Geochemistry and Geomicrobiology of Black Manganese Oxide Cave Deposits in Monroe and Greenbrier Counties, West Virginia

Home , Birnessite, Greenbrier Group

GEOCHEMISTRY AND GEOMICROBIOLOGY OF BLACK MANGANESE OXIDE CAVE DEPOSITS IN MONROE AND GREENBRIER COUNTIES, WEST VIRGINIA

A Thesis Presented to The Graduate Faculty of The University of Akron

In Partial Fulfillment of the Requirements for the Degree Master of Science

Alex T. Dalla Piazza August, 2016 GEOCHEMISTRY AND GEOMICROBIOLOGY OF BLACK MANGANESE OXIDE CAVE DEPOSITS IN MONROE AND GREENBRIER COUNTIES, WEST VIRGINIA

Alex T. Dalla Piazza

Thesis

Approved: Accepted:

______Advisor Interim Dean of the College Dr. Ira D. Sasowsky Dr. John Green

______Faculty Reader Dean of the Graduate School Dr. James McManus Dr. Chand Midha

______Faculty Reader Date Dr. John M. Senko

______Department Chair Dr. James McManus

ii ABSTRACT

The process and distribution of manganese oxide deposition was analyzed throughout Scott Hollow and Maxwelton Sink Caves in Monroe and Greenbrier Counties, West Virginia to (1) explain why manganese oxide is preferentially depositing in caves, (2) determine what locations are ideal within caves for deposition, (3) determine what mineral form of manganese oxide is depositing, and (4) determine if manganese oxidizing bacteria are present within the caves. Collectively, 48 water samples, 16 oxide coated lithic samples, and 10 microbiological samples were analyzed. Water samples were processed utilizing ion chromatography, atomic absorption spectroscopy, and titration. Water data were additionally processed in PHREEQC Version 3 to analyze the thermodynamic favorability of birnessite precipitation in cave waters. PHREEQC was also utilized to calculate charge balance errors to ensure an accurate dataset. X-ray diffraction (XRD) and environmental scanning electron microscopy (ESEM) analyses were carried out on pulverized oxide coating samples to determine the mineralogy of the coatings. Cave bacteria were grown on K-medium to determine the presence of manganese oxidizing bacteria. Furthermore, manganese deposits were categorized and mapped along sections of the main stream passage of both caves. Leucoberbelin blue (LBB), a redox indicator, was utilized to confirm the presence of both manganese oxidizing bacteria and manganese oxide deposits. Water analyses revealed that physical and chemical parameters showed little variability along the cave stream passages. PHREEQC analyses determined that the cave iii waters were supersaturated in respect to birnessite. XRD and ESEM analyses show the mineral form of manganese oxide deposited within the caves is likely birnessite. Bacteria grown on K-medium were determined to be manganese oxidizing bacteria. Bacteria were found growing on surface and cave sample plates as well as on both plates grown in the light and dark. The presence of manganese oxidizing bacteria was confirmed by testing the colonies with LBB. Manganese deposit mapping shows manganese oxide deposition occurs throughout the surveyed cave passages. It appears that manganese oxide deposition occurs almost everywhere along the cave stream passages. Birnessite was found to be the mineral phase of manganese oxide present in both cave systems and was determined to be supersaturated in the waters of both caves with saturation indices ranging from 7.08 to 10.78. Manganese oxidizing bacteria were present throughout the cave stream passages and are believed to be catalyzing manganese oxide deposition. From visual observations, manganese oxide deposition preferentially occurs in areas of turbulent stream flow, and upon siliceous substrates.

iv ACKNOWLEDGEMENTS

I would like to first thank my thesis advisor, Dr. Ira D. Sasowsky, for the guidance, encouragement, and countless hours spent assisting this research. I would not be where I am today without your help. I also would like to thank my thesis committee members Dr. John Senko and Dr. James McManus for their assistance in the completion of this project. Furthermore, I would like to extend my gratitude to Research Associate Tom Quick for always taking time out of his busy day to help me with all of my laboratory analyses. Thank you also to Annie Hartwell for her assistance with the ICP and analyzing the corresponding data. Thank you to Dr. Sarah Carmichael of Appalachian State University for the assistance with the Leucoberbelin Blue (LBB) recipe, as this was essential to my research study. I would also like to thank Michael Dore for the use of Scott Hollow Cave and the multiple hours spent guiding our research team through the cave passages. This research would have been impossible without you. Furthermore, thank you to Dave Socky for the guidance in Maxwelton Sink Cave. Thank you to the West Virginia Cave Conservancy (WVCC) for permission to conduct research within Maxwelton Sink Cave and thank you to the West Virginia Association for Cave Studies (WVACS) for the use of your field house during our research trips. To my field and laboratory assistants; Zachary Strong, Hunter Campbell, Kelsey Budahn, Shagun Sharma, and Nick Wander, thank you for sacrificing multiple weekends, as I could not have accomplished all I set out to do without your help. Thank you also to Paul Krasner for his GIS expertise and overall input on the maps within this project and

v to my fellow graduate students for making graduate school a memorable experience I will never forget. I would like to extend a special thank you to the Hall family for opening up their home to me as I set out to begin my professional career. I would also like to thank Nicole Hall for all of her love and support, even when it seemed like there was no end in sight. Finally, I would like to thank my grandparents, parents, brother, and sister for instilling the hard-working attitude I have built my academic foundation upon and for always pushing me to be the best I can be, not only in school but in life. Without your support and encouragement none of this would have been possible.

vi TABLE OF CONTENTS Page LIST OF FIGURES ��x LIST OF TABLES �� xii CHAPTER I. INTRODUCTION ��1 1.1 Scientific Importance of Cave Research ��1 1.2 Study Overview ��2 1.3 Background Geology ��2 1.4 Background of Studied Caves ��5 1.5 Geochemistry of Manganese ��8 1.6 Geomicrobiology of Manganese Precipitation ��9 1.7 Manganese Coated Sediments ��12 1.8 Hypothesis ��14

II. METHODS ��16 2.1 Site Selection ��16 2.2 Field Methods ��19 2.2.1 Mapping of Manganese Oxide Deposits...... 19 2.2.2 Quantification of Steam Discharge...... 22 2.2.3 Collection of Geochemical Field Parameters...... 22 2.2.4 Collection of Water Samples...... 23 2.2.5 Collection of Microbiological Samples...... 24 2.2.6 Collection of Oxide Coating Samples...... 25

vii 2.3 Laboratory Analyses ��25 2.3.1 Stream Water Alkalinity Titrations...... 26 2.3.2 Stream Water Dissolved Anions...... 26 2.3.3 Stream Water Dissolved Cations/Metals...... 27 2.3.4 Manganese Oxidizing Bacteria Culturing...... 28 2.3.5 Manganese Coating Analysis...... 30 2.4 Streamwater Geochemical Modeling and Charge Balance Calculation ��31

III. RESULTS ��33 3.1 Scott Hollow Cave ��33 3.1.1 Manganese Oxide Distribution...... 33 3.1.2 Stream Discharge...... 37 3.1.3 Geochemical Analysis ...... 39 3.1.4 Microbiological Analysis...... 43 3.1.5 Manganese Coating SEM/EDAX and XRD Analysis ...... 46 3.2 Maxwelton Sink Cave ��46 3.2.1 Manganese Oxide Distribution...... 46 3.2.2 Geochemical Analysis...... 51 3.2.3 Microbiological Analysis...... 55 3.2.4 Manganese Coating SEM/EDAX and XRD Analysis ...... 55

IV. DISCUSSION ��61 4.1 Manganese Oxide Distribution ��61 4.2 Geochemistry ��62 4.3 Microbiology ��63 4.4 Manganese Coating Mineralogy ��68

V. CONCLUSIONS ��71 REFERENCES ��73

viii APPENDICES ��78 APPENDIX A - LABORATORY EQUIPMENT ��79 APPENDIX B - CAVE PHOTOGRAPHS ��87 APPENDIX C - WEATHER DATA ��96 APPENDIX D - DISCHARGE DATA ��100 APPENDIX E - STREAMWATER PHYSICAL AND CHEMICAL DATA ��105 APPENDIX F - PHREEQC DATA ��111 APPENDIX G - XRD DIFFRACTOGRAMS ��112

ix LIST OF FIGURES Figure Page 1 Map of West Virginia Counties, with the study area highlighted, showing locations of Scott Hollow and Maxwelton Sink Caves in Monroe and Greenbrier counties 3 2 Photograph of karst landscape displaying a massive sinkhole in Pocahontas County, West Virginia 4 3 Landform diagram displaying the various features associated with karst topography, such as sinkholes, caves, and springs 6 4 Stratigraphic column depicting the limestones and shales of the Greenbrier Group 7 5 Eh/pH diagram showing the stability of manganese solids and dissolved manganese at standard temperature and pressure 10 6 Photograph of steam pebbles coated in manganese oxide on top of an eroded manganese oxide coated limestone cave ledge 13 7 Map showing sampling location of Mastodon Avenue infeeder stream (SH13) in Scott Hollow Cave 17 8 Map showing sampling locations along Mystic River in Scott Hollow Cave 18 9 Map showing sampling location of the Heaven Passage infeeder stream (MX1) in Maxwelton Sink Cave 20 10 Map showing sampling locations along Cove Creek in Maxwelton Sink Cave 21 11 Map of various types of Mn deposits observed along and in Mystic River of Scott Hollow Cave 35 12 Photographs of the various types of manganese deposits mapped 36 13 Photograph of a manganese oxide crust, confirmed by the LBB spot test 38 14 Dissolved oxygen, conductivity, pH, and alkalinity measurements taken along Mystic River and infeeder streams 40 15 Photograph showing colonies of manganese oxidizing bacteria grown on K-media 44

x 16 Diffractogram of manganese coating sample taken from SH12 48 17 Map of various types of Mn deposits observed along and in Cove Creek of Maxwelton Sink Cave 50 18 Dissolved oxygen, conductivity, pH, and alkalinity measurements taken along Cove Creek and Heaven Passage infeeder stream 52 19 Photograph showing colonies of manganese oxidizing bacteria grown on K-media 56 20 Diffractogram of manganese coating sample taken from MX1 60 21 Histogram of average physical and chemical parameters between Scott Hollow and Maxwelton Sink Cave measured in November 2015 64 22 Histogram of average physical and chemical parameters between Scott Hollow and Maxwelton Sink Cave measured in February 2016 65 23 Histogram of surface and subsurface colony forming units (log CFU/g) from Scott Hollow Cave and Maxwelton Sink Cave 67 24 ESEM image of an oxide coating from site SH4 in Scott Hollow Cave 69 25 ESEM image of an oxide coating from site MX4 in Maxwelton Sink Cave 70

xi LIST OF TABLES Table Page 1 K - Medium recipe for Mn(II)-oxidizing bacteria, modified from Templeton et al., 2005 29 2 Date, activity, and surface conditions during Scott Hollow research trips 34 3 Saturation index of birnessite in Scott Hollow Cave 42 4 Enumerations of plated cave and surface bacteria samples from Scott Hollow Cave 45 5 Percent elemental weights of Scott Hollow Cave manganese oxide crust/coating samples 47 6 Date, activity, and surface conditions during Maxwelton Sink research trips 49 7 Saturation index of birnessite in Maxwelton Sink Cave 54 8 Enumerations of plated cave and surface bacteria samples from Maxwelton Sink Cave 57 9 Percent elemental weights of Maxwelton Sink Cave manganese oxide crust/coating samples 59

xii CHAPTER I INTRODUCTION

1.1 Scientific Importance of Cave Research

Cave and karst research is important in a broad range of scientific disciplines, such as in the fields of archeology, chemistry, geology, hydrology, microbiology and paleoclimatology (Kambesis, 2007). With approximately one fourth of Earth’s landscape composed of karst, hundreds of millions of people in these regions depend on karst aquifers for their drinking water supply (Doerfliger et al., 1999). Research in the areas of water quality, groundwater pollution, and biological integrity (Poulston, 1991) sheds light on the vulnerability of these karst aquifers. Research on iron and manganese oxidizing bacteria, bacteria that are known to be found in many cave systems (Spilde et al., 2005), is beneficial in the remediation of acid coal mine drainage systems (Akcil and Koldas, 2006). Additionally, these iron and manganese oxidizing bacteria may also have water treatment implications in the case of metal contaminated water (Tani et al., 2012). Paleoclimatology research involves the use of cave sediments and speleothems to recreate the climate of the Late Quaternary period, the age of the last ice age, in an effort to model climate evolution (Bar-Matthews et al., 1997). Research in the petroleum industry has found that collapsed carbonate cave systems may act as a reservoir for oil and gas (Graven, 2016). The research presented within this study focuses primarily in the fields of chemistry, geology, hydrology and microbiology.

1 1.2 Study Overview

Manganese oxide coatings, although found in surface streams, seem quite more common in limestone caves (Sasowsky, personal communication, 2015). As precipitation continues, cave stream sediments can become cemented together in a process that is hypothesized to be mediated by microbial activity (Carmichael et al., 2013; Northup et al., 2000; Tebo et al., 2005). The specific locations where this precipitation takes place and what conditions make caves ideal for this precipitation to occur, have not previously been explored and will be the scope of this study. The conclusions drawn from this study will help to (1) explain why manganese oxide deposition is preferentially occurring in caves, (2) determine what locations are ideal for manganese oxide mineral precipitation to occur and if photochemistry is a contributing factor to this precipitation, (3) identify the mineral form of the manganese oxide precipitate, and (4) determine if manganese oxidizing bacteria are present in the caves. Scott Hollow and Maxwelton Sink caves in Monroe and Greenbrier Counties, West Virginia (Figure 1) were selected as research locations based upon access, extensive underground stream segments, availability of cave maps, and observable black manganese oxide deposition.

