly broken
apart by fast flowing conduit water. The Welsh Run Conococheague Spring contained several sections of Meridon
diatom colonies, but no identified algae. As no Meridon diatoms were identified in the Welsh Run surface stream, it has
been hypothesized that the Meridon diatoms present in the Cave and resurgence were from the Welsh Run Pond
swallet (Appendix D). Because more of the Meridon diatoms were found in the Conococheague resurgence than in the
Cave, it is also hypothesized that the water from the pond swallet takes a more direct route to the resurgence than to
the Cave. This was also suggested from the previous dye traces from the Welsh Run Stream and an adjacent sinkhole
with flowing water that may have been originating from the pond swallet (Hurd et al., 2011) (Appendix E). This study
confirms two distinct sources contributing to the cave and the resurgence shown by the algae only found in the known
Welsh Run Stream contributor and the Meridon diatoms not present in the Welsh Run Stream.
Again, the abundances of organisms in the Welsh Run and Green Spring conduit systems are hypothesized to be
a result of either direct transport of the organisms into the systems via losing surface water sources, or the past
colonization of the organisms in the subterranean systems that are maintained by the transport of allochthonous
surface POM and DOM. The former hypothesis is expected for the Welsh Run Cave system as surface water sources,
with one confirmed contributor, are close in proximity with the furthest source in speculation approximately one mile
away. The latter hypothesis may be probable for the Green Spring system as surface water sources are greater
distances (approximately six miles away) from the resurgence, and all nearby potential sources at the times of sampling
were not flowing. Note that this study did not sample nearby ponds or lagoons that may have an influence on Green
Spring. The contributors and subterranean characteristics of the Green Spring system are unknown except for what is
monitored at the resurgence. There is a possibility that this system contains a cave system of riffles and pools that are
ideal for the colonization and evolution of subterranean fauna.
Green Spring contained a few obligate surface water organisms including various types of diatoms as well as
many organisms of unknown origin (Appendix C). Sampling at Green Spring revealed several types of diatoms in the
P a g e | 30
months of March and April which show a connection to an unknown surface water source or sources. The lack of these
organisms from samples in the months of January and February suggest the diatoms were not in bloom as a result of
colder surface water temperatures or fewer daylight hours that were not favorable for photosynthesis. A total of three
diatom types were found in the groundwater of the Green Spring resurgence including Melosira, Tabellaria, and
Meridon (Appendix B). The Melosira and Tabellaria diatoms were also found on the same sampling dates to be present
in Burd Run, Branch Creek, and Middle Spring Creek approximately six miles away and No Name Stream directly across
from Green Spring (Appendix C). The presence of these diatoms in large numbers in the sampled surface streams,
however, cannot confirm a connection of these streams to Green Spring. Interestingly, the Meridon diatoms sampled
directly from Green Spring were not found in the Burd Run, Branch Creek, or Middle Spring Creek surface waters.
Meridon diatoms were only found in the No Name Stream. Due to the large abundances of Meridon diatoms sampled
from the Welsh Run Pond Swallet and lack of these diatoms in the flowing Welsh Run surface stream, it is hypothesized
that these diatoms bloom in still waters such as a pond, wetland, or possibly a lagoon. Based on this hypothesis, a losing
pond or wetland which may be continuously losing as a result of being spring fed, or a losing lagoon may be contributing
to the Green Spring groundwater as well as to No Name Stream. This hypothesis, however, would need to be supported
by sampling nearby ponds, wetlands, and lagoons that are up gradient of Green Spring. All diatoms are made of silica
forming a protective shell called a frustule and are expected to be robust to transport over long distances. Regardless of
not knowing exact contributors of water to Green Spring, it can be suggested that even if the proposed contributors of
Burd Run, Branch Creek, and Middle Spring Creek are supplying water to Green Spring, that there is also another source
shown by the presence of Meridon diatoms in the resurgence that were not found in the said surface waters (Appendix
C).
