Reference Diatom Assemblage Response to Transplantation into a Stream Receiving
Treatment for Acid Mine Drainage in Southeastern Ohio
A thesis presented to
the faculty of
the College of Arts and Sciences of Ohio University
In partial fulfillment
of the requirements for the degree
Master of Science
Jonathon B. Gray
November 2011
© 2011 Jonathon B. Gray. All Rights Reserved.
2 This thesis titled
Reference Diatom Assemblage Response to Transplantation into a Stream Receiving
Treatment for Acid Mine Drainage in Southeastern Ohio
by
JONATHON B. GRAY
has been approved for
the Program of Environmental Studies
and the College of Arts and Sciences by
Morgan L. Vis
Professor of Environmental and Plant Biology
Howard Dewald
Interim Dean, College of Arts and Sciences 3 ABSTRACT
GRAY, JONATHON B., M.S., November 2011, Environmental Studies
Reference Diatom Assemblage Response to Transplantation into a Stream Receiving
Treatment for Acid Mine Drainage in Southeastern Ohio
Director of Thesis: Morgan L. Vis
Acid mine drainage (AMD) is a prevalent legacy of coal mining within
Appalachia. Streams receiving AMD effluent are drastically altered both chemically and biologically. Hewett Fork, a stream in southeastern Ohio, is one such affected stream.
Although treatment methods have reduced acidity considerably downstream, the ability of Hewett Fork to sustain a biological community compared to those found in reference conditions remains unclear. To assess this, tiles colonized with diatom assemblages from an unimpacted stream were transplanted into Hewett Fork along a stream health gradient, from poor to good, and sampled after one, three, and six weeks in the treated stream.
Chlorophyll a concentrations and species diversity metrics were calculated to compare reference assemblages to transplanted assemblages. Results suggested that after an initial one week acclimation period, assemblages at the uppermost and lowermost sites along the reach were relatively similar to those found in reference conditions, while sites within the middle region continued to show signs of impairment, although the factor(s) causing this impairment remain unknown. These findings suggest that although treatment has been effective on a site-specific basis, the expected linear-response to treatment may not be achieved due to underlying factors that are inhibiting reference-like biological communities from reestablishing within the affected stream reach.
4 Approved: ______
Morgan L. Vis
Professor of Environmental and Plant Biology 5 ACKNOWLEDGMENTS
First and foremost, I would like to thank Dr. Morgan Vis for the uncountable hours she has invested in my success as a graduate student. She was always there to provide me with the encouragement and advice that I needed whenever uncertainty reared its ugly head. I am most grateful for the opportunity to join her lab. It is also imperative that I thank my thesis committee members, Drs. Kelly Johnson and Brian McCarthy for their assistance in the development of my research project as well as their continued support as my project moved forward. I must also acknowledge Nate Smucker for his role in showing me the ropes upon arriving in the lab and for continuing to put up with my inquiries into…everything. Also, Justin Pool for taking me out into the world of southeastern Ohio and sharing with me the sage-like advice of an experienced fellow tile scraper. Furthermore, I would like to thank Lisa DeRose, Dr. Daryl Lam, Mariah Thrush, and Lauren Fuelling for assisting me in sampling the wilds of Hewett Fork; we came, we saw, we sampled! And I can’t forget my lab mates, Emily Johnston, Eric Salomaki,
Lauren F. (as previously mentioned) for making our little office into so much more than a workspace; you made it feel like a home. Not to mention the entirety of the
Environmental Studies Program and the Department of Environmental and Plant Biology.
Thanks to the Phycological Society of America for awarding me the Croasdale
Fellowship so that I might attend Diatom Camp, as well as Drs. Edlund and Potapova for introducing me to diatom ecology. And for that matter, thank you diatoms! Thank you to all of the following funding sources for their generosity and support: Graduate Student
Senate Original Work and Travel Grants, the Voinovich School of Leadership and Public 6 Affairs (especially Jen Bowman!), the Department of Environmental and Plant Biology, and the OU Research and Creativity Expo.
Finally, there is no way any of this would have been possible without the support of my wife, Mindy. Is it any wonder that the last ten years of my life have been the best years thus far? I love you, Minders. 7 TABLE OF CONTENTS
Page
Abstract...... 3 Acknowledgments...... 5 List of Tables ...... 9 List of Figures...... 10 Introduction...... 12 Materials and Methods...... 20 Results...... 30 Discussion...... 37 Conclusions and Future Directions...... 43 References...... 44 Appendix A: Diatom Growth Forms and the Representative Genera, with Descriptions of Growth Form and Potential Costs/Benefits of that Growth Form...... 71 Appendix B: Acid Mine Drainage Diatom Index of Biotic Integrity (AMD-DIBI) Component Metrics from Zalack et al. (2010)...... 73 Appendix C: Habitat Characteristics at the Time of Initial Tile Colonization, Transplantation, and 1-, 3-, and 6-Weeks After Transplantation. N/A in Water Velocity Represents Below Detectable Levels of Measurement, yet Definite Flow was Visible... 75 Appendix D: Water Chemistry for Each Site at 1, 3, and 6 Weeks After Transplantation...... 77 Appendix E: Number of Cumulative (n = 3) Taxa Identified at Each Site for Week 1 Sampling...... 80 Appendix F: Bray-Curtis Dissimilarity Scores for Each Site and Sampling Time Using Pooled Replicates (n = 3). Mean is the Mean Dissimilarity Between Clear Creek and Hewett Fork Sites...... 94 Appendix G: Bray-Curtis Dissimilarity Scores for Each Site and Sampling Time, Rounded to Four Decimal Places...... 95 Appendix H: Number of Cumulative (n = 3) Taxa Identified at Each Site for Week 3 Sampling...... 98 Appendix I: Number of Cumulative (n = 3) Taxa Identified at Each Site for Week 6 Sampling ...... 112 Appendix J: Number of Cumulative (n = 3) Taxa Identified at Each Site from the Comparative Study of Clear Creek, HF039 and HF090...... 129 8 Appendix K: Bray-Curtis Dissimilarity Scores for Clear Creek and the Most Downstream (HF039) and Upstream (HF090) Sites on Hewett Fork After a 6-Week Colonization Period Using Pooled Replicates (n = 3). Mean is the Mean Dissimilarity Between Clear Creek and Hewett Fork Sites...... 142 2 Appendix L: The χ 0.05,1 Values from Comparisons of Growth Forms Between Clear Creek and HF039, and Clear Creek and HF090 After a 6-Week Colonization Period... 143 9 LIST OF TABLES
Page
Table 1: Mean and range (in parentheses) historical water chemistry data for Hewett Fork for months June - September, with n = number of samples available for each parameter from www.watersheddata.com ...... 52 Table 2: Mean and range (in parentheses) historical biodiversity data for Hewett Fork for sampling months June – September, with n = the number of samples available for each parameter from www.watersheddata.com and Pool (2010). N/A represents no data available due to limited samples available (in the case of data ranges) or a lack of samples for that site ...... 53 Table 3: Mean water chemistry (n = 3; range in parentheses) from each site for the duration of the study. Bold results are significantly different from the Clear Creek reference site with asterisks representing significance level (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001). Power analysis of sites not found to be significantly different found that in order to achieve an acceptable power level (0.8), n = 15...... 54 Table 4: Row-wise Spearman rank correlations between water chemistry samples from each site for the duration of the study. Significant results in bold, with asterisks representing significance level (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001 ...... 55 Table 5: Z-scores and associated p-value for comparison of current and historical Hewett Fork water chemistry data. Scores in bold indicate no significant (P ≤ 0.05) differences between current and historical data...... 56 Table 6: Mean (n = 3) Acid Mine Drainage-Diatom Index of Biotic Integrity (AMD- DIBI) scores for each site at 1, 3, and 6 weeks after transplantation. Metric scores are derived from methods developed by Zalack et al. (2010). Scores that increase with impairment are divided by the 90th percentile of site values, subtracted from 1, and multiplied by 10, while scores that decrease with impairment are divided by the 90th percentile of site values and multiplied by 10. Acidophilic sp. metric scores were multiplied by -1 to correspond with the increased level of impairment associated with greater abundances of acidophilic taxa ...... 57 Table 7: Mean (n = 3) Acid Mine Drainage-Diatom Index of Biotic Integrity (AMD- DIBI) scores for Clear Creek and the most downstream (HF039) and upstream (HF090) sites on Hewett Fork. Metric scores are derived from methods developed by Zalack et al. (2010). Scores that increase with impairment are divided by the 90th percentile of site values, subtracted from 1, and multiplied by 10, while scores that decrease with impairment are divided by the 90th percentile of site values and multiplied by 10. Acidophilic sp. metric scores were multiplied by -1 to correspond with the increased level of impairment associated with greater abundances of acidophilic taxa ...... 58
10 LIST OF FIGURES
Page
Figure 1. Map of Hewett Fork drainage basin. Sampling sites are marked by black diamonds and labeled as designated by the Raccoon Creek Watershed Group. River mile is noted in parentheses. Acid inputs to Hewett Fork are located at sites HF131, HF190, and HF120. The doser is located at HF131. The study reach consisted of sites HF090, HF075, HF060, HF045, and HF039...... 59 Figure 2. Example of the tile arrays used for diatom colonization...... 60 Figure 3. One-way ANOVAs of species richness, species evenness, Shannon diversity, and percent relative abundance of dominant taxon for each site 1-week after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Sites are labeled as in Figure 1. Arrow shows longitudinal position of Hewett Fork sites. Bars with the same letters are not significantly different (P ≤ 0.05)...... 61 Figure 4. Chlorophyll a (mg m-2) for each site 1, 3, and 6 weeks after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical dashed line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Arrow shows longitudinal position of Hewett Fork sites. Sites are labeled as in Figure 1. Bars with the same letters are not significantly different (P ≤ 0.05). Power analysis found that in order to achieve an acceptable power level (0.8), n = 15...... 62 Figure 5. Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarities (stress = 7.1732) of diatom assemblage compositions for all sites...... 63 Figure 6. One-way ANOVAs of species richness, species evenness, Shannon diversity, and percent relative abundance of dominant taxon for each site 3 weeks after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Sites are labeled as in Figure 1. Arrow shows longitudinal position of Hewett Fork sites. Bars with the same letters are not significantly different (P ≤ 0.05)...... 64 Figure 7. Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarities (stress = 8.0813) between diatom assemblage compositions for all sites 3 weeks after transplantation, with an overlay of water chemistry variables that significantly correlate with AMD-DIBI scores. Ellipses were placed around replicates using the ordiellipse sub-routine of the Vegan package in order to visualize variation within sites, as larger ellipses represent greater variation of within-site taxonomic composition...... 65
11
Figure 8. One-way ANOVAs of species richness, species evenness, Shannon diversity, and percent relative abundance of dominant taxon for each site 6 weeks after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Sites are labeled as in Figure 1. Arrow shows longitudinal position of Hewett Fork sites. Bars with the same letters are not significantly different (P ≤ 0.05)...... 66 Figure 9. Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarities (stress = 7.0136) between diatom assemblage compositions for all sites 6 weeks after transplantation, with an overlay of water chemistry variables that significantly correlate with AMD-DIBI scores. Ellipses were placed around replicates using the ordiellipse sub-routine of the Vegan package in order to visualize variation within sites, as larger ellipses represent greater variation of within-site taxonomic composition...... 67 Figure 10. Comparison of species richness, evenness, and Shannon diversity and percent relative abundance after 6-week colonization period at Clear Creek (CC) and the most downstream (HF039) and upstream (HF090) sites within the Hewett Fork study reach. Bars represent mean (n = 3) diversity index score per site, with lines representing the mean +/- 1 SE (n = 3). Bars with the same letters are not significantly different (P ≤ 0.05). Sites are labeled as in Figure 1...... 68 -2 Figure 11. Log10 transformed chlorophyll a (mg m ) after ...... 69 Figure 12. Comparison of mean (n = 3) proportional composition of diatom growth forms at Clear Creek, HF039, and HF090 after a six-week colonization period. Bars may not sum to 1 due to the use of mean proportion values...... 70
12
INTRODUCTION
Acid mine drainage (AMD) is a prevalent legacy of intense mining within the
Western Allegheny Plateau (WAP) ecoregion of the United States, affecting an estimated
1,300 stream miles in Ohio alone (Omernik, 1987; Ohio Environmental Protection
Agency [OEPA], 2000; Farley and Ziemkiewicz, 2005). AMD is the result of a series of oxidation reactions occurring when remnant sulfur-rich minerals, commonly associated with coal seams, are exposed to oxygen, sulfidogenic bacteria, and water, all flowing through (often abandoned) mine complexes or over mine tailings (Warner, 1971). The resulting mine effluent, i.e. AMD, is a solution containing elevated levels of sulfuric acid
(H2SO4), iron oxides (FeO2), and a host of other metals that greatly impact receiving waters (Warner, 1971).