1.3 Background Geology

The study area is situated in the southeastern part of the state, in the Appalachian Plateau. These counties are underlain by sedimentary deposits ranging in age from Paleozoic to Quaternary (Reger and Price, 1926; Dasher et al., 2012). The terrain is mountainous and rugged (Figure 2). Long periods of fluvial erosion have created dendritic river systems through the mature plateau, with rivers flowing into the Ohio 2 Figure 1: Map of West Virginia Counties, with the study area highlighted, showing locations of Scott Hollow and Maxwelton Sink Caves in Monroe and Greenbrier counties. Base data from West Virginia Geological and Economic Survey, 2009.

3 Figure 2: Photograph of karst landscape displaying a massive sinkhole in Pocahontas County, West Virginia. Courtesy of Ira D. Sasowsky.

4 River to the west or north (Dasher et al., 2012). Deposits of alluvium, limestone, sandstone and shale are common in this region (Price and Heck, 1939; Dasher et al., 2012). The study area exhibits karst topography (Figure 3) and is dominated by the Greenbrier Group, a Mississippian age (350-340 million years ago), pure calcium, marine limestone group that was deposited in a shallow ocean basin. The Greenbrier Group (Figure 4) trends East-Northeast (ENE) along east-central West Virginia, ranging in thickness from 305 meters to 122 meters (Jones and Tudek, 2012). It conformably overlies the Maccrady Shale (Price and Heck, 1939; McCue et al., 1939), a shale series that acts as an aquiclude (Jones and Tudek, 2012). Above the Maccrady Shale is the Hillsdale Limestone, the bottom formation of the Greenbrier Group. The Hillsdale Limestone is overlain by the Sinks Grove Limestone, followed by the Patton Limestone, the Taggard Formation, the Pickaway Limestone, the Union Limestone, the Greenville Shale, and the Alderson Limestone at the top of the section. The Lillydale Shale overlies the Alderson Limestone and represents the beginning of the Mauch Chunk Group. Structurally, this region was exposed to tremendous stresses coming from the east and south east during the Alleghenian orogeny, creating joints that trend northeast and southwest (Price and Heck, 1939; Jones and Tudek, 2012). Sinkholes and cave systems are found throughout the study area due to the dissolution of limestone.

1.4 Background of Studied Caves

Scott Hollow Cave, located in Monroe County, West Virginia, is a dendritic conduit system that follows the contact of the Hillsdale formation with the underlying Maccrady Shale (Dore, 1995). The Mystic River cave passage within Scott Hollow cave is approximately 15 meters high and 12 meters wide, and is about 8 kilometers long. The 5 Figure 3: Landform diagram displaying the various features associated with karst topography, such as sinkholes, caves, and Figure 3: Landform diagram displaying the various features associated with karst topography, springs. Figure from Kenny and Hayward, 2009.

6 7 Lillydale (Mauch Chunk Shale Group) 240 Alderson Limestone Legend 210 Greenville Shale Massive Limestone

180 Union Bedded limestone Limestone

Shaley limestone 150 Bioclastic Limestone

120 Pickaway Bedded Chert Limestone

Sandy limestone Thickness in meters

90 Group Greenbrier

Taggard Cherty limestone Formation

Oolitic limestone 60 Patton Limestone Shale

30 Sinks Grove Limestone Pickaway Joints

Hillsdale Limestone 0 Maccrady Shale

Figure 4: Stratigraphic column depicting the limestones and shales of the Greenbrier Group. FromFigure Shank 1-3: (2002)Generalized after Heller stratigraphic (1980). column of the Greenbrier Group Carbonates in southern Greenbrier County (After Heller, 1980). 7 overall gradient of Mystic River was calculated to be 8.7 meters/kilometer (Davis, 1999). Breakdown boulders in Mystic River are described to be as large as “house sized” and are found throughout the length of the passage (Dore, 1995). The cave has multiple infeeder streams, such as the Middle Earth stream and North-South stream, which eventually flow into Mystic River. Maxwelton Sink Cave is located in Greenbrier County, West Virginia near the Greenbrier Valley Airport (Handley and Pearson, 2012). The cave is approximately 16 kilometers in length and is about 95 meters deep. Cove Creek, the main stream within the cave, is located northeast from the entrance. The calculated gradient of Cove Creek is 30 meters/kilometer. The Cove Creek passage ranges in height from approximately 1 meter in the upstream passage section to 15 meters in the downstream section. Passage widths range from 3 meters to up to about 7 meters. The downstream section of the Cove Creek passage has multiple waterfalls and larger rooms (Handley and Pearson, 2012).

1.5 Geochemistry of Manganese

Manganese is the 10th most abundant element in the Earth’s crust (Post, 1999) and is widely found in sediments, soils, groundwater, and biological specimens. The chemistries of manganese and iron are quite similar, as both metals undergo reduction- oxidation (redox) processes in weathering environments (Hem, 1985). The behavior of manganese is also geochemically similar to magnesium, cobalt, and nickel (Post, 1999). Manganese may substitute for iron, magnesium, and calcium in silicates such as

rhondonite, MnSiO3, and in carbonates, such as rhodochrosite, MnCO3 (Hem, 1985). In stream water, manganese may come from igneous and metamorphic rocks, such as mafic silicates like basalt, manganese carbonates, and from minerals such as olivine, amphibole and pyroxene (Hem, 1985; Tebo et al., 2005). In this study it is likely that the manganese 8 is from limestone or dolomite (Hem, 1985), as igneous and metamorphic rocks are not found in the headwaters of this study region. Near the surface of the Earth, manganese is easily oxidized (Post, 1999). As manganese becomes depleted from rocks by surface water interactions, it takes the highly mobile form of Mn2+ (Post, 1999; Hem, 1985). Manganese can also have oxidation states of 3+ and 4+ (Post, 1999; Hem, 1985). At a pH of 10.5 and above, manganese takes the principal hydroxide form of MnOH+ (Hem, 1985). The form manganese takes in natural waters follow the Eh/pH (Pourbaix) diagram (Figure 5). Low Eh values are representative of reducing environments and high Eh values represent oxidizing environments. Manganese, although only a minor chemical component of fresh and marine waters, plays an important role in aquatic geochemical cycles through the form of oxides (Bratina et al., 1998). The surface area and charge distribution of manganese oxides allow adsorption, making them a reservoir for metal cations (Bratina et al., 1998), as well as electron acceptors for bacterial respiration (Tebo et al., 2005). When these oxides become reduced and soluble, they release the adsorbed metal cations (Bratina et al., 1998). Microorganisms may play a major role in the geochemical cycle of manganese as well as the adsorbed metal cations (Bratina et al., 1998).

1.6 Geomicrobiology of Manganese Precipitation

The formation of manganese oxide minerals, in general, was originally thought to be controlled mainly by abiotic processes (Carmichael et al., 2013; Barton and Northup, 2007). More recent research, however, has led to a new hypothesis that includes microbial mediation as a catalyst in the formation of manganese oxides (Carmichael et al., 2013; Northup et al., 2000). When Mn (II) oxidation occurs at a rate too high to be explained by abiotic processes alone, microbial mediation is inferred (Tebo et al., 2005). 9 1.2

1.0 Water Oxidized

0.8

0.6 MnO2(s)

0.4 V) ( Mn(II) h

E 0.2

0.0

-0.2 MnCO3(s)

-0.4 Water Reduced -0.6 2 4 6 8 10 pH

Figure 5: Eh/pH diagram showing the stability of manganese solids and dissolved manganese at standard temperature and pressure. Redrawn from Klinchuch and Delfino, 2000.

10 The rate of manganese mineralization is accelerated with the presence of manganese oxidizing bacteria. While microbially mediated oxidation of Mn(II) is observed to be widespread, it is not well understood why these bacteria oxidize Mn(II) (Tebo et al., 2005). Microbial activity in caves from microorganisms such as algae, bacteria, archaea, fungi, protozoa, and viruses, aids in the formation of various different minerals (Northup and Boston, 2005). Manganese oxidizing bacteria also lead to the dissolution of carbonate material in caves and the formation of the punk rock underneath, as these reactions can produce acidity (Northup and Boston, 2005). Factors such as seasonality, temperature, and pH may play an important role in abiotic and biotic manganese oxidation. Rapid abiotic oxidation of manganese occurs when pH is above 9.0 with water temperatures near 0 degrees Celsius (Hem, 1963; Johnson et al., 1995). The oxidation rate of manganese is strongly temperature dependent, with lower temperatures more favorable (Hem, 1985). At higher temperatures, abiotic oxidation should be slow but can be increased by oxidizing bacteria (Johnson et al., 1995). Optimal water temperature for active manganese oxidizing bacteria is approximately 20 degrees Celsius (Johnson et al., 1995; Tebo, 1985). In has been suggested that if water temperatures dip below 19 degrees Celsius, microbially mediated manganese oxide precipitation becomes slow, even with high concentrations of dissolved oxygen (Johnson et al., 1995), however, this may not be the case in all situations. Common methods of analysis to determine the presence of manganese oxidizing bacteria include culturing and scanning electron microscopy (SEM). A culturing experiment by Parchert et al. (2012) was conducted by placing rock fragments into four types of ampicillin containing media. Each day the plates were sub-cultured to produce the purest possible cultures. These cultures were then examined for manganese

11 oxides using light microscopy. Supporting evidence was found that fungi play a role in manganese oxide formation (Parchert et al., 2012). Photochemistry may also play a role in manganese oxide precipitation. Research has shown that manganese oxide precipitation may be enhanced in the presence of light (Hansel and Francis, 2006). Contrary to this, more recent studies have shown non-photochemical precipitation of manganese oxides may lead over photochemical precipitation (Learman et al., 2011). Dissolution rates of manganese oxide have also been found to be 6 to 70 times higher in the light compared to in the dark (Sunda and Huntsman, 1994).

1.7 Manganese Coated Sediments

Manganese coated sediments (Figure 6) are found in both surface streams (Hem, 1985) and in cave streams (White, 2007). In a study by Carpenter and Hayes (1978), ceramic plates were placed in a surface stream both upstream and downstream from the Magruder Mine located in Lincoln County, Georgia. Cu, Pb, Au, and Ag were extracted from this mine prior to 1938. After a 36 day period, the ceramic plates were found to have brown to black speckled manganese oxide precipitate, with the most precipitation occurring on the downstream ceramic plate closest to the mine. Robinson (1981) found manganese and iron coatings on surface stream cobbles and stream alluvium on samples collected from the Ridge and Valley Province in Virginia. It was believed these coatings form “at an interface between oxidizing and reducing conditions such as where groundwater percolates into a streambed” (Robinson, 1981). Once precipitation of these oxide coatings occur, it creates a catalytic effect that enhances further oxide precipitation as long as there is a supply of dissolved oxygen and Mn(II) (Crerar and Barnes, 1974; Hem, 1978; Morris and Bale, 1979). As stated by Robinson (1981) and supported by 12 Figure 6: Photograph of steam pebbles coated in manganese oxide on top of an eroded manganese oxide coated limestone cave ledge.

13 Carpenter and Hayes (1980), oxide coatings can be removed by sediment burial, abrasive processes, the breaking of larger deposits into smaller deposits, and by biological erosion. The mineralogy of various manganese deposits was studied by Potter and Rossman (1979). They compared desert varnish, manganese dendrites, river deposits, and cave deposits. They found the desert varnish to be composed of birnessite, (Na,Ca)

Mn7O14 · 2.8 H2O, and clay minerals, and their manganese dendrite samples to be

4+ 3+ composed of the manganese mineral romanechite, (Ba,H2O)2 (Mn ,Mn )5O10. River and stream samples were taken from the streambed, splash zone, and from stream alluvium and were composed mainly of birnessite. They also found that cave deposits closely resemble river deposits and are also mainly composed of birnessite. In caves, manganese is found deposited on similar substrates to those in surface streams. White et al. (2009) found coatings on stream cobbles, chert ledges, and other silica substrates. They examined the mineral form of manganese oxide forming samples taken from caves such as Jewel Cave in South Dakota to Matt’s Black Cave in West Virginia. The most common mineral form found was birnessite, the same form found in surface streams. Additional studies by Moore (1981) and Kashima (1983) support this conclusion. Not all black coatings found in caves are manganese oxides however, as some may be organic matter that has been carbonized (Hill, 1982).

1.8 Hypothesis

I hypothesize that black manganese oxide deposition is occurring along cave streams with observably higher gradients and higher turbulence, such as in areas where water emerges from cave breakdown or around waterfalls, and on a preferred substrate. I also propose that manganese oxidizing bacteria are present along the cave stream passages and are aiding in manganese oxide deposition. 14 The hypothesis will be evaluated by identifying and assessing the locations in select cave streams where the deposition of manganese is occurring. It will be determined if deposition is occurring in areas of higher gradient through visual observations and extensive cave mapping of manganese oxide deposits. The composition of the substrate the oxide coatings have deposited on will be assessed. Microbiological samples will be taken throughout the cave passages to be cultured for manganese oxidizing bacteria.

The literature shows that birnessite, (Na,Ca)Mn7O14 · 2.8 H2O, is typically the mineral form of manganese oxide found on cave fluvial sediments (Hill and Forti, 1997) and this will be tested for in the surveyed cave stream passages. Birnessite is structurally

composed of MnO6 octahedra (Post, 1999). Another common form of manganese oxide

4+ 3+ found is todorokite, (Ca,Na,K)(Mn ,Mn )6 O12 · 3.5 H2O. Todorokite is composed of triple chained MnO6 tunnel structures (Post, 1999). The mineral form can be confirmed through X-ray diffraction (Post, 1999). If deposition is found to be occurring in areas observed to be of higher gradient, along with the presence of manganese oxidizing bacteria, this will support the hypothesis. Should this hypothesis not be supported, re-framing of the hypothesis will be needed and additional research would need to be completed to deduce why the precipitation of manganese oxide occurs in certain locations, what makes the specific location ideal for mineral precipitation, what mineral form is precipitating, and if microbial mediation is catalyzing this precipitation.