The single midge larvae found in the Green Spring groundwater does not necessarily suggest a surface water
source even though midge larvae are most often associated with surface waters. Midge larvae can also be present in
moist soils (Hurd and Stewart pers. comm, 2015) therefore, there is a possibility that this larvae may have originated
from soil and epikarst areas anywhere above the subterranean conduit stream of Green Spring and cannot be a definite
identifier as a surface water source. There were also thousands of Type 1 rotifers as were present in the Welsh Run
Pond swallet water in Burd Run, Branch Creek, and Middle Spring Creek with only one such rotifer found in Green
P a g e | 31
Spring. As was the case in the Welsh Run system, these rotifers may be unreliable as tracers. The copepods in the
Green Spring system were not confirmed as being stygobitic or from surface water origin although other evidence
suggests that some organisms have colonized the Green Spring system and have become obligate. This was shown by
the presence of stygobitic unidentified isopods displaying no pigment or eyes (Figure 11). This evidence, however, does
not rule out the possibility of the other organisms found from the resurgence originating from a surface water source as
the constant supply of water to the resurgence remains unknown.
Figure 11: Lateral view of a stygobitic isopod found from Green Spring displaying no eyes or pigmentation (the dark
appearance of the isopod is the result of the dilution of light from the microscope) (A), compared to a non-stygobitic
isopod with eyes and pigmentation (dorsal view) sampled from a surface water source (B).
7.
Conclusions
Sampling for aquatic organisms is time consuming and it is often difficult to determine the taxonomy of
organisms beyond order and family. The sampling of organisms in this study to determine if they are affected by
changes in hydrologic condition and various water characteristics showed no effect as hydrologic conditions throughout
most of the study were at baseflow conditions with very few precipitation events to alter hydrologic condition.
Determining effects that fluctuation in hydrology has on the abundances and diversity of organisms would need to be
monitored in a system long term. The importance of monitoring the types and abundances of organisms within a
P a g e | 32
system would be related to studying water quality perhaps for a location or budget where it is not feasible to do in
depth water quality tests. Pioneer studies using invertebrates may help for future monitoring of water quality when
certain subsurface invertebrates are identified as indicators, as they are for measuring integrity of surface streams
(Danielopol, 1981; Malard et al., 1996; Simon and Buikema, 1997; Fox et al., 2010). To determine sensitivities of
invertebrates related to fluctuations in hydrology and water quality, it is important to do an overall inventory of the
system seasonally as abundances and types of invertebrates may change dependent on flow within the system or the
influence that a contributing surface source may have in introducing organisms into groundwater flow. After organisms
present are identified, it is important in terms of monitoring water quality to identify how the abundances and diversity
of the organisms in the system change in relation to a known pollution event (Malard et al., 1996). This would require a
before-pollution state to be observed as well as an after-pollution state. After the pollution event occurs, the organisms
can continue to be monitored to determine recovery rate and if other organisms begin to colonize the system
(Danielopol, 1981; Simon and Buikema, 1997; Hancock, 2006). This must be done on a system by system basis for
groundwater as not all systems contain the same organisms depending on what organisms were able to colonize the
system (Culver, 1976; Malard et al., 1996). This is contrary to surface systems that have standard biotic indices of
integrity (IBI’s) dependent on the type of surface stream being analyzed. Future studies should examine the use of
invertebrates as groundwater quality monitors in caves, resurgences, wells, etc. long term and provide baselines of
aquatic subterranean organisms present, especially in systems where pollution events have the possibility to occur or
have occurred in the past.