As seeps of AMD reach previously unaffected waters, they have a detrimental impact on the ecosystem, drastically altering the chemical, and therefore biological characteristics of the receiving waters in what could be considered a biphasic manner
(Parsons, 1977; McKnight and Feder, 1984; Johnson, 2003). During the first phase, the elevated H2SO4 concentrations are responsible for reducing the pH of receiving waters, which in turn, increase metal solubility and soluble metal cation concentrations thus increasing conductivity (Filipek et al., 1987; Sabater et al., 2003; Bray et al., 2008). The second phase occurs upon the dilution of the H2SO4, thereby neutralizing its capacity to affect stream pH. At this juncture, the previously dissolved metals may form metal oxide precipitates (most commonly Fe(OH)3) that may coat the stream bottom in a fine metal- 13 laden sediment, greatly reducing habitat availability for fish, macroinvertebrates, and periphyton (McKnight and Feder, 1984; Niyogi et al., 2002; Lear et al., 2009).
Ultimately, AMD-related alterations to stream chemistry and physical habitat contribute to the decline of biotic integrity within the impacted stream. Associated acidity
(Gerhardt et al., 2004; Lear et al., 2009), metal solubility/deposition (Niyogi et al., 2001;
Sabater et al., 2003; Butler, 2009), and the resulting multi-level trophic disruption have detrimental effects on the biological assemblages (i.e. bacteria, algae, macroinvertebrates, and fish) and their functionality within the affected stream (Niyogi et al., 2002; Bray et al., 2008; Simon et al., 2009; Smucker and Vis, 2011). Bacterial components of biofilms may experience phosphorus (P) limitation due to P sorption to iron, thereby limiting P retention in affected streams (Smucker and Vis, 2011). Furthermore, deposition of metal oxides may decrease bacterial respiration rates, thereby reducing the effectiveness of these communities to decompose leaf litter, further reducing the available nutrient pool in the impacted stream (Niyogi et al., 2001; Simon et al., 2009). Periphyton community structure becomes altered due to the replacement of acid/metal sensitive species with more tolerant taxa, resulting in decreased biological diversity in primary producers
(Lampkin and Sommerfeld, 1982; Verb and Vis, 2000; Niyogi et al., 2002). Stresses caused by increased osmotic pressure due to higher conductivity can affect diatom growth rates and nutrient uptake (Potapova and Charles, 2003). Thus AMD pollution has contrasting effects on algal biomass in impacted streams, albeit at differing phases of impairment. Nearer to the source of the AMD input, at lower pH, algal biomass can increase as specialist taxa dominate. As acidic waters are diluted, there is a negative correlation between biomass and metal oxide precipitation (Bray et al., 2008). 14 Macroinvertebrate communities are similarly affected, as species diversity is lost through replacement by more specialized and tolerant taxa (Gerhardt et al., 2004).
Further alteration of trophic dynamics may be observed as plasticity in macroinvertebrate feeding behaviors may serve to reduce leaf litter degradation rates (Dangles, 2002). Fish, the most capable of escaping the impacts of AMD, are either found in limited numbers or are completely absent from the degraded site (Parsons, 1977; Karr, 1991).
Prior to 1977, limited government regulation on mining activities allowed companies to expend little effort in reclamation of mined sites (ODNR, 2009). The passage of the Surface Mining Control and Reclamation Act (SMCRA) in 1977 drastically altered the practices associated with mining. Henceforth, all mining permits were bonded to ensure that mine land restoration to governmental standards was an integral part of the operations. Furthermore, SMCRA created an abandoned mine land fund in order to address the degradation that had occurred prior to its passage (SMCRA,
1977). Since that time, increasing efforts have been undertaken not only to address sources of AMD pollution but also to mitigate the effects of AMD stemming from abandoned and closed mining operations. To achieve these goals, a number of methods have been adopted. Passive and active abiotic and biotic treatments have had some degree of success in the neutralization of acidity in AMD impacted streams (Johnson and
Hallberg, 2005). Abiotic treatment methods, such as dosers, anoxic limestone drains, and steel slag leach beds add alkaline materials to neutralize the acidity (Simmons et al.,
2002). Dosers (large storage tanks installed near AMD seeps) dispense a predetermined quantity of alkaline reagent at a controlled rate into affected waters (Johnson and
Hallberg, 2005). Anoxic limestone drains, a passive remediation option, use similar 15 techniques of adding a buffering reagent to acidified water in the form of limestone gravel. The dissolution of the limestone increases the stream pH without the need for additional inputs of lime, while the anoxic drain prevents the oxidation of ferric iron and aluminum (Clyde et al., 2010). Steel slag leach beds are a series of ponds along an AMD seep that consist of a limestone gravel bottom and a high-alkaline (CaCO3) steel slag check dam (Simmons et al., 2002). Biotic treatment systems utilize organisms that produce alkaline by-products and/or immobilize metals to remediate AMD affected streams (Johnson and Hallberg, 2005; Clyde et al., 2010). Active biological methods, such as off-line sulfidogenic bioreactors, utilize sulfur-reducing bacteria (SRB) to reduce
2- SO4 , allowing for the formation of alkaline buffers (Johnson, 2003; Clyde et al., 2010).
In these systems, metal recovery is also a possibility, reducing the potential for the precipitation of metals back into the remediated stream (Johnson and Hallberg, 2005).
Passive biotic treatments such as aerobic and/or anaerobic wetlands are also commonly employed to mitigate AMD streams. These treatments rely on either SRB (aerobic) or organic waste materials (anaerobic) to reduce the sulfate in acidified waters (Johnson and
Hallberg, 2005), and will often include limestone drains in order to polish the effluent
(Clyde et al., 2010). Although each of these methods does contribute to the abatement of the chemical alterations associated with AMD, each treatment also results in the creation of a ‘sacrifice zone’, a length of stream extending for a mile or more downstream from the treatment site, in which the precipitation of metal oxides precludes the reestablishment of the structure and function of biological communities (McKnight and
Feder, 1984; Farley et al., 2004; Bray et al., 2008; Borch, 2010). 16 While previous reclamation efforts have primarily been concerned with alleviating the impacts of AMD on water chemistry, the ever-growing body of literature acknowledging the link between species diversity and overall ecosystem functionality has shifted the focus of restoration towards a more holistic, ecological approach (Karr, 1991;
Battaglia et al., 2005; Dodds et al., 2008; Sabater and Stevenson, 2010). Recent studies have observed significant losses in ecosystem functionality with a decrease in species diversity, indicating that measures of species diversity in and of itself may therefore be an effective means of assessing ecosystem health (Benayas et al., 2009; Cardinale et al.,
2011). Moreover, given the chemical variability of natural and impaired waters, assessing the biological components of an ecosystem can provide greater insight into the longer- term impacts that stressors may have on an ecosystem (Karr, 1981). Currently, there are a number of methods available in which ecological measures can be used as a means of assessing water quality. Community structures of fish, macroinvertebrates, and periphyton have all been used to assess biological integrity of aquatic systems (Karr,
1981; USEPA, 2003; Wang et al., 2005; Zalack et al., 2010). Most often the biological information is summarized in indices, which are composed of metrics that convey biological information about stream health. These structural multi-metric indices are the primary means of utilizing biological information to infer stream health and provide invaluable assistance to resource managers and the implementation of management plans along stressor gradients (USEPA, 2003). Surveys of biological communities are completed to assess the response of communities to a number of chemical/physical variables. Metrics such as species richness (S), species evenness (Jʹ), Shannon diversity
(Hʹ), and abundance of taxa with known species-specific tolerance/sensitivity to a 17 stressor can be determined and given a score (Karr, 1981; Wang et al., 2005; Zalack et al., 2010). The summation of all scores/metrics is the biological index used to infer stream health (Karr, 1981; Wang et al., 2005; Zalack et al., 2010).
Diatoms, microscopic algae that are ubiquitous in aquatic habitats, have become more widely recognized as a highly effective in assessing aquatic health (Lowe and Pan,
1996; Stevenson and Pan, 1999; Hill et al., 2000). Diatoms are good bioindicators due to their close relationship to the stream chemistry, rapid rate of reproduction, and their role in trophic dynamics (Mayer and Likens, 1987; Stevenson and Pan, 1999; Shieh et al.,
2002; Hirst et al., 2004). Successional assemblage development (from adnate, to erect, and finally to stalked growth forms) allows for biofilm maturity to be assessed, such that structural components of assemblages, based on growth form abundances, can aid in assessing site-specific stressors (Appendix A) (Hoagland et al. 1982; Stevenson, 1983).
In conjunction with known species-specific tolerances for an array of environmental variables, diatom assemblages can provide significant insight into assemblage health, allowing for quantification of site quality based on the taxa present at the site (Lowe,
1974; van Dam, 1982; Dixit et al., 1992; Potapova and Charles, 2002).
Due to their abundance and response time to water variations, several American states and European countries have adopted diatom-based metrics as standard water quality measuring methods (Barbour et al., 1999; European Union, 2000; Kelly et al.,
2008). The development of diatom indices further adds to the relevance of diatoms as biomonitoring tools, as it can assess ecosystem health using the diatom assemblages that are present (Hill et al., 2000; Wang et al., 2005, Zalack et al., 2010). Zalack et al. (2010) developed the AMD-DIBI, an index specifically designed for AMD impacted systems, 18 allowing for a comprehensive assessment of stream health along an AMD gradient.
The AMD-DIBI is an index comprised of nine metrics that utilize structural aspects of diatom assemblages (Appendix B) to assess environmental conditions. Assemblage data from study sites are compared to reference data in order to quantify assemblage structure for each metric. These comparisons are then scaled to 0-10, allowing for each metric to be given a score based on its similarity to that of the reference assemblage. These scores are summed to provide an AMD-DIBI score for each site. This final score allows for the sites to be characterized into four narrative classes, each representing stream quality (≥ 59 excellent, 42-58 good, 23-41 fair, and ≤22 poor) (Zalack et al., 2010). The AMD-DIBI was shown to be similarly responsive to a stream receiving treatment for AMD as well, qualifying the robustness of such an index (Pool, 2010).