15 CHAPTER II METHODS

2.1 Site Selection

Potential research sites were identified through observations made by previous cave explorers and from previous research pertaining to manganese oxide precipitation. From this information, Monroe and Greenbrier Counties in West Virginia were selected as the main area of interest to conduct this research. A reconnaissance trip in August 2015 was used to examine multiple caves within these counties. Scott Hollow Cave, Maxwelton Sink Cave, Matt’s Black Cave, and Breakdown Cave were examined for lengthy stream passages, presence of black oxide crusts/coatings, accessibility, and feasibility of conducting research in order to assess the hypothesis. Ultimately, Scott Hollow Cave and Maxwelton Sink Cave were selected as the study sites. Sampling location selection within each cave stream passage took place during the first sampling trip in November 2015, with the intent to resample the exact marked locations in future trips. Prior to the trip, it was determined that a maximum of 15 locations per cave would be examined in order to provide broad coverage while keeping data at a manageable size. Locations were selected within each stream passage based on criteria such as an observable change in stream gradient, change in stream turbidity, presence of black oxide crusts and or coatings, distance from previous location, and presence of infeeder streams that could contribute to a change in water chemistry. Thirteen locations were selected in the Scott Hollow Cave system (Figure 7 and Figure 8)

16 SH13

Figure 7: Map showing sampling location of Mastodon Avenue infeeder stream (SH13) in Scott Hollow Cave. Sampling locations SH1-SH12 are within the boxed area and are shown in Figure 8. Base map from Dore, 1995

17 Figure 8: Map showing sampling locations along Mystic River in Scott Hollow Cave. Base map from Dore, 1995. flow is to the right (North).

18 and 12 sites within Maxwelton Sink Cave system (Figure 9 and Figure 10). Each location was marked using either flagging tape or pin flags, and labeled with an alphanumeric code and date. Locations were labeled in sampling sequence from SH1 to SH13 in Scott Hollow Cave and MX1 to MX12 in Maxwelton Sink Cave. One surface soil sample was also taken near each cave entrance for biological analysis.

2.2 Field Methods

All field measurements were recorded in feet in order to be consistent with the cave maps that are scaled in feet. These measurements have been converted to metric throughout this study and are shown as such in tables. All samples were labeled following the same naming convention; Cave abbreviation, followed by sampling location and ending with the sampling trip. Scott Hollow was abbreviated to SH and Maxwelton Sink to MX. Sample locations were designated by numbers 1 through 13 and sampling trips were labeled either A or B for November 2015 and February 2016, respectively. For example, a sample taken in Scott Hollow Cave in November 2015 at location 4 would read: SH4-A.

2.2.1 Mapping of Manganese Oxide Deposits Manganese oxide deposits, in the form of coatings and cemented sediments, were mapped along the main stream passage of each cave. The presence of oxide coatings and manganese cemented sediment was confirmed using a 0.04 % solution of leucoberbelin blue (LBB), a colorimetric dye that reacts with Mn(III) and Mn (IV), following the methods of previous studies such as Krumbein and Altmann (1973), Templeton et al. (2005), and Carmichael et al. (2013). The 0.04% solution of LBB was made by taking 0.02 g of Sigma Aldrich leucoberbelin blue (CAS# 52748-86-5) and adding 0.5 mL of 19 MX1

Figure 9: Map showing sampling location of the Heaven Passage infeeder stream (MX1) in Maxwelton Sink Cave. Sampling locations MX2-MX12 are within the boxed area and are shown in Figure 10. Base map from Handley and Pearson, 2012.

20 Figure 10: Map showing sampling locations along Cove Creek in Maxwelton Sink Cave. Base map from Handley and Pearson, 2012. River flow is to the left (Southwest).

21 acetic acid. The final volume was brought to 50 mL by adding de-ionized water. The mixture was filtered using a sterile 0.20 µm filter. The categories of mapped deposits were decided upon based on observable differences in precipitation location and color during the first sampling trip and are described in detail in the results section.

2.2.2 Quantification of Steam Discharge Channel cross sections were measured and velocity readings were taken in select areas of Mystic River in Scott Hollow using a meter stick, measuring tape, and flow meter in order to calculate stream discharge. A FP101 – FP201 Global Flow Probe was used to record stream velocity. Due to complications, the probe was not used at site SH9. Instead, velocity was calculated using the float method. A small piece of paper was timed using a stopwatch over a distance of ten feet. Velocity and stream geomorphology measurements were taken during both sampling trips to assess variations and discharge was calculated.

2.2.3 Collection of Geochemical Field Parameters Dissolved oxygen, pH, temperature, and conductivity field meters were brought to each sampling location to record these parameters. Dissolved oxygen, pH, temperature, and conductivity were measured from a beaker full of stream sample. The beaker was rinsed with fresh sample before each parameter was taken to minimize cross contamination from field meter probes. Dissolved oxygen was recorded using aVWR Symphony H10P meter and VWR electrode 89231-608. Temperature and pH were measured using a Cole Palmer pH meter, model 59002-00 and the conductivity was measured using an Oakton Model CON 11 (s/n 1371046) conductivity meter. All meters were calibrated immediately before taking samples at the first sampling location and recalibrated every four hours. The pH meter was calibrated using both the pH 7 buffer

22 solution and the pH 4 buffer solution. The dissolved oxygen probe was plugged in the night prior to sampling to allow for polarization of the probe. Procedures of Sasowsky and Dalton (2005) were followed to accurately measure pH. Observations along with date and time were recorded at each location.

2.2.4 Collection of Water Samples A total of 30 sample kits were prepared, 15 for each cave. 13 were used in Scott Hollow and 12 in Maxwelton Sink, leaving 7 extra sample kits in case of error or other unforeseeable circumstance. Each bag contained five sterile 50 mL VWR polypropylene centrifuge tubes with printed graduations and flat caps (number 89039-656).Two centrifuge tubes were labeled for alkalinity samples, one for an anion sample, one for a metal/cation sample and one in case it was determined a biological sample was needed. Metals/cations centrifuge tubes were filled with 50 mL of filtered sample, using a 20 mL soft-jet sterile syringe attached with a 25 mm VWR syringe filter with 0.45 µm cellulose acetate membrane. Five drops of ultra-pure nitric acid were added to the 50 mL metal/ cation centrifuge tube after the filtered water sample was added in order to keep metals dissolved until transport to the laboratory for further analysis. Initially, the ultra-pure nitric acid was added to the metals/cations centrifuge tube prior to water sampling. This proved to be problematic, as the acid melted the centrifuge tube, causing a last minute change in the sampling procedure. Centrifuge tubes for collection of anion analysis were filled with a minimum of 10 mL of filtered sample. The amount of anion sample collected was dependent on the performance of the filter, as suspended sediment in turbid sections of stream clogged the filters and prevented a larger volume of sample. For alkalinity samples, 100 mL of unfiltered sample were collected using two 50 mL centrifuge tubes from each site. Alkalinity samples were filled to the top of the centrifuge tube to prevent

23 degassing of the sample during transport to the laboratory. Samples were stored in a cooler during transport to the laboratory and stored in a refrigerator upon arrival. In the Scott Hollow system, the 13 samples were collected beginning at the sump, labeled SH1, and ending above the double waterfall, SH11. Eleven samples were taken from the main stream passage, Mystic River (Figure 8). The remaining three samples, SH6, SH9, and SH13, were collected from the infeeder streams that flow into the main stream passage. SH6 was collected from Craig’s Creek and both SH9 and SH13 were collected from the Middle Earth stream. SH9 was collected where the Middle Earth stream discharges into Mystic River (Figure 8), while SH13 was taken near the cave entrance (Figure 7). In the Maxwelton Sink system, 10 samples were collected during the first sampling trip and 12 samples were collected during the second trip. Sampling began at MX1 (Figure 9), the Heaven’s passage infeeder stream. The first sample in the main stream passage, Cove Creek, was taken furthest upstream at MX7, with the furthest downstream sample taken below the second waterfall at MX12 (Figure 10). The sample naming convention that was previously explained was applied throughout both sampling trips for continuity purposes.

2.2.5 Collection of Microbiological Samples A total of ten biological samples were collected, with four samples taken in Scott Hollow and four samples taken in Maxwelton Sink Cave. Samples consisted of oxide coating scrapings and attached sediment. Samples were taken at sites SH1, SH6, SH9, and SH11 in Scott Hollow (Figure 8) and at sites MX3, MX5, MX8, and MX10 in Maxwelton Sink (Figure 10). A surface soil sample was taken around the entrance of each cave system, accounting for the final two samples. Samples were collected by sanitizing a metal spatula with ethanol and allowing the spatula to dry. Sample was then collected and

24 placed in a 50 mL centrifuge tube. These samples were stored in a cooler for transport and placed in the laboratory refrigerator until needed for analysis.

2.2.6 Collection of Oxide Coating Samples Suspected manganese oxide coatings were collected in November 2015 throughout the Scott Hollow and Maxwelton Sink cave systems. Samples were taken along Mystic River in Scott Hollow Cave at sites SH3, SH4, SH6, SH7, SH8, SH9, and SH12, and along Cove Creek in Maxwelton Sink Cave at sites MX1, MX2, MX3, MX4, MX5, MX7, MX9, and MX10. Samples were collected by scraping oxide coatings along with the accompanying substrate using a metal spatula. Samples were stored in sterile 50 mL VWR polypropylene centrifuge tubes with printed graduations and flat caps (number 89039-656). The presence of manganese was confirmed by the LBB test, as positive samples are indicated by the LBB turning dark blue when in contact with manganese oxide. The mineral form present, birnessite or todorokite, was determined through additional laboratory analysis as described below.

2.3 Laboratory Analyses

All laboratory analyses were completed at the University of Akron, and took place in the geochemistry, chemistry, and geomicrobiology laboratories over the course of several months. Laboratory work was conducted immediately upon returning from the field to ensure the most accurate data possible. All samples were kept under refrigeration while not in use, for preservation purposes.

25 2.3.1 Stream Water Alkalinity Titrations To calculate the proton accepting capacity of the samples, alkalinity titrations were performed using the setup in Appendix A (Figure A1). Twenty six total water samples from Scott Hollow Cave (13 from trip A and 13 from trip B) and 22 total water samples from Maxwelton Sink Cave (10 from trip A and 12 from trip B) were analyzed. 25 mL of sample was measured using a volumetric pipette and placed into a 50 mL glass beaker. A known volume of 0.01639N H2SO4 (sulfuric acid), a strong acid, was slowly added via burette to the sample at 20° Celsius until a pH of 4.5 was reached. This endpoint indicates that the bicarbonate/carbonate species has been completely converted to carbonic acid. To monitor the pH, an Oakton 35620-Series pH/mV/°C meter with a Cole-Parmer 5992-40 GG3 electrode was placed into the sample being titrated. During titration, the sample was stirred using a controllable magnetic stirring device with magnetic stir bar. Glass beakers and stir bars were cleaned using DI water between each titration to prevent cross contamination of samples. Initial and final volumes of sulfuric acid were recorded. Alkalinity was then calculated by dividing 1000 by the sample volume (25 mL) and multiplying by the final volume Vf( ) minus the initial volume (Vi) of sulfuric acid. Calculated alkalinity values will be used to look for any trends in the Scott Hollow/ Mystic River and Maxwelton Sink/Cove Creek cave stream systems.

2.3.2 Stream Water Dissolved Anions

- 2- - Samples were analyzed for major anions, Cl , SO4 , and NO3 using a Dionex (DX 120) ion chromatograph. Software used for the analyses was Thermo Scientific’s Dionex Chromeleon 7 version 7.2.2.6394. Samples were placed into glass 0.5 mL vials via syringe and run against a master standard of chloride, sulfide, nitrate, and phosphate. 10 mL of the master standard was put into 200 mL, 100 mL, 50 mL, and 25 mL volumetric flasks with DI water to create a working set of standards with the concentrations of 100

26 mg/L, 40 mg/L, 20 mg/L, 10 mg/L, and 5 mg/L. The master standard consists of a 1000 mg/L of each chloride, sulfide, nitrate, and phosphate, and was prepared by laboratory associate Thomas Quick. Dionex Seven Anion Standard Check was also analyzed against the unknown samples for quality assurance and a blank was analyzed last to assure that the water used to make up the working set of standards did not contribute to the ions in the unknown samples. Scott Hollow Cave and Maxwelton Sink Cave samples were run separately due to limitations in laboratory equipment scheduling.

2.3.3 Stream Water Dissolved Cations/Metals Samples were also analyzed for Ca, Mg, Na, K, Fe and Mn using an Atomic Absorption Spectrometer AAnalyst 700 (Appendix A). Samples were analyzed for Fe and Mn against standard curves of 0.05, 0.10, 0.25, 0.50, and 1.0 mg/L. Ca and Mg samples were diluted to 1/5 to account for concentrations that might exceed the detection limit of the Atomic Absorption Spectrometer. The dilution was accomplished by pipetting 10 mL of the sample in to a 50 mL volumetric flask and then filling it with 40 mL of DI water. 10 mL of this diluted sample was placed in a 100 mL beaker. 1 mL of lanthanum chloride was added to prevent the sample from ionizing. These diluted and acidified samples were run against standard curves of 1, 2, 5, 10, 20, and 40 mg/L. Values were multiplied by a factor of 5 to calculate the actual value of Ca and Mg in the sample. Samples analyzed for K and Na were not diluted and were run against standard curves of 1, 2, 5, 10, 20, and 40 mg/L. Additional processing of select Mn and Fe samples was completed as a quality control/quality assurance to ensure low concentrations were measured accurately. Five samples from Scott Hollow Cave and five samples from Maxwelton Sink Cave were selected from the November 2015 sampling trip. MX 1 – 5A and SH 1 – 5A were selected and run on an Agilent Technologies 700 Series ICP-OES and processed using ICP Expert

27 II Agilent 710-ES Version 1.1 instrument software in the University of Akron’s chemistry laboratory located in Knight Hall. Samples MX2 and SH2 were run in duplicate to ensure standardization curves were correct. Fe samples were run against a wavelength of 238.204 nm and Mn samples were run against a wavelength of 257.610 nm.