The feasibility of using aquatic organisms as basic tracers proved to be somewhat effective depending on the
system for determining surface water contributors. The Welsh Run Cave system showed two distinct sources by
sampling groundwater and surface water. Diatoms and algae were crucial in determining the two sources of this system
which will in the future be confirmed with dye traces. The Green Spring system was less clear in terms of specific
surface contributors, but showed direct surface connections by the presence of obligate photosynthesizing surface
water organisms. The photosynthetic diatoms present in the system were found during baseflow conditions suggesting
a permanent flowing source is contributing to Green Spring and that the diatoms present at the end of sampling were a
result of seasonal surface conditions favorable for blooms. The diatoms found in Green Spring also suggest that there is
P a g e | 33
more than one source contributing to the flow as one diatom type was not identified in any of the sampled surface
streams that were suspected contributors. Future research on this system will sample nearby ponds and possibly
lagoons to determine if the diatom type not found in the flowing surface sources is present in still surface waters as was
the case in the Welsh Run system. The lack of algae found in Green Spring suggests that the surface contributor may be
long distance and the algae was degraded, whereas the surface water diatoms, which are all composed of a silica shell
known as a frustule, were more robust and able to be transported through the system. This suggests that the length of
the system may be critical in the feasibility of using organisms as tracers. This study showed that using organisms as
basic tracers to identify surface water sources can be used for determining a specific source as was the case in the Welsh
Run system, or as the case with Green Spring can be used to confirm surface water contributors during baseflow
conditions when only permanent surface water sources are flowing. Future sampling should be continued on the Green
Spring system on a regular basis to find seasonal trends in the presence and types of organisms and how they compare
with the types of organisms in various surface waters. Using organisms as basic tracers may be feasible for a system,
especially if the system is conduit, but cannot be determined until sampling is done for the groundwater and
surrounding surface waters. Traces using dyes can often be difficult as coloring surface water sources and possibly
domestic well water is a risk. Using organisms as basic tracers may be an alternative for determining sources or may at
least be a way to narrow down the possibilities of potential surface contributors in which dyes can be used.
P a g e | 34
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Appendix A: Fauna counts from Welsh Run Cave and Green Spring on all sampling dates. Green Spring separated into
two figures to show large numbers of nauplii on a separate graph from other fauna.
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Appendix B: All organisms sampled in groundwater and surface water sources. Scales are shown in millimeters. Color
dots on images show locations where captured: Green, Green Spring; Blue, Welsh Run Cave; Purple, Welsh Run
Conococheague Spring; Orange, potential Green Spring surface water sources; Red, Welsh Run Cave surface water
sources.
A, B, Nauplius; C,D, Harpacticoid copepod; E,F, Cyclopoid
copepod; G, gravid cyclopoid copepod; H, Daphniidae; I,
Ostracoda; J, stygobite isopod; K, surface water isopod; L,
surface water amphipod; M,N, Type 1 rotifer; O,
unknown; P, Type 2 rotifer; Q, Type 3 rotifer; R, Type 4
rotifer; S, Oligochaeta; T, Tardigrada; U, Nematoda; V,
Gastropoda; W, X, Diptera larvae; Y, Carchesium; Z,
Plecoptera; AA, Spirogyra; BB, Meridon; CC, Tabellaria;
DD, Closterium; EE, Pediastrum; FF, Melosira
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Appendix C: Organisms sampled at Green Spring and possible contributors including Burd Run, Branch Creek, and
Middle Spring Creek. Although not a suspected contributor, No Name Stream was sampled to identify organisms in the
only stream that continuously flows in the vicinity of Green Spring. Organisms circled in green show diatoms, which are
obligate surface organisms, sampled from Green Spring as well as Burd Run, Branch Creek, and Middle Spring Creek.
The diatom circled in blue was only found in Green Spring and No Name Stream.
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Appendix D: Organisms sampled in Welsh Run Cave and the Welsh Run Conococheague Spring as well as the known
contributor of the Welsh Run Stream Swallet and the suspected contributor of the Welsh Run Pond Swallet. Organisms
circled in red show algae, which are obligate surface organisms, sampled from the Welsh Run Cave and the Welsh Run
Stream. The diatom circled in orange was found in the Welsh Run Cave, Welsh Run Conococheague Spring, and Welsh
Run Pond Swallet. Note that algae was not sampled from the pond swallet and the diatom colonies from the pond
swallet were not found in the Welsh Run Stream.
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Appendix E: Welsh Run System dye trace from the Welsh Run Stream swallet using Sulpho Rhodamine Blue (SRB) dye
which showed the stream swallet water contributed to the Cave and to the resurgence (blue line). Dye trace from the
sinkhole adjacent to the Cave using Tinopal optical brightener which was shown to contribute only to the resurgence
(black line). The proposed contribution of water from the Pond Swallet is shown by the dashed orange line.