Since diatom communities are readily used as indicators of water quality and ecological health, and indices have been developed, they seem to be well suited to assess stream restoration. The purpose of this research is to examine the effects of AMD remediation on diatom assemblages along a treatment gradient as a means of assessing the ability of treated sites to provide habitats for assemblages indicative of the targeted habitat quality of the restoration project. In order to examine treatment effectiveness, this project was divided into two objectives: 1) to determine the efficacy of acid mine drainage (AMD) mitigation on restoring an AMD-impacted stream’s ability to sustain transplanted reference diatom assemblages, and 2) to compare the diatom assemblage structure of a reference assemblage to assemblages from the most upstream and downstream sites within a stream reach receiving treatment for AMD. 19 To achieve the first objective, I studied a diatom assemblage that had been colonized in Clear Creek, an unimpaired reference site, and transplanted to five sites along an AMD-treatment gradient in a small southeastern Ohio stream, Hewett Fork (Fig.
1) (OEPA, 1997a). It is my hypothesis that a) reference taxa will be extirpated and replaced with taxa more representative of the stress gradient, such that stress-tolerant taxa will dominate the most upstream/impaired site and will gradually shift to a more stress- sensitive and diverse assemblage with downstream distance from the doser, and b) that species diversity indices, reduced biomass (chlorophyll a concentrations), and lowered
AMD-DIBI scores will reflect an increasing trend in stream health with downstream distance from the doser.
For the second objective, I examined diatom assemblages that had been colonized for 6-weeks at Clear Creek and the most upstream and downstream sites within the
Hewett Fork study reach. I hypothesize that a) after a 6-week colonization period, mature biofilms will be present, and that b) species diversity indices, as well as shifts in growth form composition, algal biomass, and AMD-DIBI scores, will reflect an increasing trend in stream health with downstream distance from the doser.
20
MATERIALS AND METHODS
Site Description
Hewett Fork is the primary tributary of Raccoon Creek and drains approximately
105 km2 of primarily (~ 70-75%) forested landscape within the Western Allegheny
Plateau (WAP) Level III Ecoregion in southeastern Ohio (Rice et al., 2002). Within
Hewett Fork, a number of AMD inputs have been identified. The primary input of AMD occurs at two seeps near river mile 11, the site of the abandoned Rice Hocking underground mine (hereafter HF131) (Farley et al., 2004). Two additional AMD inputs are located further downstream, one at the confluence of Hewett Fork with Carbondale
Creek (hereafter HF120), and the other at the confluence of Hewett Fork and Trace Run
(hereafter HF190), both occurring near river mile 10 (Fig. 1). The cumulative effects of these seeps have dramatically diminished the stream quality for the length of the Hewett
Fork, resulting in the stream receiving a limited resource water – acid mine drainage
(LRW-AMD) designation from the Ohio Environmental Protection Agency (OEPA)
(OEPA, 1997b). The LRW-AMD designation is assigned to streams in which biological communities are comprised of species tolerant of AMD conditions, specifically lowered pH and increased metal concentrations (OEPA, 1997b).
To address these diminished stream conditions, the Ohio Department of Natural
Resources (ODNR) developed a plan for restoration in which treatment wetlands would be placed at HF131, the site of the primary influx of acidity. The targeted goal of this restoration project was to ameliorate the impacts of AMD on stream biota in order to 21 increase biological diversity and to bring the stream to warm water habitat status, the designation commonly assigned to unimpacted sites within the region (Rice et al., 2002).
Although the treatment wetlands were responsible for the removal of a considerable amount of the targeted pollutants; i.e., iron and aluminum, approximately
328.9 kg day-2 of acid continued to discharge from the wetlands into Hewett Fork (Farley et al., 2004). In 2004, ODNR Division of Mines and Reclamation replaced the treatment wetlands with an Aqua-Fix® calcium oxide (CaO) doser and a concrete mixing channel at HF131 in hopes of actively treating the primary AMD input. The doser and subsequent mixing channel are able to continuously introduce alkaline materials into the mine waste stream prior to its confluence with Hewett Fork, allowing for an immediate buffering of the AMD. While this alkaline addition has substantially increased the pH downstream of the doser, including an increase in the buffering capacity at both HF120 and HF190
(Farley et al., 2004), the concomitant metal precipitation associated with this pH shift has resulted in a “sacrifice” zone; i.e. a reach approximately one-mile designated to absorb the precipitated metals, consequentially inhibiting localized biotic recovery to facilitate recovery further downstream, occurring immediately downstream of the doser and extending for approximately one-mile downstream (Farley et al., 2004; Borch, 2010).
The specific study area within Hewett Fork consists of five sites (Fig. 1) in what appears to be a transition zone from poor to good habitat quality for fish, macroinvertebrates, and diatoms along an AMD treatment gradient. Three of these sites, river miles 8.3 (HF090), 6.4 (HF060), and 4.0 (HF039), are current sampling points used by the Raccoon Creek Partnership for biological (fish and macroinvertebrates) and chemical monitoring and have been given designations of “impaired,” “intermediate,” 22 and “good,” respectively based on their biological and chemical characteristics (Tables
1 and 2). To better study the biological and chemical aspects of this reach, two additional sites were established summer 2010, one between river miles 7.4 and 7.2 (hereafter
HF075), and the other at river mile 5.0 (hereafter HF045), to allow for finer scale monitoring of the AMD treatment gradient and its effect on fish, macroinvertebrate, and diatom assemblage compositions.
Objective 1: Comparison of reference diatom assemblage structure after transplantation from Clear Creek into Hewett Fork
In order to assess AMD-treatment efficacy on Hewett Fork’s ability to sustain reference-like diatom assemblages, it was first necessary to colonize artificial substrate so that diatom assemblages representative of reference conditions could be transported to
Hewett Fork. To maximize diatom colonization, six tile arrays consisting of 48 25.8 cm2 unglazed ceramic tiles were attached to wire-mesh with pipe cleaners and affixed parallel to the substratum with rebar (Fig. 2) within a riffle of the reference site, Clear Creek, during late June 2010 (OEPA, 1988; OEPA, 1997a). A 6-week colonization period was observed to allow development of a mature biofilm (Gale et al., 1979; Hoagland et al.,
1982). Measurements of water velocity, depth, temperature, conductivity, pH, and percent canopy cover were recorded, as well as qualitative substratum characteristics, presence of woody debris/leaf litter, and potential grazers. Water velocity and depth were recorded with a FP101 Global Flow Probe (Global Water, Gold River, California), and percent canopy cover was determined using a handheld densiometer (Spherical
Densiometer Model-C, Forest Densiometers, Bartlesville, Oklahoma). In situ conductivity and pH were determined using handheld probes (Oakton Waterproof 23 ECTestr low and Waterproof pH Testr 30, Oakton Instruments, Vernon Hills, Illinois).
- 2- In addition, water samples were collected for nitrate (NO3 ), orthophosphate (PO4 ), total
2- iron (Fe) (required digestion), total aluminum (Al), and sulfate (SO4 ) analysis and were transported back to the lab on ice.
At the end of the six-week colonization period, tile arrays were each randomly assigned to one of the six sites used in this study. Five of these arrays were placed at sites along the AMD-treatment gradient on Hewett Fork (sites HF039, HF045, HF060, HF075, and HF090, from downstream to upstream, respectively). The sixth tile array, which was designated as the control array, was removed, transported with the other tile arrays, and replaced at the reference site (Clear Creek). Transport of the control array was deemed necessary in order to mimic the potential disturbance experienced by the other arrays during transport. The transport process was to remove all tile arrays (Clear Creek, HF039,
HF045, HF060, HF075, and HF090) from Clear Creek and placed them in 38.8 L
Sterilite™ containers with stream water to prevent desiccation and to minimize biofilm disturbance during transport. Upon reaching the transplant sites, specific microhabitat selection for tile placement was determined by choosing riffles most representative of the reference colonization site conditions, i.e. water depth, water velocity, substratum, and canopy cover, in an attempt to minimize habitat heterogeneity between sites. After adequate microhabitats were chosen, the tile arrays were again affixed parallel to the substratum.
After 1, 3, and 6 weeks, 3 replicates of 4 tiles each were randomly selected from each of the six sites (Clear Creek, HF039, HF045, HF060, HF075, and HF090) and were cut from the tile arrays. Biofilm was removed via scraping with a razor blade and 24 scrubbing with a hard-bristled brush into acid washed Nalgene™ bottles containing stream water, which were transported to the lab on ice and stored in the dark at 4o C until they were processed for diatom enumeration and chlorophyll a analysis. In the case that the randomly selected replicate was too over laden with silt or debris, the next replicate was chosen instead. Measurements of water depth, water velocity, pH, conductivity, temperature and water samples for analysis were collected in a similar manner as described above each time tiles were sampled.
Within 24 hours of returning to the laboratory, water samples were shaken to suspend solutes, and 10 mL subsamples were removed for turbidity measurements using a HACH 2100P™ portable turbidimeter (HACH 2100P™ Turbidimeter, HACH
Company, Loveland, Colorado). The remaining water samples were filtered with
Milipore© 0.45 µm white gridded filters prior to chemical analysis. Filtered water was
- adjusted for pH if required and analyzed for nitrate (NO3 ) (method 8192),
2- orthophosphate (PO4 ) (method 8048), total iron (Fe) (required digestion; method 8008),
2- total aluminum (Al) (method 8012), and sulfate (SO4 ) using EPA approved methods via a HACH DR/890™ colorimeter (HACH DR/890™ Colorimeter, HACH Company,
2- Loveland, Colorado) and HACH powder pillows (Hach Company, 2009). As SO4 concentrations for the Hewett Fork sites exceeded readable limits of the colorimeter, the
2- Hewett Fork samples used for SO4 were diluted at a 5:1 distilled H2O to sample ratio for weeks 1 and 3, and 10:1 distilled H2O to sample ratio for week 6.
All biofilms were processed within 24 hours of collection. After homogenizing the contents, 1 mL aliquots of each sample were filtered onto Whatman GF/G 0.6 µm filters, which were then wrapped in aluminum foil and frozen until chlorophyll a analysis 25 could be performed. For analysis, filters were steeped in 8 mL of 90% acetone at 4o C for 20 hours in the dark, after which time the leachates were analyzed for chlorophyll a concentrations using a Turner TD-700 fluorometer (TD-700 Laboratory Fluorometer,
Turner Designs, Sunnyvale, California) (Arar and Collins, 1997).