2.3.4 Manganese Oxidizing Bacteria Culturing To identify possible microbial influence, samples of crusts and coatings were analyzed for manganese oxidizing bacteria. Four cave samples (SH1, SH6, SH9, and SH11) and one surface sample from Scott Hollow Cave and four cave samples (MX3, MX5, MX8, and MX10) and one surface sample from Maxwelton Sink Cave were analyzed. Sampling for manganese oxidizing bacteria enumerations was completed using methods similar to those of Templeton et al., 2005 to determine bacteria presence and abundance between the surface and subsurface. Rock scrapings were tested with a 0.04% solution of LBB to confirm the presence of manganese oxide. Positive samples were cultured using K-medium (Table 1) to grow the oxidizing bacteria. The medium was prepared by mixing the constituents in Table 1 in three 2 liter flasks and stirring with a magnetic stirrer on low to avoid producing air-bubbles. One flask was mixed with all the ingredients except the noble agar and poured into 78 five mL VWR, disposable, 16 x 125 mm, borosilicate glass culture tubes to be used for dilutions. The media and dilution mixtures were sealed with foil or caps, taped with autoclave tape, and placed in the autoclave for 15 minutes to kill any bacteria. The mixture was then poured onto 78 sterile plates to solidify into the medium. Each plate was poured with 15 mL of medium. Four samples, MX10, SH1, and both surface samples, were diluted out to 10-8, with each dilution sampled in duplicate and grown in both light and dark conditions. 0.50 g of sediment was spread per 5.0 mL of suspension medium to quantify the bacteria and compare cave and surface bacterial differences. The plates were incubated for two weeks

28 Table 1. K - Media recipe for Mn(II)-oxidizing bacteria, Tablemodified 1: K from - Medium Templeton recipe et for al., Mn(II)-oxidizing 2005. bacteria, modified from Templeton et al., 2005.

Media Compostion Carbon Source MnCl2 100% Deionized water 2 g/L Peptone 1 mM 20 mM HEPES (pH 7.5) 0.5 g/L Yeast Extract 15 g/L Noble agar

29 and again tested with LBB to confirm the presence of manganese oxide.This method is similar to the one outlined by Parchert et al. (2012). Colonies were enumerated as colony forming units per gram (CFU/g) by staff of the University of Akron geomicrobiology lab run by Dr. Senko. Only manganese oxidizing bacteria colonies were enumerated. The remaining samples, SH6, SH9, SH11, MX3, MX5, and MX10 were plated only to detect the presence of manganese oxidizing bacteria.

2.3.5 Manganese Coating Analysis Sediment samples and oxide coatings were analyzed using laboratory techniques such as X-ray diffraction (XRD) and the environmental scanning electron microscope (ESEM). An attempt was made to isolate pure coatings from the substrate, but this was unsuccessful as the substrate and coating remained attached. The only samples that can be quantified as a pure coating are MX3, MX5, and MX9. The laboratory techniques required samples to be in powdered form. To accomplish this, samples were first dried in a drying oven at 105° Celsius for 36 hours to remove all water. Prior to placing sediment and oxide coating samples into the drying oven, detailed descriptions and observations were noted. Samples were placed into aluminum sample boats and dried at 65° Celsius for two and a half days. Dried samples were then ground using a porcelain mortar and pestle. Samples that were unable to be ground using the mortar and pestle were pulverized in a SPEX rock mill (Appendix A), powered by an Emerson Electric S60AAW-6118 1/3 horsepower motor. Sample was placed into a tungsten lined aluminum canister with two tungsten-carbide mixing balls and placed in the SPEX mill for ten minutes. All usable powder was stored in plastic bags labeled with sample name and trip collected (Appendix A). Any rock fragments too large to be powdered were removed. The canister and tungsten-carbide balls were cleaned with hot water, scrubbed with a brush,

30 and dried with acetone between the powdering of each different sample to prevent cross contamination. X-ray powder diffraction was used to determine the mineral phases present in the samples. Powdered samples were packed into Rigaku 900005 X-ray slides with a 2.0 mm indent and analyzed with the Rigaku Ultima IV X-ray Diffractometer (Appendix A) from 5 degrees to 70 degrees 2-theta. CuK-alpha radiation was used with a 1 minute scan speed per degree. Rigaku PDXL modeling software version 2.0.3.0 was then used to match curves and determine the mineral phases present in each sample. Petrographic analyses of the sediment and oxide coating samples were accomplished through the use of SEM microscopy. The environmental scanning electron microscope (ESEM) with energy dispersive x-ray (EDX) can shed insight into crystal structure and composition of sample in addition to what has been determined through XRD analysis. Samples were analyzed with an FEI Quanta 200 Environmental Scanning Electron Microscope (Appendix A) running in low vacuum mode. In addition to imaging, energy dispersive x-ray (EDX) was performed using the EDAX Inc. attachment to calculate weight percent of the elements present in each coating sample. Double-sided carbon tape was used to mount the powdered sample which was then placed under the microscope. Due to the use of the carbon mounting tape, elemental weight percentages had to be normalized by removing the carbon percentages and then recalculating the remaining elemental percentages.

2.4 Streamwater Geochemical Modeling and Charge Balance Calculation

Cation, anion, and alkalinity data from both Scott Hollow and Maxwelton Sink were analyzed using water resources groundwater software from the United States Geological Survey (USGS). The data were input into PHREEQC Version 3 (Charlton and Parkhurst, 31 2011; Parkhurst and Appelo, 2013) to perform geochemical calculations. Redox potential of both cave streams were calculated within PHREEEQC using the O(0)/O(-2) redox couple, as Eh was not measured in the field. Saturation indices, molality, molarity, and the charge balance error were then calculated to determine the potential for birnessite to precipitate. Data were run using the WATEQ4F database (Ball and Nordstrom 1991) as birnessite is not present in the PHREEQC database. Percent charge balance error (%CBE) was calculated using the PHREEQC software.

32 CHAPTER III RESULTS

3.1 Scott Hollow Cave

Photographs taken during sampling in Scott Hollow Cave can be viewed in Appendix B. Trip dates, sampling conditions, and activities are listed in Table 2. Weather data from the months in which sampling took place are in Appendix C.

3.1.1 Manganese Oxide Distribution The distribution of black manganese oxides were mapped throughout approximately 600 meters of the Mystic River passage in Scott Hollow Cave (Figure 11). Deposits were broken into three categories based on color, thickness, and substrate and labeled as; cemented cobbles and pebbles, eroded coatings, and fine grained coatings.The absence of an observable manganese deposit was mapped as bedrock. Cemented cobbles and pebbles (Figure 12A) were the darkest of all deposits and thickest from visual observation, although this was not measured. These deposits were also the hardest and could only be broken apart with the use of a rock hammer. Only coarse grained material was found to be cemented together in these deposits. The cemented cobbles and pebbles were black in color, had an observably smooth appearance, and were only located along the stream banks or in the stream. These deposits were found around waterfalls and in areas where water of higher turbulence emerges from breakdown and on siliceous material.

33 F ° F, No precipitation F, F, No precipitation F, ° ° Trace snow, 29 Trace Surface Conditions Sunny, 85 Sunny, Cloudy, 54 Cloudy, Activity mapping of Mn deposits mapping Preliminary research survey research Preliminary 5 bio samples, 13 water samples, samples, 13 water 5 bio samples, coating samples, 4 discharge measurments. 4 discharge samples, coating 13 water samples, 4 discharge measurements, 4 discharge samples, 13 water Date 17 August 2015 17 August 13 February 2016 13 February 20 Novemeber 2015 20 Novemeber B A Trip Reconnaissance Table 2: Date, activity, and surface conditions during Scott Hollow research trips. 2: Date, activity, Table

34 Figure 11: Map of various types Mn deposits observed along and in Mystic River Scott Hollow Cave. Mapping Figure 11: took place during the February 2016 sampling trip. Base map from Dore, 1995.

35 Figure 12: Photographs of the various types of manganese deposits mapped. Photograph A) is representative of cemented cobbles and pebbles, B) is an eroded coating, and C) is a fine grained coating. Photographs courtesy of Ira D. Sasowsky and Hunter J. Campbell.

36 Eroded deposits (Figure 12B) ranged in color from light brown to dark brown and appear to have been eroded and thus named accordingly. These deposits could be scraped off the substrate it had deposited on using a metal spatula. Eroded deposits were found almost everywhere throughout the Mystic River cave passage. These deposits were most commonly above the water’s edge, coating the limestone cave walls and cave breakdown up to heights of approximately 3 meters. These deposits were also observed in the stream channel on silicious material. Fine grained coatings (Figure 12C) were light brown with interbedded black manganese oxide and clay sediments. These deposits were soft, as one could easily push a finger through the coatings, and were only composed of fine grained sediments. Fine grained coatings were observed near infeeder streams, where these streams deposit fine grained sediments. Fine grained coatings are also found along the main stream passage, as they wash downstream from an infeeder stream. Bedrock was mapped as areas where the stream was flowing on top of rock with no manganese oxide deposits present. The mapped manganese oxide deposits were confirmed as such using the leucoberbelin blue (LBB) spot test (Figure 13).

3.1.2 Stream Discharge Stream discharge varied between sampling trips, likely due to weather variability on the surface. Higher discharge was observed during the November 2015 sampling trip compared to the February 2016 sampling trip (Appendix D). Discharge was calculated to be 1.03 m3/s at SH1 (sump) in November 2015 compared to 0.87 m3/s in February 2016. Upstream at SH8, November 2015 discharge was calculated to be 0.87 m3/s compared to 0.60 m3/s in February 2016. This discrepancy is likely due to precipitation in November 2015, increasing discharge (Appendix C). Snowfall in February 2016 would not increase discharge as the water was frozen in the form of snow on the surface.

37 Figure 13: Photograph of a manganese oxide crust, confirmed by the LBB spot test. Scale is 30 cm across. Photograph from February 2016.

38 3.1.3 Geochemical Analysis November 2015 (Trip A) physical and chemical data are summarized in Appendix E. Sampling locations can be viewed on Figures 8 and 9. Dissolved oxygen values in the Mystic River ranged from 8.70 at SH2 to 9.50 mg/L at SH10, and from 8.76 mg/L to 9.63 mg/L in the infeeder streams. Moving downstream from the double waterfall (SH11), D.O values increased and slightly fluctuated until reaching Craig’s Creek (SH6), where D.O dropped to 8.76 mg/L. Moving downstream from Craig’s Creek, D.O. began to slightly rise, ending at 9.14 mg/L at the sump (Figure 14). Mystic river water temperature remained relatively constant at 11.8 to 11.9 degrees C. The Mastodon Avenue infeeder near the cave entrance (SH13) was colder, with a water temperature of 10.1 degrees C. Both the Middle Earth infeeder (SH9) and Craig’s Creek infeeder (SH6) were warmer, with water temperatures of 14.9 and 12.2 degrees C, respectively. Conductivity readings in Mystic River remained in the 441 – 451 µS range (Figure 14). Conductivity values taken from the entrance infeeder and Middle Earth stream were both lower with values of 225 and 357 µS, respectively. A conductivity reading from Craig’s Creek was the highest at 454 µS. The input of the Middle Earth stream reduced the conductivity of Mystic River slightly, but it returned to its normal range at the next downstream sampling point. The pH of Mystic River ranged from 7.50 at SH12 to 7.78 at SH1 along the surveyed stream section (Figure 14). The lowest pH reading was taken from the Middle Earth infeeder stream at 6.97. Of all major cations analyzed, Ca was the highest measured, ranging from 32.6 mg/L at SH13 to 87.1 mg/L at SH6. Fe and Mn were the lowest measured cations, with the highest values measured at 0.281 mg/L and the lowest at 0.001 mg/L. Low concentrations of Mn and Fe were confirmed by ICP analysis (Appendix E). The alkalinity of Mystic River ranged from 232 mg/L at SH8 to 244 mg/L at SH2, SH5, SH6, SH7, SH11, and SH12, remaining relatively constant moving downstream (Figure 14), with the only fluctuations occurring near the input of infeeder streams.

39 14.0

13.0

12.0

11.0

D.O (mg/L) 10.0 SH7 SH9 SH10 SH1 SH3 SH4 SH12 9.0 SH8 SH6 SH2 SH5 SH11 8.0 0 100 200 300 400 500 600

1200 1100 1000 900 800 700 600

Conductivity (µS) 500 400 300 0 100 200 300 400 500 600 8.4 8.2 8 7.8 7.6 7.4

pH 7.2 7 6.8 0 100 200 300 400 500 600 260

240

220 Sump Above 200 waterfall

180

Alkalinity (mg/L) Alkalinity 160

140 0 100 200 300 400 500 600 Distance Upstream (m) Nov 2015 Mystic River Feb 2016 Mystic River Nov 2015 Craig's Creek Feb 2016 Craig's Creek Nov 2015 Middle Earth Feb 2016 Middle Earth

Figure 14: Dissolved oxygen, conductivity, pH, and alkalinity measurements taken along Mystic River and infeeder streams. Measurements begin downstream at SH1 and end upstream above the waterfall at SH11.

40 The two lowest alkalinity readings were in the Mastodon Avenue infeeder and the Middle Earth infeeder, with alkalinities of 120 and 148 mg/L, respectively. Alkalinity of Craig’s Creek was similar to the values measured in Mystic River (244 mg/L). Anion concentrations measured in Mystic River were roughly the same throughout.

- - 2- Cl was measured to be around 7.9 mg/L, NO3 measured around 11.8 mg/L and SO4 measured around 10.5 mg/L. Anion concentrations of the infeeder streams varied. The

- - 2- Mastodon Avenue infeeder had Cl , NO3 , and SO4 concentrations of 4.06 mg/L, 3.92

- - 2- mg/L, and 7.83 mg/L, respectively. The Middle Earth infeeder had Cl , NO3 , and SO4 concentrations of 7.74 mg/L, 13.69 mg/L, and 28.27 mg/L, respectively, and Craig’s

- - 2- Creek had Cl , NO3 , and SO4 concentrations of 7.13 mg/L, 17.60 mg/L, and 9.29 mg/L, respectively. Birnessite was found to be supersaturated in Mystic River and the Cove Creek, Mastodon Avenue, and Middle Earth Infeeder streams (Table 3). Supplementary PHREEQC analyses of the measured geochemical parameters are available in Appendix F. February 2016 (Trip B) physical and chemical data are summarized in Appendix E. Dissolved oxygen values ranged from 11.60 at SH12 to 13.45 mg/L at SH1, all higher than the previous sampling trip, and showed no distinct trend moving upstream to downstream in Mystic River (Figure 14). Water temperature remained relatively consistent at 10.9 degrees Celsius, slightly colder than the first sampling trip.The infeeder streams were coldest, as temperatures ranged from 8.5 to 10.9 degrees Celsius. Conductivity of Mystic River ranged from 1087 to 1119 µS (Figure 14). Conductivity measured in the infeeder streams ranged from 475 to 1090 µS. These values were much higher than the first sampling trip. The pH in Mystic River ranged from 7.94 at SH10 to 8.02 at SH1, SH2, and SH12 (Figure 14), slightly higher than the previous sampling trip. The pH measurements taken in the infeeder streams ranged from 8.15 to 8.18, also slightly higher than the previous sampling trip. Of all major cations analyzed, Ca was

41 Table 3: Saturation index of birnessite in Scott Hollow Cave.

Site Date CBE % SI birnessite SH1 11/20/2015 -7.82% 8.07 SH2 11/20/2015 -7.08% 7.52 SH3 11/20/2015 -4.83% 7.80 SH4 11/20/2015 -2.90% 7.72 SH5 11/20/2015 -7.35% 8.93 SH6 11/20/2015 -6.89% 7.18 SH7 11/20/2015 -10.13% 7.61 SH8 11/20/2015 -10.96% 7.75 SH9 11/20/2015 -8.19% 7.08 SH10 11/20/2015 -8.77% 8.85 SH11 11/20/2015 -8.23% 9.07 SH12 11/20/2015 -9.18% 7.57 SH13 11/20/2015 -4.27% 9.01 SH1 2/13/2016 -11.84% 8.02 SH2 2/13/2016 -14.82% 9.55 SH3 2/13/2016 -9.71% 7.98 SH4 2/13/2016 -4.91% 7.93 SH5 2/13/2016 -7.74% 7.91 SH6 2/13/2016 -5.22% 8.27 SH7 2/13/2016 -7.64% 7.91 SH8 2/13/2016 -11.59% 10.78 SH9 2/13/2016 -7.02% 8.44 SH10 2/13/2016 -10.84% 7.88 SH11 2/13/2016 -5.75% 7.90 SH12 2/13/2016 -8.69% 8.01 SH13 2/13/2016 19.73% 9.92

42 again the highest measured, ranging from 71.6 mg/L at SH1 to 82.6 mg/L at SH4 in Mystic River and from 22.9 to 83.9 mg/L in the infeeders (Appendix E). Fe and Mn were again the lowest measured cations, with the highest values measured at 0.16 mg/L and the lowest at 0.001 mg/L. The alkalinity in Mystic River was lower during this sampling trip compared to the previous measurements taken in November 2015 (Figure 14). The alkalinities of the Craig’s Creek, Middle Earth, and Mastodon Avenue infeeders were 232 mg/L, 152 mg/L, and 88 mg/L, respectively. Cl- concentrations measured in Mystic River were roughly

- the same throughout. NO3 decreased gradually downstream, likely due to dilution.

2- SO4 concentrations were measured from 11.50 mg/L to 17.81 mg/L in Mystic River. The highest concentration was measured in the SH9 infeeder at 39.57 mg/L and the lowest concentration was measured in the Craig’s Creek infeeder with a concentration of 9.36 mg/L (Appendix E). Supplementary PHREEQC analyses of the measured geochemical parameters are available in Appendix F. Birnessite was again calculated to be supersaturated in Mystic River and all measured infeeder streams (Table 3).

3.1.4 Microbiological Analysis Laboratory samples began to develop brown colonies over the course of a week and a half, indicative of manganese oxidizing bacteria (Figure 15A). These suspected manganese oxidizing bacteria colonies were tested with a 0.04% solution of LBB to confirm the presence of manganese oxides, indicating that manganese oxidizing bacteria were present (Figure 15B). Manganese oxidizing bacteria were confirmed to be present on cultures from surface and subsurface samples, as well as on plates grown in the dark and in the light (Table 4). Bacteria were more abundant on the surface than in the cave.

43 Figure 15: Photograph showing colonies of manganese oxidizing bacteria grown on K-media. A) shows brown Figure 15: Photograph showing colonies of manganese oxidizing bacteria grown on K-media. colonies forming (white arrows) with a scale of 15 cm across and B) shows brown that are positively idenitifed as manganese oxidizing bacteria through the LBB spot test (white box) with a scale of 8 cm across. Dark black/purple spots are mold.

44 Table 4: Enumerations of plated cave and surface bacteria samples from Scott Hollow Cave.

Sample Dilution log CFU/g 10-1 3.78 10-2 4.00 10-3 0.00 10-4 0.00 SH1 Light 10-5 0.00 10-6 0.00 10-7 0.00 10-8 0.00 10-1 4.48 10-2 5.17 10-3 3.78 10-4 0.00 SH1 Dark 10-5 0.00 10-6 0.00 10-7 0.00 10-8 0.00 10-1 5.12 10-2 6.79 10-3 6.67 10-4 7.25 SH Surface Light 10-5 7.47 10-6 8.00 10-7 0.00 10-8 0.00 10-1 5.00 10-2 5.72 10-3 6.92 10-4 6.14 SH Surface Dark 10-5 8.51 10-6 0.00 10-7 0.00 10-8 0.00

45 3.1.5 Manganese Coating SEM/EDAX and XRD Analysis SEM/EDAX analysis shows that samples coated in black oxides taken from Scott Hollow cave were most commonly composed of O, Mg, Al, Si, K, Ca, Ti, Mn, and Fe (Table 5). All samples were composed of less than 1% of Mn and less than 7% of Fe. Oxygen and Silicon made up the majority of the sample composition. Two samples (SH3 and SH4) had traces of titanium (< 1%) and magnesium (< 2%). Sample SH3 was the only Scott Hollow sample to have a measured concentration of calcium. XRD analysis shows the coatings to take the mineral form of birnessite (Figure 16, Appendix G). A high quartz spike is observed on all Scott Hollow diffractograms (Appendix G). Illite and kaolinite were also found to be present.

3.2 Maxwelton Sink Cave

Photographs taken during sampling in Maxwelton Sink Cave can be viewed in Appendix B. Trip dates, sampling conditions, and activities are listed in Table 6. Weather data from the months sampling took place are in Appendix C.

3.2.1 Manganese Oxide Distribution Depositional patterns of black manganese oxides were mapped throughout 700 meters of the Cove Creek passage in Maxwelton Sink Cave (Figure 17). Deposits were broken into the same three categories as described in section 3.1.1; Cemented cobbles and pebbles (Figure 12A), eroded coatings (Figure 12B), and fine grained coatings (Figure 12C). Locations with no observable manganese deposition were mapped as bedrock. As in Scott Hollow Cave, manganese oxide is observed to be almost everywhere. Cemented cobbles and pebbles were again observably the thickest of deposits and only located along the stream banks or in the stream. These deposits were found around waterfalls 46 - Fe 1.7 2.82 1.65 6.47 6.28 2.14 - - 0.8 Mn 0.46 0.26 0.11 0.11 - - - - - Ti 0.77 0.59 ------Ca 4.89 K 0.94 1.21 0.71 1.28 0.93 0.83 0.57 Normalized Wt % Normalized Si 17.8 16.3 16.99 18.88 23.59 22.46 17.23 Al 4.41 5.17 3.76 5.39 3.43 3.73 2.52 - - - - - 1.8 Mg 0.71 O 71.28 68.75 70.29 68.61 72.77 71.14 77.45 Sample # Sample SH3 SH4 SH6 SH7 SH8 SH9 SH12 Table 5: Percent elemental weights of Scott Hollow Cave manganese oxide crust/coating samples. Table

47 Figure 16: Diffractogram of manganese coating sample taken from SH12.

48 F ° F, No Precipitation F, F, No precipitation F, ° ° Surface Conditions Below freezing, 17 freezing, Below Sunny, 80 Sunny, Cloudy, 45 Cloudy, Activity Preliminary research survey research Preliminary 12 water samples, mapping of Mn deposits of Mn deposits mapping samples, 12 water 5 bio samples, 10 water samples, coating samples coating samples, 10 water 5 bio samples, Date 16 August 2016 16 August 14 February 2016 14 February 21 Novemeber 2015 21 Novemeber B A Trip Reconnaissance Table 6: Date, activity, and surface conditions during Maxwelton Sink research trips. 6: Date, activity, Table

49 Figure 17: Map of various types Mn deposits observed along and in Cove Creek Maxwelton Sink Cave. Mapping took place during the February 2016 sampling trip. Base Map from Handley and Pearson, 2012.

50 and in areas where water of higher turbulence emerges from breakdown. Eroded coatings covered the passage walls and were observed up to heights of 3 meters, similar to those in Scott Hollow. Fine grained coatings were observed near the Heaven Passage infeeder. Bedrock was mapped as areas where the stream was flowing on top of rock with no manganese oxide deposits.

3.2.2 Geochemical Analysis November 2015 (Trip A) chemistry data are summarized in Appendix E. Cove Creek dissolved oxygen values ranged from 8.24 mg/L to 9.71 mg/L. The only infeeder stream sampled, the Heaven Passage stream, was measured at 9.14 mg/L. D.O values fluctuate moving downstream throughout the system (Figure 18). Cove Creek water temperatures remained relatively constant, ranging from 11.5 to 11.9 degrees Celsius. The infeeder stream had the lowest recorded water temperature at 11.4 degrees Celsius. Conductivity of Cove Creek ranged from 394 to 490 µS. The infeeder stream had a conductivity of 379 µS, the lowest of all sample locations. Upstream, conductivity was recorded at its highest values of 490 µS, dropping off significantly to 394 µS moving downstream to the flowstone. The conductivity then rose continuing downstream where it remained around 415 µS, ending at the last sampling location below the first waterfall (Figure 18).The pH values in Cove Creek ranged from 7.32 at MX4 to 7.58 at MX6 and MX7. A pH of 7.56 was recorded in the Heaven Passage infeeder stream. Moving downstream through the system, pH dropped until reaching the flowstone, where it increased slightly and then dropped slightly at the first waterfall (Figure 18). Of all major cations analyzed, Ca was the highest measured, ranging from 55.8 mg/L (MX4) to 73.1 mg/L (MX9). Mg, Na, and K had the next highest measured values, respectively. Fe and Mn were the lowest measured cations, as all values were less than 0.1 mg/L.

51 13.00 MX11 12.00 MX12 11.00

10.00 Heaven MX2 Passage MX3 MX6 MX10 MX9 D.O (mg/L) 9.00 MX8 MX4 MX5 8.00 MX7

7.00 0 100 200 300 400 500 600 700 800 1400

1200

1000

800

600 Conductivity (µS) 400

200 0 100 200 300 400 500 600 700 800

8.2

7.8

7.6 pH

7.4

7.2

7 0 100 200 300 400 500 600 700 800

220

210

200

190

180 Alkalinity (mg/L) Alkalinity 170

160 0 100 200 300 400 500 600 700 800 Distance Upstream (m) Nov 2015 Cove Creek Feb 2016 Cove Creek Nov 2015 Heaven Passage Feb 2016 Heaven Passage

Figure 18: Dissolved oxygen, conductivity, pH, and alkalinity measurements taken along Cove Creek and Heaven Passage infeeder stream. Measurements begin downstream at MX12 and end upstream at MX7. 52 The alkalinity of Cove Creek ranged from 168 mg/L at MX4 and MX5 to 208 mg/L at MX9 (Figure 18). Alkalinity of the Heaven Passage infeeder stream was similar to the values measured in Cove Creek (204 mg/L). Anion concentrations were slightly variable throughout the stream passage. Cl- concentrations measured in Cove Creek generally decreased downstream. The most upstream sample locations (MX7 and MX6) measured

- 11.2 mg/L, dropping to 8.2 mg/L below the first waterfall (MX10). NO3 concentrations varied throughout Cove Creek, ranging from 7.63 mg/L to 9.96 mg/L with an anomalous

2- concentration of 48.48 at sampling location MX8. Cove Creek SO4 concentrations were the most variable, ranging from 19.06 mg/L to 58.77 mg/L and displaying a distinct decreasing downstream trend. The Heaven Passage infeeder stream had the lowest