For diatom enumeration, 25 mL subsamples of the homogenized biofilm solution were boiled in 30% H2O2 for 1 hour, followed by an additional 30 minutes of boiling in
50% HNO3 to remove all organic matter (Stoermer et al., 1995). Treated composite samples were placed in a 250 mL beakers, diluted with approximately 200 mL of distilled water, and allowed to settle before removing the supernatant. This process was repeated until the samples reached a circumneutral pH. Cover slips were placed into evaporation tray cells and immersed in 10 to 25 ml of each sample, dependent upon diatom density, and left to settle in an evaporation chamber until dry, allowing for random dispersal of diatoms (Battarbee, 1973). Finished cover slips were removed with forceps and mounted on slides using Naphrax™, a highly reflective mounting medium, and heated on hotplates to remove excess solvent. Once completed, slides were viewed with a light microscope at
1000× magnification. Counts of 500 valves per sample were performed along an 18 mm transect, with each valve identified to the lowest taxonomic level using multiple taxonomic references (Patrick and Reimer 1966, 1975; Krammer and Lange-Bertalot
1986, 1988, 1991a, 1991b), with nomenclature updated to reflect the current taxonomic status as per definitions from AlgaeBase (www.algaebase.com, accessed 08/01/11). For samples in which there were fewer than 500 valves present, all transects were counted in their entirety and all valves were identified to the lowest taxonomic level. 26 Objective 2: Comparison of in situ diatom assemblages at the most upstream and downstream site on Hewett Fork with the reference site, Clear Creek
In order to compare the in situ diatom assemblages at Clear Creek versus the most upstream and downstream sites on Hewett Fork (HF090 and HF039), tile arrays consisting of 48 25.8 cm2 unglazed ceramic tiles were attached to the substratum at Clear
Creek in late June (06/24/10) and sites HF039 and HF090 in early August (08/08/10), as previously described. A 6-week colonization period was observed at each site (Clear
Creek: 06/24/10 - 08/08/10; HF039 and HF090: 08/08/10 – 09/23/10) to allow for the development of a mature biofilm (Gale et al., 1979; Hoagland et al., 1982).
At the end of the 6-week colonization period, 3 replicates of 4 tiles each were randomly selected from each of the three sites (Clear Creek, HF039, and HF090) and were cut from the tile arrays. Biofilms were collected and processed as previously described for diatom enumeration and chlorophyll a analysis.
Data Analysis
Exploratory data analysis was performed for water chemistry and diatom assemblages for all sites and sampling times using version 2.12.2 of R statistical software
(R Foundation for Statistical Computing, Vienna, Austria).
One-way analysis of variance (ANOVA) tests were performed between reference site water chemistry variables and those of Hewett Fork to determine if there were any significant chemical differences between the reference and treatment sites based solely on cumulative water chemistry data. The pH data were transformed to H+ concentrations prior to making comparisons. Data were tested for violations of assumptions of normality and variance prior to running ANOVAs using Shapiro-Wilks and Bartlett tests, 27 2- 2- respectively. The parameters PO4 , SO4 , and Al failed to meet these assumptions and were transformed (1/x). Furthermore, current water chemistry data were compared to available historical data (www.watersheddata.com) for samples collected between June and September. Comparisons of temperature, total Al, total Fe, pH, conductivity, and
2- SO4 were examined using a Ζ-test to determine if collected data significantly differed from historical data.
Total diatom taxa were enumerated to determine the mean (n = 6) Bray-Curtis dissimilarity score between Clear Creek and Hewett Fork sites, as well as to derive diatom assemblage structure metrics, including species evenness (J´), species richness
(S), relative abundance of dominant taxon, and Shannon diversity (H´) for each site and sampling time for both objectives using version 1.17-6 of the Vegan package for R
(Oksanen et al., 2011). Chlorophyll a concentrations and assemblage metrics were tested for violations of assumptions of normality and variance using Shapiro-Wilks and Bartlett tests, respectively. Chlorophyll a from the non-transplanted tiles (objective 2) failed to meet the tests for normality; therefore non-transplanted chlorophyll a concentrations were log transformed to meet normality assumptions. One-way ANOVAs were performed to assess variation of the means of assemblage metrics and chlorophyll a concentrations among treatment sites and Clear Creek. Fisher’s Least Significant Difference (LSD) test from the Agricolae package (de Mendiburu, 2010) was utilized to discern significant differences between sites, with the results being plotted using base R graphing tools or the Sciplot package (Morales et al., 2010). In cases in which ANOVAs find no significant differences, power analyses were performed using the pwr package
(Champley, 2009). 28 To further explore overall similarity of the study assemblage structure to those of Clear Creek, AMD-Diatom Index of Biotic Integrity (AMD-DIBI) scores, as developed by Zalack et al. (2010), were calculated for each site at each sampling date.
AMD-DIBI scores are derived from the summation of 9 metric scores (Appendix B) that either increase or decrease with AMD-associated impairment, allowing for the multi- metric approach to the characterization site quality based on diatom assemblages
(KYDOW, 2002; Wang et al., 2005; Zalack et al., 2010). Scores that increase with impairment are divided by the 90th percentile of site values, subtracted from 1, and multiplied by 10, while scores that decrease with impairment are divided by the 90th percentile of site values and multiplied by 10 (Wang et al., 2005; Zalack et al., 2010).
Acidophilic sp. metric scores were multiplied by -1 to correspond with the increased level of impairment associated with greater abundances of acidophilic taxa. The final score for each metric was rounded to the nearest integer prior to summation. Final AMD-DIBI scores were used to classify each site as excellent (score ≥ 59), good (42-58), fair (23-41), or poor (≤ 22) (Zalack et al., 2010). Due to multiple replicates collected for each sample site/time, metric scores were calculated for each replicate with the mean score used as the overall site score for each sampling time. Spearman rank correlations were performed with the Hmisc package (Harrell et al., 2010), to examine potential relationships between
AMD-DIBI scores and water chemistry for each sampling time.
Bray-Curtis (BC) dissimilarity coefficients were calculated for each site and sampling time as well (Bray and Curtis, 1957) and ordinated using non-metric multidimensional scaling (NMDS) in the Vegan package. Scree plots were utilized to determine the lowest number of dimensions necessary to achieve an acceptable stress 29 level. The NMDS was set to run a maximum of 500 times and used random starting configurations until the same least-stress solution was reached twice (Kruskal, 1964).
Ellipses were placed around replicates using the ordiellipse sub-routine of the Vegan package in order to visualize variation within sites, as larger ellipses represent greater variation of within-site taxonomic composition.
Tiles colonized at Clear Creek and HF039 and HF090 for six weeks without undergoing transplantation (objective 2) were compared to examine natural assemblage composition. Comparisons were made using one-way ANOVAs to determine significant differences in the community metrics as well as chlorophyll a concentrations. AMD-DIBI scores were calculated for all three sites at the end of the six weeks. Mean (n = 3) Bray-
Curtis dissimilarity score were calculated for each site. Assemblage growth form composition was examined as a means of inferring potential stressors that are not necessarily AMD-specific; i.e. grazing pressure (Hoagland et al., 1982; Stevenson, 1983).
Qualitative comparisons of assemblage growth form composition were determined by summing present diatom genera based on growth form and motility for each site (Appendix A) as in Wang et al. (2005). A χ2 goodness of fit test was utilized to examine discrepancies between frequencies of growth forms for Clear Creek and HF039, and Clear Creek and HF090. Summed growth form proportions were rescaled to sum to 1 and visualized using stacked bar plots.
30
RESULTS
Site Selection and Habitat Characteristics
Characteristics used to choose sites for tile transplantation varied among sites for the duration of the study, with the exception of canopy cover, which was only measured at initial tile placement (Appendix C). Water temperature for the duration of the study varied only slightly for Hewett Fork sites (mean: 22 C; range: 20.5-25 C) while Clear
Creek maintained a relatively lower temperature (mean: 21.1 C; range: 18-23). Water depth and velocity had the greatest degree of variation for the duration of the study
(Appendix C). During week 3 sampling, water depth at HF045 was below the measureable limit of equipment. Although the tiles remained submersed in the stream, the tile array at HF045 was moved approximately 2 m further downstream to ensure continued submersion. Water velocity varied throughout the study. During weeks 1 and
3 stream velocity fell below detectable limits for Clear Creek (weeks 1 and 6) and HF045
(weeks 3 and 6) (Appendix C). Despite the low flow conditions, definite flow was visible at each sampling time. Sites HF045 and HF075 each had a higher degree of woody debris and leaf litter present for the duration of the study. Clear Creek was the only site in which there was visual confirmation of the presence of macroinvertebrates.
Water chemistry
Although water chemistry values (Appendix D) fluctuated among sites and sampling dates, one-way ANOVA showed significant differences (P ≤ 0.05) between the
2- reference and all treated sites for only two variables, pH and SO4 (Table 3). Clear Creek had a significantly higher pH (mean: 7.97, range: 7.8-8.2) for the duration of the study. 31 - Elevated SO4 concentrations, a common indicator of AMD, were also present at all
Hewett Fork sites (mean 619 mg L-1, range 145-1400 mg L-1). Conductivity, Al, and Fe, parameters that are also often associated with AMD pollution when they are elevated, did not differentiate reference from treatment stream conditions. The concentration of Al was not significantly (P ≤ 0.05) different across sites and sample dates, while conductivity did not differ significantly (P ≤ 0.05) between Clear Creek and the two furthest downstream sites (HF039 and HF045). Fe was not significantly (P ≤ 0.05) different between Clear
Creek and sites HF060 and HF075 (Table 3). Subsequent power analysis concluded that in order to achieve a desirable level of power (0.8), with p = 0.05 and an effect size of
0.4, the number of replicates would have to be increased to 15 per site.
There was a marked trend of decreasing conductivity with distance from the doser
2- site (HF131). However, both SO4 and conductivity continually increased in Hewett Fork
2- each sampling date, with SO4 nearly tripling at all sites between weeks 3 (range 250-395
-1 -1 2- mg L ) and 6 (range 900-1400 mg L ). The Clear Creek SO4 levels did not exceed 21
-1 - -1 2- -1 mg L (Table 3). The NO3 (range: 0.04-0.55 mg L ) and PO4 (range: 0.03-2.63 µg L )
- 2- measurements for all sites were below the recommended threshold (NO3 : 0.083; PO4 :
10) for reference streams in the WAP ecoregion (USEPA, 2000). Turbidity for Clear
Creek remained below USEPA recommendations for the duration of the study while
HF045 was the only site to continually exceed the 5.2 NTU reference threshold (USEPA,
2000). Similarly, this site also had the highest mean Fe among all sites (0.84 mg L-1), followed closely by HF090 (mean: 0.76 mg L-1). The turbidity readings for the remainder of the sites continued to fluctuate between sampling dates (range: 1.85-10.5 NTUs). 32 Four significant correlations were found between water chemistry data; Fe2+ was positively correlated with turbidity (r = 0.61, p < 0.01), and negatively correlated
2- - with pH (r = -0.47, p < 0.05) and PO4 (r = -0.54, p < 0.05), and conductivity and SO4 were also significantly positively correlated (r = 0.95, p < 0.001) (Table 4).
Comparisons of site-specific water chemistry data from this study with historical
Hewett Fork data showed a number of significant differences (P ≤ 0.05) based on z- scores (Table 5) for temperature, Fe, and Al. Temperature measurements from this study were consistently higher for all sites for the duration of the current study. Conversely, Fe and Al concentrations were markedly lower at all sites within the current study, with the exception of HF045, which was found to have a significantly higher concentration of Fe.
2- No significant differences (P ≤ 0.05) were shown between SO4 concentrations for any of the current of historical site-specific water chemistry data. There were also no significant differences found between current data for HF039 (z = 1.90, p = 0.02872) and HF045 (z =
1.34, p = 0.09176) and historical data. The pH was significantly different for a single site,
HF090 (z = 2.82, p = 0.0024).