2- measured SO4 concentration at 15.03 mg/L. Birnessite was found to be supersaturated in Cove Creek and the Heaven Passage infeeder stream (Table 7). Supplementary PHREEQC analysis outputs are available in Appendix F. February 2016 (Trip B) chemistry data are summarized in Appendix E. Dissolved oxygen values ranged from 11.80 at MX2 to 12.12 mg/L at MX7 and showed no distinct trend moving upstream to downstream in Cove Creek (Figure 18). A dissolved oxygen reading at the Heaven Passage infeeder was measured to be 12.59 mg/L, the highest reading during this sampling trip. Water temperature remained relatively consistent at 11.4 degrees Celsius, slightly colder than the first sampling trip. Conductivity of Cove Creek ranged from 990 at MX9 to 1277 µS at MX6 (Figure 18). Conductivity measured in the Heaven Passage infeeder stream was the lowest, at 974 µS. These values were more than two times higher than the first sampling trip. The pH in Cove Creek ranged from 7.79 at MX5 to 8.05 at MX7, slightly higher than the previous sampling trip (Figure 18). A pH measurement taken in the Heaven Passage infeeder was 8.03, also slightly higher than the previous sampling trip. Of all major cations analyzed, Ca was measured to be the highest in Cove Creek with an anomalous concentration of 160.7 mg/L below

53 Table 7: Saturation index of birnessite in Maxwelton Sink Cave.

Site Date CBE % SI birnessite MX1 11/21/2015 -3.48% 8.38 MX2 11/21/2015 -8.05% 8.46 MX3 11/21/2015 -3.63% 8.34 MX4 11/21/2015 -5.65% 8.03 MX5 11/21/2015 -5.07% 8.16 MX6 11/21/2015 -3.89% 8.36 MX7 11/21/2015 -3.60% 8.35 MX8 11/21/2015 -15.14% 8.07 MX9 11/21/2015 -3.89% 7.70 MX10 11/21/2015 -4.95% 7.18 MX1 2/14/2016 -2.47% 7.97 MX2 2/14/2016 -6.95% 7.91 MX3 2/14/2016 -4.64% 7.84 MX4 2/14/2016 -6.56% 8.86 MX5 2/14/2016 -6.20% 7.58 MX6 2/14/2016 -6.35% 7.88 MX7 2/14/2016 -8.39% 8.90 MX8 2/14/2016 -8.80% 7.86 MX9 2/14/2016 -6.33% 7.94 MX10 2/14/2016 33.65% 9.16 MX11 2/14/2016 -3.67% 7.93 MX12 2/14/2016 -7.16% 7.93

54 the first waterfall (Appendix E). Ca was measured to be 67.0 mg/L in the Heaven Passage infeeder. Fe and Mn were measured to be the lowest. Mn ranged from 0.001 mg/L in the Heaven Passage infeeder to 0.019 below the waterfall at MX 10 (Appendix E). The alkalinity of Cove Creek ranged from 180 at MX4 to 200 mg/L at MX6 and MX7 and remained relatively constant throughout the stream (Figure 18). Alkalinity of

- - 2- the Heaven’s Passage infeeder stream was 192 mg/L. Cl , NO3 , and SO4 concentrations all decreased moving downstream throughout Cove Creek from 10.8 to 8.3 mg/L, 11.3 to 10.8 mg/L, and 54.4 to 19.4 mg/L, respectively (Appendix E). Birnessite was again found to be supersaturated in Cove Creek and the Heaven Passage infeeder stream (Table 7). Supplementary PHREEQC analysis from the February 2016 data are available in Appendix F.

3.2.3 Microbiological Analysis Samples left to grow over the course of two weeks began to develop the same brown colonies observed growing from the Scott Hollow samples after about a week and a half. These suspected manganese oxidizing bacteria colonies were tested with a 0.04% solution of LBB to confirm the presence of manganese oxides, indicating manganese oxidizing bacteria were present (Figure 19). Manganese oxidizing bacteria were confirmed to be present on both surface and subsurface plates, as well as on plates grown in the dark and in the light (Table 8).

3.2.4 Manganese Coating SEM/EDAX and XRD Analysis SEM/EDAX analysis show samples coated in black oxides taken from Maxwelton Sink Cave had a similar composition to the samples taken in Scott Hollow cave (O, Mg, Al, Si, K, Ca, Mn, and Fe) with the exception of Ti. Most samples were composed of less

55 Figure 19: Photograph showing colonies of manganese oxidizing bacteria grown on K-media. Blue rings indicate positive identification of manganese oxidizing bacteria from LBB spot test. Scale is 20 cm across.

56 Table 8: Enumerations of plated cave and surface bacteria samples from Maxwelton Sink Cave.

Sample Dilution log CFU/g 10-1 3.60 10-2 4.00 10-3 0.00 10-4 0.00 MX10 Light 10-5 0.00 10-6 0.00 10-7 0.00 10-8 0.00 10-1 4.40 10-2 5.18 10-3 5.00 10-4 6.69 MX10 Dark 10-5 7.30 10-6 8.30 10-7 0.00 10-8 0.00 10-1 5.15 10-2 5.73 10-3 5.72 10-4 7.67 MX Surface Light 10-5 7.60 10-6 0.00 10-7 0.00 10-8 0.00 10-1 4.90 10-2 5.72 10-3 5.48 10-4 7.00 MX Surface Dark 10-5 7.78 10-6 0.00 10-7 0.00 10-8 0.00

57 than 2% of Mn and less than 7% of Fe. Oxygen and silicon made up the majority of the composition weight percent (Table 9). XRD analysis of selected samples shows the common mineral phases were illite and quartz as well as manganese oxide in the form of birnessite (Figure 20, Appendix G).

58 10 Fe 3.7 3.04 6.01 2.48 5.97 2.79 1.99 2.32 1.3 Mn 4.22 0.38 0.33 0.41 3.91 0.24 0.21 0.29 ------Ti - - Ca 0.65 5.95 0.25 0.52 0.24 0.32 1.01 K 0.9 1.09 0.78 0.68 1.15 1.14 1.18 0.94 1.49 Normalized Wt % Normalized Si 7.16 13.8 15.62 17.16 11.73 15.42 11.73 13.62 11.61 Al 6.15 3.37 3.59 2.45 4.36 6.88 4.28 3.72 4.85 - - 3.1 1.7 Mg 0.76 1.02 0.67 0.92 0.91 O 68.47 68.34 77.45 76.89 71.41 72.34 75.65 78.30 76.73 Sample # Sample MX9 MX2 MX1 MX3 MX4 MX5 MX6 MX7 MX10 Table 9: Percent elemental weights of Maxwelton Sink Cave manganese oxide crust/coating samples. Table

59 Figure 20: Diffractogram of manganese coating sample taken from MX1.

60 CHAPTER IV DISCUSSION

4.1 Manganese Oxide Distribution

Manganese oxide deposition has occurred almost everywhere along the surveyed areas in both the Mystic River passage of Scott Hollow Cave (Figure 11) and the Cove Creek passage of Maxwelton Sink Cave (Figure 17). Coatings are found on walls, on stream floors, and on pebbles within the streams, consistent with findings locally (White et al., 2009), nationally (Peck, 1986), and globally (Hill and Forti, 1997; Gâzquez et al., 2011). The walls of both caves are dominated by a brown to black coating that appears to have been eroded over time due to its patchy appearance (Figure 12B). In both Scott Hollow Cave and Maxwelton Sink Cave, in-stream coatings were either of the fine grained coating type (Figure 12C) or of the cemented cobbles and pebbles type (Figure 12A). Cemented cobbles and pebbles in both Mystic River and Cove Creek were in areas where finer grained sediments had not settled out, allowing for the manganese surface from previous deposition to remain exposed, allowing for a catalytic effect that enhances further precipitation as long as there is there is a supply of dissolved oxygen and manganese (Crerar and Barnes, 1974; Hem, 1978; Morris and Bale, 1979). While detailed gradient measurements were not acquired, visual observations suggest cemented cobbles and pebbles are found in areas of steeper stream gradient and more turbulent stream flow, such as in areas around waterfalls and where stream water emerges from breakdown boulders (Figure 11 and Figure 17). From geochemical measurements and PHREEQC

61 analysis there is sufficient dissolved oxygen and manganese for precipitation to continue in both caves to support these claims (Table 3, Table 7, and Appendix F). Though cobbles and pebbles are found throughout the majority of the surveyed stream passages, the observation of less manganese cemented cobbles and pebbles in areas where fine grained coatings were mapped is likely due to the absence of a stable preferential substrate. It is also possible that removal of oxide coatings due to burial, and lower Eh, inhibits the pebbles and cobbles from becoming cemented together, as mentioned by Robinson (1981) and Carpenter and Hayes (1980). Thus, ideal locations for thick manganese coatings, in the form of cemented cobbles and pebbles, are where manganese and dissolved oxygen are sufficient, such as in areas of steeper stream gradient and more turbulent stream flow, and where a stable preferential substrate is found.

4.2 Geochemistry

PHREEQC analysis confirms that the cave stream water of both Mystic River and Cove Creek are supersaturated with respect to birnessite (Table 3, Table 7, and Appendix F). Birnessite saturation ranged from 7.08 (SH9) to 9.07 (SH11) in Scott Hollow Cave in November 2015 and from 9.55 (SH2) to 10.78 (SH8) in February 2016 (Table 3). Birnessite saturation ranged from 7.18 (MX10) to 8.46 (MX2) in Maxwelton Sink Cave in November 2015 and from 7.58 (MX5) to 9.16 (MX10) in February 2016 (Table 7). This supersaturation of birnessite in these cave stream waters indicates that precipitation is a thermodynamically favorable reaction, as confirmed by the observed manganese oxide coatings found throughout the surveyed reaches in both caves. Palmer (2007) also suggests caves are a favorable location for manganese precipitation due to the mixing of anoxic waters in an oxic cave environment, creating a redox gradient. This redox gradient created by a cave’s oxic environment may explain why manganese oxide deposits are 62 more commonly observed in caves than on the surface. Deposited manganese in caves may also be protected from dissolution due to the lack of ultraviolet (UV) light, as UV light increases the reductive dissolution rate of manganese 6 to 70 times, compared to in the dark (Sunda and Huntsman, 1988; Sunda and Huntsman, 1994). Geochemically, the waters of Scott Hollow Cave and Maxwelton Sink Cave exhibit similar characteristics. Dissolved oxygen, temperature, conductivity and pH readings followed similar trends (Figure 14 and Figure 18). From the November 2015 data, D.O, water temperature, pH, potassium, chloride, and nitrate were all measured to be higher on average in Mystic River of Scott Hollow, relative to Cove Creek of Maxwelton Sink (Figure 21). The February 2016 data show that the dissolved oxygen, pH, and potassium are, on average, the same in both systems (Figure 22). Water temperature, magnesium, chloride, and sulfate were higher in Cove Creek in Maxwelton Sink, with nitrate measured to be higher in Mystic River of Scott Hollow (Figure 21 and Figure 22). The higher nitrate concentrations and seasonal variability in Mystic River of Scott Hollow Cave as compared to Cove Creek of Maxwelton Sink Cave suggest differences in surface land use practices. From visual observations, Scott Hollow Cave is situated beneath an area with a higher density of agriculturally developed land which has a direct influence on groundwater quality compared to Maxwelton Sink Cave. These findings agree with those of Bishop (2010).

4.3 Microbiology

Manganese oxide and manganese oxidizing bacteria were present in the cave and were confirmed by the LBB spot test (Figure 15 and Figure 19), as the deep blue rings indicate the presence of manganese oxide (Krumbein and Altmann, 1973). During field mapping, the LBB spot test was successfully used throughout the stream passage 63 - 2 4 SO (mg/L) - 3 NO (mg/L) - Cl K (mg/L) Maxwelton Sink - Cove Creek Na (mg/L) Mg (mg/L) pH Scott Hollow - Mystic River Mystic - Hollow Scott Selected Novemeber 2015 Physical and Chemical Parameters and Chemical 2015 Physical Novemeber Selected (C) Water Temperature D.O (mg/L)

5 0

15 10 40 35 30 25 20 Values Figure 21: Histogram of average physical and chemical parameters between Scott Hollow Maxwelton Sink Cave measured in November 2015.

64 - 2 4 SO (mg/L) - 3 NO (mg/L) - Cl (mg/L) K (mg/L) Maxwelton Sink - Cove Creek Na (mg/L) Mg (mg/L) pH Scott Hollow - Mystic River Mystic - Hollow Scott Selected February 2016 Physical and Chemical Parameters (C) Water Temperature D.O (mg/L)

5 0

35 30 25 20 15 10 Values Figure 22: Histogram of average physical and chemical parameters between Scott Hollow Maxwelton Sink Cave measured in February 2016.

65 and confirmed the presence of manganese oxides and ongoing manganese oxidation throughout the entire length of the passages surveyed. Sediment, cave walls, in-stream pebbles and rocks from the rivers edge all tested positive in both caves. Bacteria cultured from both Scott Hollow Cave and Maxwelton Sink Cave were confirmed to be manganese oxidizing bacteria by also utilizing the LBB spot test. Bacteria from the surface near the entrance of both caves were also confirmed to be manganese oxidizing bacteria. The presence of manganese oxidizing bacteria was expected, as manganese oxidizing bacteria are known to deposit manganese in caves (Peck, 1986). Manganese oxidizing bacteria are spatially widespread throughout both the Scott Hollow and Maxwelton Sink cave systems based on these findings. Surface samples grown in the light and dark from both caves (SH Surface and MX Surface) were observed to preferentially grow in the light, based on a comparison of colony forming units per gram of K-medium (Figure 23). Cave samples grown in the light and dark (SH1 and MX10) were observed to preferentially grow in the dark (Figure 23). SH1 cave samples grown in the light were enumerated to be 3.78 log(CFU/g) compared to 4.48 log(CFU/g) in the dark on the 10-1 plates. MX10 caves samples grown in the light were enumerated to be 3.60 log(CFU/mL) compared to 4.40 log(CFU/g) in the dark on the 10-1 plates (Table 4 and Table 8). A higher accumulation of manganese oxide was observed to be produced by bacteria on the surface samples, as compared to the samples taken from within both cave systems (Figure 23). The higher production of manganese oxide by cave bacteria growing in the dark may be due the protection from UV light as UV light may damage manganese oxidizing microbes (Tebo, 2004).

66 MX Surface MX10 Dark Sample Light SH Surface SH1

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 log (CFU/g) log Figure 23: Histogram of surface and subsurface colony forming units (log CFU/g) from Scott Hollow Cave and Maxwelton Sink Cave. Bacteria were grown on K-media enumerated by members of the Akron. Senko Lab at the University of

67 4.4 Manganese Coating Mineralogy

ESEM imaging of oxide coatings from this study show no distinct shape (Figure 24 and Figure 25). The dominant presence of silicon and oxygen in the samples from ESEM analysis suggest that quartz and clay minerals are likely the preferred substrate for metal oxide precipitation, supported by the findings of Sasowsky et al., (2000). ESEM analyses also show that low concentrations of manganese are present (Table 5 and Table 9). These low concentrations are likely due to the inability to separate a pure manganese oxide coating from its substrate. From the high amount of silicon and oxygen observed in the ESEM data, the presence of quartz and clay minerals were examined during XRD analyses as well as the mineral phase of manganese oxide. X-ray diffraction analyses suggest that the form of manganese oxide found in both Scott Hollow and Maxwelton Sink Cave is birnessite (Figure 16 and Figure 20), consistent with the findings of White (2009) in which he was able to confirm the presence of birnessite in nearby Matt’s Black Cave through XRD techniques. White (2009) also found birnessite to be present in multiple caves in the Appalachians, strengthening the findings of this study, as the study location falls within this region. Birnessite has also been found to be the mineral form of manganese in caves globally, such as in El Soplae Cave, Spain (Gâzquez et al., 2011). Birnessite peaks, while present, were suppressed by other minerals that composed the majority of the samples, such as quartz and clay minerals. The suppressed birnessite curves are likely due to sampling techniques. Sampled coatings were not completely separated from the substrate upon which they were found, leading to dominance of quartz peaks in the diffractograms. High quartz peaks and illite peaks were observed in the XRD diffractograms, as the ESEM data suggests (Figure 16, Figure 20, and Appendix G).

68 Figure 24: ESEM image of an oxide coating from site SH4 in Scott Hollow Cave.

69 Figure 25: ESEM image of an oxide coating from site MX4 in Maxwelton Sink Cave.

70 CHAPTER V CONCLUSIONS

Scott Hollow Cave and Maxwelton Sink Cave in Monroe and Greenbrier Counties, West Virginia have main stream passages with observable black manganese oxide deposition coating and cementing cobbles and pebbles, fine grained sediments, walls, and breakdown, similar to observations made in caves locally, nationally, and globally. From extensive field mapping, geochemical and microbial analyses, and aqueous geochemical modeling, the major findings of this research are: • Manganese oxide deposition occurs almost everywhere throughout the studied cave stream passages (Figure 11 and Figure 17). • Ideal locations for cemented manganese oxide deposits are suggested to be in areas of observable turbulent flow, allowing for reactant mixing (Figure 11 and Figure 17). These areas include waterfalls and where water emerges from breakdown boulders, determined through mapping and visual observations. • The preferred substrate for manganese oxide deposition was observed to be quartz and clay minerals (Table 5 and Table 9), not limestone. • The main stream waters and infeeder stream waters of both Scott Hollow Cave and Maxwelton Cave were supersaturated with respect to birnessite (Table 3, Table 7, and Appendix F). • The mineral form of manganese oxide in Scott Hollow Cave and Maxwelton Sink Cave is birnessite (Figure 16, Figure 20, and Appendix G). High quartz peaks and illite peaks were also observed in the analyzed diffractograms.

71 • Manganese oxidizing bacteria are present in the surface sediments around Scott Hollow Cave and Maxwelton Cave, as well as throughout the main stream passages of both caves (Figure 15 and Figure 19). More manganese oxide was observed to accumulate on the surface plates compared to the cave passage plates (Figure 23) and more manganese oxide was observed to accumulate on the plates grown in the light compared to those grown in the light (Figure 23). • Deposited manganese may be protected from dissolution due to the lack of ultraviolet (UV) light, as UV light increases the dissolution rate of manganese 6 to 70 times, compared to in the dark (Sunda and Huntsman, 1988; Sunda and Huntsman, 1994). This may explain why manganese oxide deposits are more commonly observed in cave streams as opposed to in surface streams. Findings from this study may have implications for acid mine drainage treatment systems as well as in drinking water purification systems as manganese oxidizing bacteria can improve water quality through the removal of toxic metals (Villalobos et al., 2005). Future studies should consider additional TEM analyses as the birnessite signal in the processed XRD diffractograms were relatively low, likely due the intensity of the quartz signal. Future studies may also want to consider DNA extraction methods to determine the exact strain of manganese oxidizing bacteria present in the examined cave systems. In stream Eh measurements should be taken so as to not rely on a mathematically derived value, in order to increase the accuracy of the study. It is also suggested that the thicknesses of the precipitated manganese oxide be measured in order to map thickness variations, as this may indicate preferential areas of deposition.

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77 APPENDICES

78 APPENDIX A LABORATORY EQUIPMENT

Photographs of laboratory equipment in the University of Akron Geochemistry Laboratory used for data analysis.

79 Figure A1: Photograph showing titration of stream water in the University of Akron Geochemistry Laboratory.

80 Figure A2: Photograph of Atomic Absorption Spectrometer AAnalyst 700 in the University of Akron Geochemistry Laboratory.

81 Figure A3: Photograph of SPEX mill used to pulverize oxide coating samples to powder for XRD anlysis.

82 Figure A4: Photograph of Scott Hollow oxide coating samples after pulverization by mortar/pestle and milling.

83 Figure A5: Photograph of Maxwelton Sink oxide coating samples after pulverization by mortar/pestle and milling.

84 Figure A6: Photograph of XRD.

85 Figure A7: Photograph of Environmental Scanning Electron Microscope (ESEM).

86 APPENDIX B CAVE PHOTOGRAPHS

Field photographs taken during the Novemebr 2015 and February 2016 sampling trips. Photographs courtesy of Ira D. Sasowsky and Dave Socky.

87 Figure B1: Photograph of sampling location SH1 in Scott Hollow Cave, located where Mystic River terminates into the sump.

88 Figure B2: Photograph of sampling location SH7 in Scott Hollow Cave. Turbulent water is observed emerging from a breakdown pile covered in manganese oxide.

89 Figure B3: Photograph of sampling location SH9 in Scott Hollow Cave.

90 Figure B4: Photograph of sampling location SH11 in Scott Hollow Cave.

91 Figure B5: Photograph of sampling location MX3 in Maxwelton Sink Cave.

92 Figure B6: Photograph of sampling location MX8 in Maxwelton Sink Cave.

93 Figure B7: Photograph of sampling location MX10 in Maxwelton Sink Cave.

94 Figure B8: Photograph of sampling location MX11 in Maxwelton Sink Cave.

95 APPENDIX C WEATHER DATA

Weather data from August 2015, November 2015, and February 2016. Data were compiled from The National Weather Service Charleston, West Virginia service office.

96 Temperature (F) Temperature 100 90 80 70 60 50 40 30 20 10 0 . th 30 29 28 , and 17 th 27 26 , 16 th 25 Low Temperature 24 23 22 21 20 19 18 17 High Temperature High 16 15 14 Days in August 2015 August in Days 13 Snow 12 11 10 9 8 7 Precipitation 6 5 4 3 2 1 0 3 2 1

0.5 2.5 1.5 Precipitation (inches) Precipitation Figure C1: Weather data from August 2015. Days of research visit were the 15 data from Weather Figure C1:

97 Temperature (F) Temperature 80 70 60 50 40 30 20 10 0 30 29 . st 28 27 and 21 26 th 25 24 Low Temperature 23 22 21 20 19 18 17 16 High Temperature High 15 14 13 Days in November 2015 12 Snow 11 10 9 8 7 6 Precipitation 5 4 3 2 1 1 0

1.2 0.8 0.6 0.4 0.2 Precipitation (inches) Precipitation Figure C2: Weather data from November 2015. Days of research visit were the 20 Weather Figure C2:

98 Temperature (F) Temperature 70 60 50 40 30 20 10 0 -10 28 27 . th 26 25 and 14 24 th 23 22 Low Temperature 21 20 19 18 17 16 High Temperature 15 14 13 12 DaysFebruary in 2016 Snow 11 10 9 8 7 6 Precipitation 5 4 3 2 1

6 5 4 3 2 1 0 Precipitation (inches) Precipitation Figure C3: Weather data from February 2016. Days of research visit were the 13 Weather Figure C3:

99 APPENDIX D DISCHARGE DATA

Discharge data from Mystic River of Scott Hollow Cave. Data were measured in ft3/s and converted to m3/s.

100 SH1 Discharge Data

Date Distance Width Depth Velocity Area Q Q (ft) (ft) (ft) (ft/s) (ft2) (ft3/s) (m3/s) 0.0 0.0 0.00 0.00 0.00 0.00 0.00 3.0 4.5 0.46 4.80 2.06 9.90 0.28 6.0 3.0 0.44 5.00 1.31 6.56 0.19 9.0 3.0 0.58 5.05 1.75 8.84 0.25 Nov 2015 12.0 3.0 0.53 3.90 1.58 6.14 0.17 15.0 3.0 0.37 3.50 1.10 3.85 0.11 18.0 2.6 0.20 2.00 0.52 1.04 0.03 19.1 0.0 0.00 0.00 0.00 0.00 0.00 Discharge 36.33 1.03 0.0 0.0 0.00 - 0.00 0.00 0.00 3.0 4.5 0.33 1.70 1.46 2.49 0.07 6.0 3.0 0.79 1.90 2.38 4.51 0.13 9.0 3.0 1.21 2.5 3.63 9.06 0.26 Feb 2016 12.0 8.6 0.71 2.40 6.09 14.62 0.41 15.0 0.0 0.00 - 0.00 0.00 0.00 18.0 0.0 0.00 - 0.00 0.00 0.00 19.1 0.0 0.00 - 0.00 0.00 0.00 Discharge 30.68 0.87

101 SH4 Discharge Data

Date Distance Width Depth Velocity Area Q Q (ft) (ft) (ft) (ft/s) (ft2) (ft3/s) (m3/s) 0 0.0 0.00 0.00 0.00 0.00 0.00 0.5 1.5 0.75 1.70 1.13 1.91 0.05 1 - 1.00 - - - - 2 - 1.21 - - - - 3 3.0 1.21 2.00 3.63 7.25 0.21 4 - 1.25 - - - - 5 - 1.37 - - - - 6 3.0 1.28 2.80 3.85 10.78 0.31 7 - 1.09 - - - - Nov 2015 8 - 0.85 - - - - 9 3.0 0.83 3.20 2.48 7.92 0.22 10 - 0.83 - - - - 11 - 0.71 - - - - 12 3.0 0.54 2.40 1.63 3.90 0.11 13 - 0.42 - - - - 14 - 0.21 - - - - 15 2.1 0.17 1.70 0.35 0.60 0.02 15.6 0.0 0.00 0.00 0.00 0.00 0.00 Discharge 32.36 0.92 0 0.00 0.13 0.00 0.00 0.00 0.00 3 4.50 1.27 2.30 5.72 13.15 0.37 6 3.00 1.00 2.00 3.00 6.00 0.17 Feb 2016 9 3.00 0.81 1.40 2.44 3.41 0.10 12 3.00 0.69 1.30 2.06 2.68 0.08 15 2.75 0.40 1.20 1.09 1.31 0.04 16.25 0.00 0.00 0.00 0.00 0.00 0.00 Discharge 26.55 0.75

102 SH8 Discharge Data

Date Distance Width Depth Velocity Area Q Q (ft) (ft) (ft) (ft/s) (ft2) (ft3/s) (m3/s) 0.0 0.00 0.00 0.0 0.00 0.00 0.00 1.0 1.25 1.88 6.1 2.34 14.30 0.40 2.0 1.00 0.38 6.0 0.38 2.25 0.06 3.0 1.00 0.38 5.1 0.38 1.91 0.05 4.0 1.00 0.38 5.9 0.38 2.21 0.06 Nov 2015 5.0 1.00 0.46 6.3 0.46 2.89 0.08 6.0 1.00 0.56 5.9 0.56 3.32 0.09 7.0 0.75 0.58 7.0 0.44 3.06 0.09 7.9 0.75 0.27 4.0 0.20 0.81 0.02 8.0 0.00 0.00 0.0 0.00 0.00 0.00 Discharge 30.75 0.87 0.0 0.0 0.00 0.0 0.00 0.00 0.00 1.5 2.0 0.19 4.1 0.38 1.57 0.04 3.0 2.5 0.60 6.4 1.50 9.60 0.27 Feb 2016 6.0 3.0 0.37 6.2 1.10 6.82 0.19 9.0 3.4 0.21 4.4 0.71 3.12 0.09 10.9 0.0 0.00 0.00 0.00 0.00 0.00 Discharge 21.11 0.60

103 SH9 Discharge Data

Date Distance Width Depth Velocity Area Q Q (ft) (ft) (ft) (ft/s) (ft2) (ft3/s) (m3/s) 0.0 0.0 0.33 0.50 0.00 0.00 0.00 1.0 1.0 0.58 2.73 0.58 1.59 0.05 2.0 1.0 0.54 2.73 0.54 1.48 0.04 Nov 2015 3.0 1.0 0.33 2.73 0.33 0.91 0.03 4.0 1.0 0.29 2.73 0.29 0.80 0.02 4.7 0.7 0.00 0.00 0.00 0.00 0.00 Discharge 4.78 0.14 0.0 0.0 0.00 0.0 0.00 0.00 0.00 1.0 1.5 0.13 1.3 0.19 0.24 0.01 2.0 1.0 0.42 1.3 0.42 0.54 0.02 Feb 2016 3.0 1.0 0.23 1.3 0.23 0.30 0.01 4.0 0.7 0.06 1.3 0.04 0.06 0.00 4.2 0.0 0.00 0.0 0.00 0.00 0.00 Discharge 1.14 0.03

104 APPENDIX E STREAMWATER PHYSICAL AND CHEMICAL DATA

Physical and chemical data measured in Scott Hollow Cave and Maxwelton Sink Cave.