Objective 1: Comparison of reference diatom assemblage structure after transplantation from Clear Creek into Hewett Fork
One week after transplantation, there was a noticeable difference in the number of taxa present at Clear Creek (77) and Hewett Fork (mean: 66.8; range: 52 – 99), which was also reflected in the mean Bray-Curtis dissimilarity score (0.4174569) (Appendices E and F). Diatom assemblage species diversity indices were analyzed using one-way
ANOVAs and Fisher’s LSD tests to identify Hewett Fork sites that were not significantly different from the Clear Creek (Fig. 3). All four of the indices for HF090 were not 33 significantly different (P ≤ 0.05) from the reference site. Percent relative abundance of dominant taxon remained similar (P ≤ 0.01) between Clear Creek and HF039, HF045.
The site HF075 scored the lowest of all sites in S, J´, H´, and had the greatest percent relative abundance of dominant taxa. Chlorophyll a concentrations did not significantly
(P ≤ 0.05) differ between the sites on Hewett Fork and Clear Creek (Fig. 4). Subsequent power analysis concluded that in order to achieve a desirable level of power (0.8), with p
= 0.05 and an effect size of 0.4, the number of replicates would have to be increased to 15 per site. Mean AMD-DIBI scores showed both Clear Creek and HF090 to be in the good narrative class (scores 58 and 56, respectively) (Table 6).
At week 1, ordination of Bray-Curtis dissimilarities (Appendix G) with NMDS showed four groupings of sites: 1) Clear Creek; 2) HF090; 3) HF045; and 4) HF039,
HF060, and HF075 (Fig. 5). All ordination stress values were within the 5-10 range, indicating that the ordinations were of good quality (Kruskal, 1964). The overlain environmental data, showed Clear Creek and HF090 to be related to higher pH while
2- HF045 appeared to be influenced by conductivity and SO4 (Fig. 5). HF039, HF060, and
HF075 were all similarly placed near the origin of the ordination, indicating similar species compositions between sites with no particular tie to the water chemistry parameters measured.
At week 3, 89 taxa were collected from Clear Creek, which was again higher than
Hewett Fork (mean: 71.2; range: 51 – 89), and was supported by the mean Bray-Curtis score (0.60331504) (Appendices H and F, respectively). Clear Creek and HF039 were significantly similar in all four species diversity metrics (all P ≤ 0.001) (Fig. 6). There were also no significant differences (P ≤ 0.05) between S for Clear Creek and HF090, 34 and the percent relative abundance of dominant taxon of HF075 and Clear Creek. The values for HF060 were lowest of all sites for S, J´, and H´, while highest in percent relative abundance of dominant taxon. Concentrations of chlorophyll a were not statistically different (P ≤ 0.05) between Clear Creek and all sites on Hewett Fork (Fig.
4). Subsequent power analysis concluded that in order to achieve a desirable level of power (0.8), with p = 0.05 and an effect size of 0.4, the number of replicates would have to be increased to 15 per site. Mean AMD-DIBI scores varied slightly, with both Clear
Creek and HF039 being in the good narrative class (54 and 44, respectively), while the rest of the sites remained classified as fair (Table 6).
The ordination of week 3 Bray-Curtis scores (Appendix G) (stress=8.0813) showed the influence of an AMD-derived gradient (Fig. 7). There was more variation in the distribution of HF090 replicates, although all were within the region opposite of the
AMD-derived water chemistry. The HF039 replicates were in a grouping near the origin of the ordination, while Clear Creek replicates grouped near the arrow representing an elevated pH, like at week 1.
At week 6, 230 total taxa were identified, with Clear Creek providing 84 species and Hewett Fork contributing a mean of 69 taxa (range: 45 – 95), with a mean Bray-
Curtis score of 0.58696414 (Appendices I and F, respectively). There were no significant differences (all P ≤ 0.001) between Clear Creek and Hewett Fork sites in J´, S, and H´ of
HF039, as well as the percent relative abundance of dominant taxon for HF039, HF045, and HF090 (Fig. 8). Like the week 1 data, HF075 obtained the lowest scores for S, J´, and
H´ while having the highest percent relative abundance of dominant taxon score. At
Week 6, one site, HF060 (range: 13.30-23.09 mg m-2) had chlorophyll a concentrations 35 that were significantly different (P ≤ 0.05) from Clear Creek (range 110.85-171.26 mg m-2) (Fig. 4). For the week 6, the mean AMD-DIBI scores from Clear Creek, HF039, and
HF090 were in the good narrative class while the other sites remained in the fair class
(Table 6).
The ordination of week 6 Bray-Curtis scores (Appendix G) (stress = 7.0316) showed the replicates from the individual Hewett Fork sites to be grouped together and separate from the other sites with the exception of HF090, which grouped around the cluster of Clear Creek replicates (Fig. 9). Like weeks 1 and 3, Clear Creek and HF090 showed strong affinities to elevated pH while the other sites exhibited more spatial variability within ordination space, with HF060 and HF075 most closely related to the
2- conductivity and SO4 .
Objective 2: Comparison of in situ diatom assemblages at the most upstream and downstream site on Hewett Fork with the reference site, Clear Creek
After the 6-week colonization period, cumulative species in Clear Creek and
Hewett Fork differed, with Clear Creek samples totaling 82 taxa, while Hewett Fork had a mean of 76 taxa (range: 62 - 90; Appendix J), with a mean Bray-Curtis dissimilarity of
0.83564165 (Appendix K). Clear Creek differed significantly from HF039 and HF090 in both J´ (P ≤ 0.001) and H´ scores (P ≤ 0.001), but did not differ from HF039 in S (P ≤
0.001) and HF090 in relative abundance of the dominant taxon (P ≤ 0.01) (Fig. 10).
Similarly, the Hewett Fork sites had significantly (P ≤ 0.01) less chlorophyll a (mean:
15.9 and 23.9 mg m-3 for HF039 and HF090, respectively) than Clear Creek (mean: 157.5 mg m-3, Fig. 11). 36 After the initial colonization period, neither of the Hewett Fork sites was found to be proportionally similar in growth form composition to Clear Creek. χ2 goodness of fit test found growth form composition for each site differing significantly (P ≤ 0.001) from Clear Creek, with χ2 = 3743.42459 and χ2 = 4413.48056 for comparisons with
HF039 and HF090, respectively (Appendix L). Growth forms (Fig. 12) showed a greater percentage of prostrate taxa at Clear Creek (64.5%) and HF039 (67.7%) and a greater percentage of erect taxa at HF090 (33.9%) after the six-week colonization. Clear Creek was also the only site to have an abundance of unattached taxa (10.3%). HF090 was equally comprised of prostrate, prostrate/motile, and erect taxa, with other growth forms having minimal abundance. The scores for AMD-DIBI varied among the three sites
(Table 7). Clear Creek scored a 58, placing it in the ‘good’ narrative class, while HF039 and HF090 each scored substantially lower, placing them in the ‘fair’ (score: 35) and
‘poor ‘(score: 12) classes, respectively. Ordination of Bray-Curtis dissimilarities
(stress=0.04) of the sites showed the emergence of three distinct patterns. Clear Creek and HF039 showed little variation among replicates, while HF090 showed much variation based on the size of the ellipse needed to encircle those replicates (Figure not shown). 37
DISCUSSION
Since the implementation of the treatment plan, Hewett Fork has continued to follow a general trend towards recovery. Water chemistry improvements, specifically a decrease in acid and metals loadings, have been attributed to the continual treatment at
HF131, the primary site of AMD on Hewett Fork (NPS, 2009). Data available from the
Non-Point Source Monitoring Project (NPSMP; www.watersheddata.com) shows a longitudinal trend within the studied reach similar to the findings of this study (Table 1),
2- in which there is a decrease in both conductivity and SO4 with distance from the site of impairment. Recent biological data (macroinvertebrate aggregated index for streams
[MAIS], index of biotic integrity [IBI] for fish, total fish species, and total fish present) obtained from NPSMP (Table 2) suggest that there is an increasing gradient of biological and habitat quality moving downstream within this reach. Similarly, Pool (2010) found diatom species diversity index scores for three sites in this reach (HF090, HF060, and
HF039) to increase with distance from the doser and the inputs of acidity. These data confirm the expectations of the presence of a linear relationship between distance from the doser and stream health as described by species diversity indices and water chemistry parameters. Yet, data collected for this study suggest that the trajectory of biological recovery within this reach is much more complex than previous findings indicate.
After a 6-week colonization period, the diatom assemblages from HF039 and
HF090 showed a number of significant variations from those of the Clear Creek tiles
(objective 2). Diversity metrics showed little similarity between Clear Creek and the two sites on Hewett Fork (Fig. 10), indicating that Hewett Fork sites are not capable of 38 reaching the degree of species diversity found in Clear Creek after six weeks, despite the high degree of species richness at HF039. Similarly, the significant (P ≤ 0.01) variation in chlorophyll a concentrations (Fig. 11) between Clear Creek and Hewett Fork suggests that there is some factor impeding biomass accrual in Hewett Fork and extending the time required for a mature biofilm to establish. Yet, growth form comparisons and AMD-DIBI scores show a marked difference in assemblage composition between HF039 and HF090 (Fig. 12), indicating that while each site is slow to accrue new biomass, the taxa present represent the assemblage structures that are expected from the stress gradient associated with AMD treatment. Erect and prostrate/motile taxa, indicators of decreased grazing pressure and increased sedimentation, respectively, dominate HF090, reflecting the impairment described by
NPSMP data and Pool (2010). The growth form percentages at HF039, on the other hand, are very similar to those at Clear Creek, implying that the growth form composition of HF039 is similar to that of a reference condition, i.e. more grazing pressure, less sedimentation. This response in growth form composition supports the findings of
NPSMP data. Moreover, these findings also support the notion that improvement in water chemistry may stimulate macroinvertebrate recovery (DeNicola and Stapleton,
2002; Battaglia et al., 2005). While AMD-DIBI scores similarly reflect a positive assemblage response with downstream distance from the doser, these scores also support the species diversity and chlorophyll a data, as neither site’s score was comparable to that of Clear Creek.
During the course of this study, much of the data suggested a parabolic gradient of health along the study reach, with the vertex located near HF060 and HF075 rather 39 than the linear gradient seen in previous research, indicating that there may be some stressor present at those sites that is inhibiting diatom accrual (NPSMP, 2009; Pool,
2010). The most upstream and downstream sites, HF039 and HF090, showed signs of high biological quality throughout the study with the reference site transplanted tile sets
(Figs. 3, 6, and 8), yet tiles that were colonized by in situ diatom assemblages in Hewett
Fork failed to reach this level of health after 6 weeks (Fig. 10), suggesting that some factor within Hewett Fork is greatly limiting diatom colonization. Similarly, the transplanted tiles maintained chlorophyll a concentrations that were markedly higher than those on tiles that were colonized in Hewett Fork for 6 weeks. Furthermore, sites HF060 and HF075 showed a steep decline in the majority of the indices used to classify stream health (Figs. 3, 6, and 8).
From the data gathered, two general trends were evident over the duration of the study that characterize not only the parabolic response, but also the diatom assemblage structure within this reach of Hewett Fork. The first trend that remained consistent throughout the study was that both HF039 and HF090 were able to preserve a high degree of species diversity after transplantation, as supported by species diversity metrics, chlorophyll a concentrations, and AMD-DIBI scores, indicating that either they are capable of sustaining reference assemblages present at the time of transplantation or that they are receiving an influx of propagules from upstream. Based on the NMDS plots
(Figs. 5, 7, and 9), it would appear that the composition of HF039 is more likely influenced by an increased rate of colonization from taxa present upstream. Conversely,
HF090 continually shows a strong taxonomic resemblance to Clear Creek in ordination space (Figs. 5, 7, and 9) for the duration of the study, signifying the ability of a number of 40 reference taxa to remain viable at HF090, with only a minimal influence occurring as the result of colonization by taxa from upstream. This finding may provide some explanation for the decreased diversity and lower AMD-DIBI scores that were seen in the in situ assemblage from HF090, as a diverse diatom assemblage may not have been available to colonize the site.