105 COMMENT Most Upstrem INFEEDER - Mastodon Avenue INFEEDER - Middle Earth Most Downsteam, Sump Downsteam, Most INFEEDER Craig's Creek -9.18% -8.23% -4.27% -8.19% -8.77% -7.82% -7.08% -4.83% -2.90% -7.35% -6.89% Charge Charge Balance Balance -10.13% -10.96% % Error 2- 4 7.83 9.29 10.33 10.28 28.27 10.24 10.21 10.27 10.40 10.48 10.45 10.51 12.45 SO (mg/L) - 3 3.92 NO 11.89 12.01 17.60 13.70 11.68 11.23 11.36 11.45 11.48 11.58 11.68 11.95 (mg/L) - Cl 7.87 7.90 4.07 7.14 7.75 7.85 7.80 7.64 7.70 7.75 7.70 7.72 7.77 (mg/L) 3 244 244 120 244 148 240 245 244 240 236 244 244 232 HCO (mg/L) Mn 0.005 0.148 0.041 0.001 0.030 0.005 0.005 0.004 0.004 0.005 0.083 0.005 0.005 (mg/L) Laboratory Parameters Laboratory Fe 0.050 0.281 0.053 0.011 0.040 0.040 0.030 0.030 0.040 0.040 0.019 0.040 0.040 (mg/L) K 1.99 2.04 2.37 2.68 2.24 2.05 1.98 1.90 1.87 1.94 1.85 1.90 1.96 (mg/L) Na 3.30 3.50 1.50 2.17 3.89 3.34 3.43 3.49 3.65 3.69 3.38 3.38 3.46 (mg/L) 6.1 6.2 9.6 5.8 6.0 6.7 6.7 6.9 7.3 6.8 6.2 6.7 Mg 11.0 (mg/L) Ca 79.7 80.9 32.6 87.1 79.1 81.2 82.2 84.8 86.5 81.8 77.7 72.2 47.2 (mg/L) 7.5 7.6 pH 7.52 7.55 7.72 7.66 7.78 7.54 7.69 7.59 7.59 7.53 6.97 S) µ 444 451 225 454 449 448 446 445 447 444 448 441 357 ( Conductivity Field Parameters Field (C) 11.8 11.8 10.1 12.2 11.9 11.9 11.9 11.9 11.8 11.9 11.9 11.9 14.9 Water Temperature 9.05 8.91 9.50 8.76 9.50 9.26 9.01 8.78 9.14 8.70 9.38 9.34 9.63 D.O (mg/L) - 0 (m) 523 350 49.1 571.8 459.0 Total Total 128.3 189.3 332.2 362.4 422.1 452.9 Distance Upstream 20:00 22:00 20:40 19:15 16:15 16:30 17:30 17:35 15:00 15:30 18:15 18:45 19:00 Time Date 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 11/20/2015 Site SH11 SH13 SH12 SH10 SH3 SH4 SH5 SH6 SH1 SH2 SH7 SH8 SH9 Table E1: Scott Hollow Cave physical and chemical data from November 2015. Table 106 COMMENT Most Downsteam, Sump Downsteam, Most INFEEDER Craig's Creek INFEEDER - Middle Earth Most Upstrem INFEEDER - Mastodon Avenue -9.71% -4.91% -7.74% -5.22% -7.64% -7.02% -8.69% -5.75% Charge Charge 19.73% Balance Balance -11.84% -14.82% -11.59% -10.84% % Error 2- 4 9.37 9.93 SO 11.73 11.96 11.97 12.07 12.07 12.07 17.81 39.58 11.50 11.57 11.59 (mg/L) - 3 - NO 13.37 13.59 13.67 13.71 13.77 15.51 13.90 14.07 14.65 14.03 14.07 14.20 (mg/L) - - Cl 7.39 7.62 7.52 7.53 7.56 6.93 7.64 7.69 7.82 7.69 7.71 7.82 (mg/L) 3 88 228 232 224 228 224 232 224 216 152 228 228 224 HCO (mg/L) Mn 0.001 0.036 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.011 (mg/L) Laboratory Parameters Laboratory Fe 0.012 0.004 0.001 0.001 0.008 0.004 0.001 0.001 0.001 0.019 0.005 0.010 0.166 (mg/L) K 1.63 1.61 1.54 1.60 1.86 2.45 1.62 1.62 1.69 1.42 1.44 1.44 1.20 (mg/L) Na 2.60 2.57 2.57 2.61 2.81 2.15 2.67 2.82 3.28 2.52 2.55 2.56 1.21 (mg/L) 6.1 5.9 6.3 7.1 6.5 6.5 6.5 8.2 6.0 6.3 6.6 Mg 14.5 20.3 (mg/L) Ca 71.6 69.2 73.9 82.6 76.7 83.9 77.2 72.0 50.0 73.9 77.2 80.7 22.9 (mg/L) pH 8.02 8.02 8.00 7.97 7.96 8.18 7.96 7.97 8.18 7.94 8.02 7.95 8.15 S) µ 991 475 ( 1095 1105 1101 1108 1108 1090 1106 1087 1113 1119 1119 Conductivity Field Parameters Field 8.5 (C) 11.0 11.0 10.9 10.9 10.9 10.9 10.9 10.9 10.5 10.9 10.9 10.9 Water Temperature D.O 13.45 12.22 11.81 11.90 11.70 12.23 11.72 11.78 12.17 11.65 11.60 11.65 12.12 (mg/L) - 0 (m) 49.1 Total Total 128.3 189.3 332.2 350.2 362.4 422.1 452.9 459.0 523.0 571.8 Distance Upstream 14:02 14:19 14:29 14:49 15:21 15:44 16:06 16:27 17:05 17:03 17:44 18:18 20:38 Time Date 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 2/13/2016 Site SH1 SH2 SH3 SH4 SH5 SH6 SH7 SH8 SH9 SH10 SH12 SH11 SH13 Table E2: Scott Hollow Cave physical and chemical data from February 2016. Table 107 COMMENT INFEEDER - Heaven Passage - Heaven INFEEDER Most Upstream Pooled water Pooled Flowstone Above waterfall Above Most Downstream, Below waterfall Below Downstream, Most -3.48% -3.60% -3.89% -5.07% -5.65% -3.63% -8.05% -3.89% -4.95% Charge Charge -15.14% Balance Balance % Error 2- 4 15.03 58.78 58.44 39.68 38.38 24.79 21.21 19.06 22.40 SO 20.96 (mg/L) - 3 9.58 8.97 8.98 7.87 7.63 8.70 9.18 9.17 9.96 NO 48.48 (mg/L) - Cl 9.05 8.99 8.90 8.36 8.93 8.21 7.49 8.20 11.24 11.27 (mg/L) 3 204 196 196 168 168 192 204 204 208 200 HCO (mg/L) Mn 0.020 0.020 0.020 0.020 0.030 0.040 0.012 0.040 0.011 0.002 (mg/L) Laboratory Parameters Laboratory Fe 0.100 0.021 0.040 0.030 0.040 0.020 0.046 0.010 0.025 0.011 (mg/L) K 1.51 1.31 1.32 1.35 1.34 1.29 1.20 1.27 1.06 1.15 (mg/L) Na 4.44 6.50 6.57 4.70 4.79 4.42 5.07 4.39 4.85 4.97 (mg/L) 9.6 9.1 8.9 9.2 Mg 10.8 18.9 18.8 13.6 13.2 10.5 (mg/L) Ca 68.7 68.6 68.0 56.8 55.8 67.7 67.7 65.6 73.1 69.1 (mg/L) pH 7.56 7.58 7.58 7.47 7.32 7.40 7.54 7.46 7.36 7.46 S) µ 379 490 488 395 394 407 415 414 415 416 ( Conductivity (C) Field Parameters Field 11.4 11.6 11.7 11.8 11.9 11.7 11.6 11.7 11.6 11.5 Water Temperature 9.14 8.24 9.12 8.78 8.65 9.10 8.46 9.71 8.80 8.84 D.O (mg/L) - (m) 674.8 617.8 607.2 547.4 495.0 330.1 412.4 141.7 Total Total 121.9 Distance Upstream Time 11:45 16:00 15:40 15:25 15:10 14:50 16:20 14:20 16:40 17:20 Date 11/21/2015 11/21/2015 11/21/2015 11/21/2015 11/21/2015 11/21/2015 11/21/2015 11/21/2015 11/21/2015 11/21/2015 Site MX1 MX7 MX6 MX5 MX4 MX3 MX8 MX2 MX10 MX9 Table E3: Maxwelton Sink Cave physical and chemical data from November 2015. Table 108 COMMENT Last Site, No data from 2015 from No data Site, Last Bottom of Big Falls INFEEDER - Heaven Passage - Heaven INFEEDER Most Upstream Pooled water Pooled Below Smaller waterfall Smaller Below Flowstone Above waterfall Above -7.16% -3.67% -2.47% -8.39% -6.35% -6.56% -6.20% -6.33% -6.95% -4.64% -8.80% 33.65% Charge Charge Balance Balance % Error 2- 4 SO 19.49 19.59 13.13 54.41 19.56 53.92 40.20 42.11 17.95 20.20 23.36 20.56 (mg/L) - 3 9.84 NO 10.84 10.83 10.99 11.33 10.81 11.30 10.04 10.56 10.72 10.23 10.90 (mg/L) - Cl 8.30 8.10 8.81 8.07 9.44 7.53 8.30 9.30 8.20 8.36 10.81 10.89 (mg/L) 3 188 188 192 188 200 184 200 188 188 180 192 188 HCO (mg/L) Mn 0.001 0.001 0.001 0.019 0.008 0.001 0.001 0.001 0.001 0.010 0.001 0.001 (mg/L) Laboratory Parameters Laboratory Fe 0.001 0.001 0.001 0.001 0.005 0.001 0.001 0.001 0.001 0.059 0.001 0.001 (mg/L) K 1.39 1.47 1.37 1.34 1.76 1.62 1.77 1.26 1.44 1.60 1.44 1.43 (mg/L) Na 4.03 4.01 3.34 3.99 7.95 5.34 5.98 5.32 4.45 4.13 3.63 4.17 (mg/L) 9.2 9.7 8.7 9.4 Mg 10.7 10.4 17.9 15.6 18.4 10.9 14.9 10.0 (mg/L) Ca 61.8 67.4 67.0 60.1 61.8 58.8 64.7 59.2 63.9 58.1 62.7 160.7 (mg/L) pH 8.00 8.00 7.98 8.03 8.05 8.01 7.79 7.98 7.98 7.95 7.95 7.99 S) µ 974 990 ( 1015 1008 1007 1116 1121 1277 1025 1032 1032 1032 Conductivity (C) Field Parameters Field 11.4 11.4 11.4 11.5 11.4 11.4 11.3 11.4 11.6 11.4 11.3 11.4 Water Temperature D.O 12.00 11.94 11.82 12.59 12.12 11.88 11.74 12.03 11.89 12.00 11.70 11.80 (mg/L) - 0.0 (m) 61.0 Total Total 121.9 674.8 141.7 607.2 617.8 330.1 495.0 547.4 412.4 Distance Upstream 13:12 Time 13:35 13:50 16:00 11:40 14:07 12:01 11:52 14:22 12:23 12:11 14:40 Date 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 2/14/2016 Site MX12 MX11 MX10 MX1 MX7 MX9 MX5 MX6 MX8 MX3 MX4 MX2 Table E4: Maxwelton Sink Cave physical and chemical data from February 2016. Table 109 Table E5: Fe and Mn ICP data for samples MX 1 - 5A and SH 1 - 5A.

Sample Linear Curve Linear Curve Fe (µg/mL) Mn (µg/mL) MX 1 0.1042 0.0202 MX 2 0.0670 0.0923 MX 3 0.0880 0.0896 MX 4 0.0697 0.0142 MX 5 0.0649 0.0135 SH 1 0.0455 0.0065 SH 2 0.0438 0.0050 SH 3 0.0425 0.0076 SH 4 0.0455 0.0076 SH 5 0.1022 0.0981

110 APPENDIX F PHREEQC DATA

Supplementary PHREEQC output data are available on CD only. This includes all PHREEQC output files from the November 2015 and February 2016 sampling trips in Scott Hollow and Maxwelton Sink Caves.

111 APPENDIX G XRD DIFFRACTOGRAMS

Additional birnessite matched XRD diffractograms from manganese oxide coating samples.

112 Figure G1: Diffractogram of SH3

113 Figure G2: Diffractogram of SH4

114 Figure G3: Diffractogram of SH6

115 Figure G4: Diffractogram of SH7

116 Figure G5: Diffractogram of SH8

117 Figure G6: Diffractogram of SH9

118 Figure G7: Diffractogram of MX2

119 Figure G8: Diffractogram of MX3

120 Figure G9: Diffractogram of MX4

121 Figure G10: Diffractogram of MX5

122 Figure G11: Diffractogram of MX6

123 Figure G12: Diffractogram of MX7

124 Figure G13: Diffractogram of MX9

125 Figure G14: Diffractogram of MX10

126