The second visible trend in the transplantation (objective 1) data was the steep decline in species diversity, chlorophyll a concentrations, and AMD-DIBI scores at
HF060 and HF075. Given the time allocated for assemblage restructuring/development, it was predicted that at the end of a 6-week period a mature biofilm would have developed at all sites, yet biofilms at HF060 and HF075 failed to respond as expected. This response indicates that not only are transplanted taxa being extirpated, but also that new taxa are failing to replace those lost to site-specific drivers of assemblage structure. Although the colonization of sites on Hewett Fork was shown to possibly require greater time for mature biofilm development, this does not explain the overall decline in assemblage species diversity at these two sites, as sites both upstream and downstream from HF060 and HF075 are able to maintain a higher degree of assemblage integrity.
Considering the response at HF060 and HF075, it seems clear that some factor, which was not identified in this study, is influencing assemblage structure at these middle sites. Examination of chemical parameters from this study suggest that there is no single site on Hewett Fork in which there is a greater degree of impairment than any other.
2- Although there is a longitudinal trend of decreasing SO4 and conductivity with distance from the doser, it is difficult to attribute the responses seen at HF060 and HF075 to this, 41 as HF090, which experiences greater levels of each of these parameters, continued to maintain a high degree of species diversity in the tile transplantation portion of this study.
The only chemical parameter that highlighted differences between HF060 and
HF075 from other Hewett Fork sites was Fe concentrations. Fe concentrations at HF060 and HF075 were not significantly different from Clear Creek, but were lower than at surrounding sites. This finding suggests that aqueous Fe is being removed at, or immediately upstream of, these sites. The significance of Fe in the development of biofilms in streams impacted by AMD is generally associated with the effects of precipitating Fe(OH)3, which is found to greatly decrease the availability of habitats, as well as its tendency to smother taxa, essentially decimating the established assemblage
(McKnight and Feder, 1984; Niyogi et al., 2002; Butler, 2009; Lear et al., 2009). In this study, HF060 was continually found to have some degree of Fe precipitate present, as characterized by an orange film covering portions of the substratum. While this may account for the decline seen in species diversity and algal biomass at HF060, it does not account for the similar response seen at HF075. DeRose (2011), in a comparison of aqueous and sediment metal concentrations in Hewett Fork, found a substantial spike in soil Fe concentrations at HF075, suggesting a greater rate of deposition and integration into the sediment matrix at this site. Although a number of studies have found aqueous rather than sediment metal concentrations to have a greater impact on benthic assemblages (DeNicola and Stapleton, 2002; Battaglia et al., 2005, Dsa et al., 2008), a number of possible implications of the DeRose (2011) findings arise. While increased soil Fe levels may not explain the diminished assemblage quality at these sites, the Fe dynamics may serve as an indicator of the transitional changes occurring with other 42 metals, some of which may have adverse effects on biota. Unfortunately, this study was not designed to address this complex issue; therefore it cannot be fully substantiated here. However, these findings could be explored in the future with a study designed to explicitly examine this issue. 43
CONCLUSIONS AND FUTURE DIRECTIONS
From this study, it is clear that there is continued impairment within Hewett Fork, and yet the treatment does appear to be aiding in recovery, despite the unexpected (i.e. non-linear) response of transplanted diatom assemblages. Both HF039 and HF090 seem to be capable of supporting diatom assemblages similar to those found in reference conditions, yet in situ biofilm development remains obstructed. Unfortunately, some underlying factor(s) continue to plague the middle section of this study reach, limiting the potential for recovery despite the availability of viable propagules. In order to fully address the complexity of the factors driving diatom assemblage composition within this reach, further research is required. Looking solely at the in situ diatom assemblages, the question remains as to whether biofilm maturity was reached after a 6-week colonization period. To address this, a longer colonization study may provide insight into the time required to achieve a mature biofilm within this reach or to determine if reduced biomass is what is to be expected due to current levels of impairment. Furthermore, a comprehensive examination of the relationship between the sediment metal concentrations and diatom assemblage structure must be undertaken in order to properly eliminate or elucidate the potential relationships suggested by the current study. Finally, the role of grazers in diatom assemblage structuring was not examined in this study.
These grazers may play a significant role and may have influenced the findings of this study, especially since macroinvertebrate communities are showing signs of recovery within Hewett Fork. 44 REFERENCES
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Table 1
Mean and range (in parentheses) in historical water chemistry data for Hewett Fork for sampling months June – September, with n = number of samples available for each parameter from www.watersheddata.com. Site Temperature Conductivity pH Sulfate Total Iron Total Aluminum -1 2- (°C) (µS cm ) (SO4 ) (Fe) (Al) mg L-1 mg L-1 mg L-1 Downstream HF039 19.6 472 6.8 227 0.66 0.123 (14.9-23.6) (230-714) (6.4-7.2) (125-320) (0.39-1.1) (0.05-0.25) n = 14 n = 14 n = 14 n = 10 n = 10 n = 10 HF045 20.4 575 6.9 269 0.53 0.08 (14.9-23.1) (473-709) (6.8-7.0) (189-319) (0.27-0.75) (0.05-0.12) n = 5 n = 4 n = 5 n = 4 n = 3 n = 3 HF060 19.6 528 6.6 271 1.05 0.524 (14.3-23.2) (240-721) (6.1-7.1) (193-375) (0.44-2.09) (0.05-3.69) n = 13 n = 13 n = 13 n = 12 n = 10 n = 10 HF075 19.3 623 6.8 310 0.48 0.067 (14.6-22.5) (528-766) (6.5-7.1) (239-412) (0.41-0.56) (0.05-0.09) n = 4 n = 4 n = 4 n = 4 n = 3 n = 3 Upstream HF090 20.1 611 5.0 342 1.46 1.583 (14.4-24.6) (290-889) (4.0-7.1) (216-470) (0.55-2.66) (0.05-8.53) n = 14 n = 14 n = 14 n = 14 n = 10 n = 10
53
Table 2
Mean and range (in parentheses)in historical biodiversity data for Hewett Fork for sampling months June – September, with n = the number of samples available for each parameter from www.watersheddata.com and Pool (2010). N/A represents no data available due to limited samples available (in the case of data ranges) or a lack of samples for that site. Site Index of Biotic Macroinvertebrate Aggregate Index Acid Mine Drainage – Diatom Index Total Fish Fish Species Integrity for Streams of Biotic Integrity Count Diversity (IBI) (MAIS) (AMD-DIBI) Downstream HF039 39 13 31 175 17 (28-48) (13-14) (N/A) (33-372) (13-20) n = 6 n = 5 n = 1 n = 6 n = 6 HF045 32 14 N/A 82 13 (N/A) (N/A) (N/A) (N/A) n = 1 n = 1 n = 1 n = 1 HF060 32 9 22 69 13 (17-40) (8-10) (N/A) (38-116) (9-16) n = 6 n = 5 n = 1 n = 6 n = 6 HF075 16 12 N/A 45 6 (N/A) (N/A) (N/A) (N/A) n = 1 n = 1 n = 1 n = 1 Upstream HF090 23 5 13 55 7 (12-30) (2-7) (N/A) (0-169) (0-10) n = 7 n = 8 n = 1 n = 7 n = 7
54
Table 3 Mean water chemistry (n = 3; range in parentheses) from each site for the duration of the study. Bold results are significantly different from the Clear Creek reference site with asterisks representing significance level (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001). Power analysis of sites not found to be significantly different showed that in order to achieve an acceptable power level (0.8), n = 15. Site Conductivity pH Turbidity Nitrate Orthophosphat Sulfate Total Iron Total Aluminum -1 - 2- (µS cm ) (NTS) (NO3 ) e (SO4 ) (Fe) (Al) -1 - -1 -1 -1 mg L (PO4 ) mg L mg L mg L mg L-1 Clear 436 7.9 4.56 0.18 0.17 20 0.18 0.016 Creek (400–460) (7.8-8.2) (4.49-4.64) (0.05-0.34) (0.12-0.23) (18-21) (0.13-0.21) (0.006-0.023)
Hewett Fork
Downstream HF039 687 6.6** 4.87 0.27 0.15 531*** 0.45*** 0.044 (490-880) (6.3-7.0) (3.12-6.55) (0.05-0.55) (0.04-0.33) (145-1200) (0.37-0.52) (0.010-0.100)
HF045 707 6.8** 7.87 0.15 0.04 472*** 0.84*** 0.011 (530-870) (6.5-7.1) (5.71-9.63) (0.09-0.24) (0.03-0.05) (200-900) (0.73-0.98) (0.007-0.013)
HF060 800* 6.9** 5.36 0.11 0.97 683*** 0.40 0.008 (610-930) (6.0-7.0) (4.41-6.28) (0.06-0.15) (0.13-2.63) (255-1400) (0.36-0.47) (0.005-0.009)
HF075 863* 6.9** 3.30 0.12 0.34 710*** 0.31 0.007 (680-990) (6.8-7.1) (1.85-5.53) (0.05-0.23) (0.04-0.93) (340-1400) (0.25-0.40) (0.003-0.011)
Upstream HF090 907* 6.6** 6.01 0.09 0.10 698*** 0.76*** 0.018 (730-1090) (6.3-7.2) (3.15-10.50) (0.04-0.15) (0.07-0.13) (320-1400) (0.56-1.12) (0.015-0.022) 55
Table 4
Row-wise Spearman rank correlations between water chemistry samples from each site for the duration of the study. Significant results in bold, with asterisks representing significance level (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001). Conductivity Turbidity Nitrate Orthophosphate Sulfate Iron Aluminum -1 - - 2- (µS cm ) pH (NTS) (NO3 ) (PO4 ) (SO4 ) (Fe) (Al) Conductivity (µS cm-1) - pH -0.07 - Turbidity (NTS) -0.39 0.27 - Nitrate - (NO3 ) -0.12 0.27 -0.01 - Orthophosphate - (PO4 ) -0.02 0.16 -0.46 -0.26 - Sulfate 2- (SO4 ) 0.95*** -0.01 -0.32 0.03 -0.02 - Iron (Fe) 0.26 -0.47* 0.61* -0.1 -0.54* 0.25 - Aluminum (Al) -0.13 -0.05 -0.09 -0.05 0.2 -0.2 0.15 - AMD-DIBI -0.49* 0.54* 0.16 0.13 0.27 -0.49* -0.14 0.38 56
Table 5
Z-scores and associated p-value for comparison of current and historical Hewett Fork water chemistry data. Scores in bold indicate no significant (P ≤ 0.05) differences between current and historical data. Site Temperature Conductivity pH Sulfate Total Iron Total Aluminum -1 2- (°C) (µS cm ) (SO4 ) (Fe) (Al) mg L-1 mg L-1 mg L-1 Downstream HF039 z = 6.30 z = 1.90 z = -0.55 z = 0.91 z = -4.70 z = -2.80 p = 0.00002 p = 0.02872 p = 0.29116 p = 0.18141 p = 0.00002 p = 0.00256
HF045 z = 3.81 z = 1.34 z = -0.37 z = 0.93 z = 4.33 z = -37.36 p = 0.00007 p = 0.09012 p = 0.35569 p = 0.17619 p = 0.00002 p = 0.00002
HF060 z = 6.48 z = 2.80 z = 1.59 z = 1.14 z = -15.50 z = -123.42 p = 0.00002 p = 0.00256 p = 0.05592 p = 0.12714 p = 0.00002 p = 0.00002
HF075 z = 15.50 z = 2.56 z = 0.38 z = 1.16 z = -3.78 z = -25.71 p = 0.00002 p = 0.00523 p = 0.35197 p = 0.12302 p = 0.00008 p = 0.00002
Upstream HF090 z = 7.42 z = 2.83 z = 2.82 z = 1.03 z = -3.19 z = -356.98 p = 0.00002 p = 0.00233 p = 0.00240 p = 0.15151 p = 0.00071 p = 0.00002
57
Table 6
Mean (n = 3) Acid Mine Drainage-Diatom Index of Biotic Integrity (AMD-DIBI) scores for each site at 1, 3, and 6 weeks after transplantation. Metric scores are derived from methods developed by Zalack et al. (2010). Scores that increase with impairment are divided by the 90th percentile of site values, subtracted from 1, and multiplied by 10, while scores that decrease with impairment are divided by the 90th percentile of site values and multiplied by 10. Acidophilic sp. metric scores were multiplied by -1 to correspond with the increased level of impairment associated with greater abundances of acidophilic taxa. Relative position in Sample Spec. # of Ref. Sim. to Ref. Total AMD-DIBI Narr. stream Site Time Rich. Tol. sp. Sens. sp. sp. Sites Acid. sp. Eut. sp. chlor. a Cymbella sp. Score Class Week 1 Clear Creek 10 4 8 9 10 0 2 9 6 58 Good Hewett Fork Downstream HF039 8 3 9 1 7 -6 0 8 10 40 Fair HF045 7 3 9 1 7 -10 2 8 2 29 Fair HF060 6 4 10 1 7 -6 5 6 6 39 Fair HF075 6 0 9 1 7 -1 4 7 2 35 Fair Upstream HF090 10 3 10 8 8 -5 4 9 9 56 Good Week 3 Clear Creek 9 3 8 9 10 -1 0 8 8 54 Good Hewett Fork Downstream HF039 9 2 10 2 6 -4 1 8 10 44 Good HF045 7 4 10 0 5 -2 1 6 10 41 Fair HF060 5 2 10 1 5 -4 4 4 6 33 Fair HF075 7 0 10 0 6 -8 3 5 7 30 Fair Upstream HF090 8 2 10 1 6 -9 2 6 6 32 Fair Week 6 Clear Creek 10 4 6 10 10 0 0 8 6 54 Good Hewett Fork Downstream HF039 10 2 10 0 6 -2 1 5 10 42 Good HF045 7 1 10 0 7 -2 0 6 10 39 Fair HF060 6 1 10 0 6 -4 7 1 6 33 Fair HF075 5 1 2 0 7 -10 7 6 2 20 Fair Upstream HF090 8 4 10 0 6 -1 5 5 6 43 Good 58
Table 7
Mean (n = 3) Acid Mine Drainage-Diatom Index of Biotic Integrity (AMD-DIBI) scores for Clear Creek and the most downstream (HF039) and upstream (HF090) sites on Hewett Fork. Metric scores are derived from methods developed by Zalack et al. (2010). Scores that increase with impairment are divided by the 90th percentile of site values, subtracted from 1, and multiplied by 10, while scores that decrease with impairment are divided by the 90th percentile of site values and multiplied by 10. Acidophilic sp. metric scores were multiplied by -1 to correspond with the increased level of impairment associated with greater abundances of acidophilic taxa. Relative position # of AMD- in stream Sample Spec. Tol. Sens. Ref. Sim. to Acid. Eut. Total Cymbella DIBI Narr. Site Time Rich. sp. sp. sp. Ref. Sites sp. sp. chlor. a sp. Score Class Clear Creek 08/08/10 9 4 8 10 10 0 0 9 8 58 Good Hewett Fork Downstream HF039 09/23/10 9 3 10 1 2 -1 0 1 10 35 Fair Upstream HF090 09/23/10 6 1 10 0 2 -9 0 1 1 12 Poor 59
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Figure 1. Map of Hewett Fork drainage basin. Sampling sites are marked by black diamonds and labeled as designated by the Raccoon Creek Watershed Group. River mile is noted in parentheses. Acid inputs to Hewett Fork are located at sites HF131, HF190, and HF120. The doser is located at HF131. The study reach consisted of sites HF090, HF075, HF060, HF045, and HF039.
60
Figure 2. Example of the tile arrays used for diatom colonization.
61 80 Downstream Upstream 0.9 Downstream Upstream 0.8 70 F=8.8459 b b,c a b,c a,b p<0.001 0.7
60 a c,d a c
b,c 0.6 S J' 50 c,d even.1 0.5
spec.num.1 d 40 F=17.245 0.4 p<0.001 30 0.3 20 0.2
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090
4.0 Downstream Upstream 0.7 Downstream Upstream
a a 0.6 3.5 F=3.5574 p<0.01 0.5
3.0 b,c b a,b a 0.4 a,b,c
H' a,b,c 2.5 c,d b,c d c 0.3 shannon.1 rel.abund.1
2.0 F=23.829 0.2 p<0.001 1.5 Rel. Abund. Dom. Taxon Abund. Rel. 0.1 1.0 0.0
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090
Figure 3. One-way ANOVAs of species richness, species evenness, Shannon diversity, and percent relative abundance of dominant taxon for each site 1-week after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Sites are labeled as in Figure 1. Arrow shows longitudinal position of Hewett Fork sites. Bars with the same letters are not significantly different (P ≤ 0.05). 62
Week 1 Week 3 Week 6 Downstream Upstream Downstream Upstream Downstream Upstream 200 a a a F=4.3026 a a p<0.05 a a 150 a a,b
) a,b 2 a,b ! m
b,c g
m a (
a
l
l a y h 100 chlor.[1:18] p chlor.[19:36] chlor.[37:54]
o a r
o a l a h C
50 c
F=1.4304 F=2.0463 p=0.2823 p=0.1437
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090 Figure 4. Chlorophyll a (mg m-2) for each site 1, 3, and 6 weeks after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical dashed line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Arrow shows longitudinal position of Hewett Fork sites. Sites are labeled as in Figure 1. Bars with the same letters are not significantly different (P ≤ 0.05). Power analysis found that in order to achieve an acceptable power level (0.8), n = 15. 63
Stress=7.1732 1.0
CC HF039 HF045 HF060 HF075 0.5 HF090 HF45 NMDS2 HF90 SO4 Conductivity 0.0 HF39 HF60 CC HF75
-0.5 pH
-1.0 -0.5 0.0 0.5
NMDS1
Figure 5. Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarities (stress = 7.1732) of diatom assemblage compositions for all sites 1-week after transplantation, with an overlay of water chemistry variables that significantly correlate with AMD-DIBI scores. Ellipses were placed around replicates using the ordiellipse sub-routine of the Vegan package in order to visualize variation within sites, as larger ellipses represent greater variation of within-site taxonomic composition. 64 80 Downstream Upstream 0.9 Downstream Upstream a 0.8 70 b b,c F=25.246 a b,c a c p<0.001 0.7
60 a
a b 0.6 S J'
50 b even.2 0.5
spec.num.2 c 40 F=15.466 0.4 p<0.001 30 0.3 20 0.2
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090
4.0 Downstream Upstream 0.7 Downstream Upstream
a 0.6 3.5 a F=12.607 p<0.001 b 0.5 3.0 a b b c 0.4 a a H' 2.5 b 0.3 b shannon.2 F=22.53 rel.abund.2 b 2.0
p<0.001 0.2 1.5 Rel. Abund. Dom. Taxon Abund. Rel. 0.1 1.0 0.0
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090
Figure 6. One-way ANOVAs of species richness, species evenness, Shannon diversity, and percent relative abundance of dominant taxon for each site 3 weeks after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Sites are labeled as in Figure 1. Arrow shows longitudinal position of Hewett Fork sites. Bars with the same letters are not significantly different (P ≤ 0.05). 65
Stress=8.0813 1.0
CC HF039 HF045 HF060
0.5 HF075 HF090 HF39
HF90 HF45 NMDS2 0.0 CC HF75 SO4 HF60 Conductivity pH -0.5
-1.0 -0.5 0.0 0.5 1.0
NMDS1
Figure 7. Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarities (stress = 8.0813) between diatom assemblage compositions for all sites 3 weeks after transplantation, with an overlay of water chemistry variables that significantly correlate with AMD-DIBI scores. Ellipses were placed around replicates using the ordiellipse sub-routine of the Vegan package in order to visualize variation within sites, as larger ellipses represent greater variation of within-site taxonomic composition. 66 80 Downstream Upstream 0.9 Downstream Upstream b 0.8
70 a a F=26.187 a a,b c
0.7 b
60 p<0.001 c b 0.6 S J'
50 b,c
c even.3 0.5 spec.num.3
40 F=16.029 d 0.4 p<0.001 30 0.3 20 0.2
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090
4.0 Downstream Upstream 0.7 Downstream Upstream a a 0.6 3.5 b F=10.884 b 0.5
3.0 p<0.001 a c b 0.4 H' 2.5 d b,c 0.3 b,c shannon.3 F=27.95 rel.abund.3 2.0 c c p<0.001 0.2 1.5 Rel. Abund. Dom. Taxon Abund. Rel. 0.1 1.0 0.0
CC HF039 HF045 HF060 HF075 HF090 CC HF039 HF045 HF060 HF075 HF090
Figure 8. One-way ANOVAs of species richness, species evenness, Shannon diversity, and percent relative abundance of dominant taxon for each site 6 weeks after transplantation. Black dots represent site replicates (n = 3), and the solid black lines connects the mean score of each site, with bars representing the mean +/- 1 SE (n = 3). Vertical line separates the unimpaired Clear Creek (CC) from Hewett Fork (HF) treatment sites. Sites are labeled as in Figure 1. Arrow shows longitudinal position of Hewett Fork sites. Bars with the same letters are not significantly different (P ≤ 0.05). 67
Stress=7.0316 1.0
CC HF039 HF045 HF060 HF075 HF39 0.5 HF090
HF45 NMDS2
0.0 HF60 SO4 CC Conductivity HF90
HF75 -0.5 pH
-1.0 -0.5 0.0 0.5
NMDS1
Figure 9. Nonmetric multidimensional scaling (NMDS) ordination of Bray-Curtis similarities (stress = 7.0136) between diatom assemblage compositions for all sites 6 weeks after transplantation, with an overlay of water chemistry variables that significantly correlate with AMD-DIBI scores. Ellipses were placed around replicates using the ordiellipse sub-routine of the Vegan package in order to visualize variation within sites, as larger ellipses represent greater variation of within-site taxonomic composition.
68 80 0.9
70 F=13.771 F=14.525 a p<0.001 a p<0.001
a 0.8 60 50 0.7 b b b 40 Species Richness Species Species Evenness Species 0.6 30 20 0.5 CC HF039 HF090 CC HF039 HF090 4.0 0.6 a F=8.9892 F=21.712
3.5 0.5 p<0.01 a p<0.001 a,b 3.0 0.4
b b 2.5 0.3 b 2.0 0.2 1.5 0.1 Shannon-Weiner Diversity Shannon-Weiner 1.0 0.0 Relative Abundance of Dominant Taxon of Dominant Abundance Relative CC HF039 HF090 CC HF039 HF090
Figure 10. Comparison of species richness, evenness, and Shannon diversity and percent relative abundance after 6-week colonization period at Clear Creek (CC) and the most downstream (HF039) and upstream (HF090) sites within the Hewett Fork study reach. Bars represent mean (n = 3) diversity index score per site, with lines representing the mean +/- 1 SE (n = 3). Bars with the same letters are not significantly different (P ≤ 0.05). Sites are labeled as in Figure 1. 69 6
) a 2 F=8.1795 ! p<0.01 m
5 g m (
a
l l y h p 4 o r o l h
c b
d e
3 b m r o f s n a r t
2 10 g o L 1
CC HF039 HF090
-2 Figure 11. Log10 transformed chlorophyll a (mg m ) after 6-week colonization period at Clear Creek (CC) and the most downstream (HF039) and upstream (HF090) sites within the Hewett Fork study reach. Bars represent mean (n = 3) chlorophyll a concentrations per site, with lines representing the mean +/- 1 SE (n = 3). Sites are labeled as in Figure 1. Bars with the same letters are not significantly different (P ≤ 0.05).
70
1.0
0.8 Prostrate/Motile Variable
0.6 Erect Stalked Form Unknown Unattached 0.4 Prostrate Growth Growth Form Proportions
0.2
0.0 CC HF039 HF090 Figure 12. Comparison of mean (n = 3) proportional composition of diatom growth forms at Clear Creek, HF039, and HF090 after a six-week colonization period. Bars may not sum to 1 due to the use of mean proportion values. 71
APPENDIX A: DIATOM GROWTH FORMS AND THE REPRESENTATIVE GENERA, WITH DESCRIPTIONS OF GROWTH FORM AND POTENTIAL COSTS/BENEFITS OF THAT GROWTH FORM.
Growth Form Related Genera Description Potential Implications Prostrate Achnanthes sp. Valve face lying parallel Diminished grazing Achnanthidium sp. to substratum pressure; less Amphipleura sp. susceptible to current; Amphora sp. more susceptible to Brachysira sp. shading and Caloneis sp. sedimentation Cavinula sp. Cocconeis sp. Denticula sp. Epithemia sp. Frustulia sp. Geissleria sp. Halamphora sp. Hippodonta sp. Karayevia sp. Navicella sp. Navicula sp. Neidium sp. Rhopalodia sp. Rossthidium sp. Sellaphora sp. Stauroneis sp.
Erect Eunotia sp. Attached to substratum Subjected to increased Meridion sp. via mucilage pad on grazing pressure and Synedra sp. apical end of valve greater susceptibility to current velocity; may experience light limitation Stalked Cymbella sp. Stalks attached to Subjected to increased Encyonema sp. substratum and vary in grazing pressure and Gomphoneis sp. length greater susceptibility to Gomphonema sp. current velocity Reimeria sp. Rhoicosphenia sp. Unattached Cyclotella sp. No attachment to May indicate pooled Diatoma sp. substratum; possibly water Melosira sp. planktonic Stephanodiscus sp. Tabellaria sp. Thalassiosira sp. Variable Fragilaria sp. Multiple growth forms N.A. observed Prostrate/Motile Cymatopleura sp. Valve face lying parallel Can withstand some Gyrosigma sp. to substratum yet sedimentation; less Hantzschia sp. possesses a great degree susceptible to grazing 72 Nitzschia sp. of motility pressure; decreased Stenopterobia sp. stress from currents Surirella sp. Tryblionella sp. Information not Actinocyclus sp. Information unavailable Information unavailable available (N.A.) Aulocoseira sp. Baccilaria sp. Capartogramma sp. Craticula sp. Delicata sp. Eolimna sp. Fallacia sp. Gomphonitzschia sp. Grunowia sp. Luticola sp. Plagiotroips sp. Planothidium sp. Pseudostaurosira sp.
73
APPENDIX B: ACID MINE DRAINAGE DIATOM INDEX OF BIOTIC INTEGRITY (AMD-DIBI) COMPONENT METRICS FROM ZALACK ET AL. (2010).
Response to Metric Description Impairment 1 Species richness Number of species present -
2 Percent tolerant species Percent of tolerant species from the Kentucky + Division of Water Pollution Tolerance Index (KYDPTI)
3 Percent sensitive species Percent sensitive species from the KYDPTI -
4 Number of reference species Number of the eight reference species present -
5 Percent similarity to reference sites Bray-Curtis similarity to the predefined - reference sites
6 Percent acidophilic species Percent abundance of acidophilic species + (Eunotia, Frustulia, and Pinnularia)
7 Percent eutraphentic species Percent abundance of eutraphentic species + (Nitzschia and Navicula) 8 Total chlorophyll a Milligrams of chlorophyll a per m2 of +/- streambed
74
9 Percent Cymbella species Relative abundance of Cymbella species - including Encyonema and Reimeria 75
APPENDIX C: HABITAT CHARACTERISTICS AT THE TIME OF INITIAL TILE COLONIZATION, TRANSPLANTATION, AND 1-, 3-, AND 6-WEEKS AFTER TRANSPLANTATION. N/A IN WATER VELOCITY REPRESENTS BELOW DETECTABLE LEVELS OF MEASUREMENT, YET DEFINITE FLOW WAS VISIBLE.
Water Velocity Depth % Canopy Temperature Site ID Sampling Time (m s-1) (cm) Cover (°C) Initial colonization prior to transplantation
Clear Creek 06/24/10 0.79 13.5 46 22
Transplantation date 08/08/10 Clear Creek 0.73 13.5 46 22 HF039 0.95 17 49 23 HF045 0.53 10.25 61 24 HF060 0.84 10.5 43 25 HF075 0.3 13.5 70 25 HF090 0.57 11 75 23
Week 1 (08/18/10) Clear Creek N/A 23 HF039 0.76 20.5 HF045 0.34 22 HF060 0.76 21 HF075 0.46 21.5 HF090 0.49 21
Week 3 (09/01/10) Clear Creek 0.45 7 20.5 HF039 0.79 12.5 20.5 HF045 0 15 21 HF060 1.2 9.5 22 HF075 0.45 9 22 HF090 0.29 10 21.5
Week 6 (09/23/10) Clear Creek N/A 7 18 HF039 0.39 3 21 HF045 N/A 15 22 HF060 0.33 9.5 21.5 HF075 0.33 9 22 76 HF090 0.07 12.5 21.5 77
APPENDIX D: WATER CHEMISTRY FOR EACH SITE AT 1, 3, AND 6 WEEKS AFTER TRANSPLANTATION.
Nitrate Orthophosphate Sulfate Iron - - 2- Conductivity Turbidity (NO3 ) (PO4 ) (SO4 ) (Fe) Aluminum (Al) Site Sampling time (µS cm-1) pH (NTS) mg L-1 mg L-1 mg L-1 mg L-1 mg L-1
Week 1 (08/18/10)
Clear Creek 450 8.2 4.49 0.15 0.17 21 0.21 0.02
Hewett Fork
HF039 490 6.3 6.55 0.55 0.04 145 0.52 0.023
HF045 530 6.5 8.26 0.09 0.03 200 0.82 0.013
HF060 610 6.7 4.41 0.15 2.63 255 0.36 0.009
HF075 680 6.9 5.53 0.08 0.06 340 0.4 0.006
HF090 730 6.9 10.5 0.07 0.13 320 1.12 0.017
78
Nitrate Orthophosphate Sulfate Iron - - 2- Conductivity Turbidity (NO3 ) (PO4 ) (SO4 ) (Fe) Aluminum (Al) Site Sampling time (µS cm-1) pH (NTS) mg L-1 mg L-1 mg L-1 mg L-1 mg L-1
Week 3 (09/01/10)
Clear Creek 460 7.9 4.55 0.05 0.23 21 0.19 0.023
Hewett Fork
HF039 690 7 4.94 0.05 0.07 250 0.46 0.01
HF045 720 6.9 9.63 0.11 0.04 315 0.73 0.007
HF060 860 6.9 6.28 0.06 0.16 395 0.47 0.005
HF075 920 6.8 1.85 0.05 0.93 390 0.25 0.011
HF090 900 6.3 4.38 0.04 0.11 375 0.59 0.022
79
Nitrate Orthophosphate Sulfate Iron - - 2- Conductivity Turbidity (NO3 ) (PO4 ) (SO4 ) (Fe) Aluminum (Al) Site Sampling time (µS cm-1) pH (NTS) mg L-1 mg L-1 mg L-1 mg L-1 mg L-1
Week 6 (09/23/10)
Clear Creek 400 7.8 4.64 0.34 0.12 18 0.13 0.006
Hewett Fork
HF039 880 7 3.12 0.21 0.33 1200 0.37 0.1
HF045 870 7.1 5.71 0.24 0.05 900 0.98 0.012
HF060 930 7 5.39 0.13 0.13 1400 0.37 0.009
HF075 990 7.1 2.52 0.23 0.04 1400 0.28 0.003
HF090 1090 7.2 3.15 0.15 0.07 1400 0.56 0.015
APPENDIX E: NUMBER OF CUMULATIVE (N = 3) TAXA IDENTIFIED AT EACH SITE FOR WEEK 1 SAMPLING.
Clear Creek HF039 HF045 HF060 HF075 HF090 Achnanthes biasolettiana var. subatomus Lange-Bertalot 3 0 0 0 0 0 Achnanthes childanos Hohn & Hellerman 66 140 63 32 60 19 Achnanthes minutissima var. gracillima (Meister) Lange-Bertalot 0 0 0 1 0 0 Achnanthes minutissima var. macrocephala Hustedt 0 0 1 0 0 2 Achnanthes ventralis (Krasske) Lange-Bertalot 0 0 0 0 0 2 Achnanthidium biasolettianum (Grunow) Round & Bukhtiyarova 0 1 4 0 1 1 Achnanthidium kranzii (Lange- Bertalot) Round & Bukhtiyarova 0 0 0 0 0 1 Achnanthidium minutissimum (Kützing) Czarnecki 58 42 85 83 141 81 81
Actinocyclus normanii (Gregory) Hustedt 23 0 1 0 2 8 Amphipleura pellucida (Kützing) Kützing 0 3 0 0 0 0 Amphora aequalis Krammer 33 92 0 81 0 19 Amphora commutata Grunow 0 0 7 0 0 45 Amphora inariensis Krammer 40 36 0 34 7 28 Amphora ovalis (Kützing) Kützing 27 4 8 4 4 3 Amphora pediculus (Kützing) Grunow ex A.Schmidt 326 221 420 467 448 379 Brachysira Brébissonii R.Ross 0 0 0 1 0 0 Brachysira microcephala (Grunow) Compère 0 18 59 21 1 1 Brachysira styriaca (Grunow) R.Ross 0 0 0 0 1 0 Caloneis hyalina Hustedt 0 0 1 0 0 0 82
Capartogramma crucicula (Grunow) R.Ross 0 0 1 0 0 0 Cocconeis pediculus Ehrenburg 11 12 4 5 13 28 Cocconeis placentula Ehrenburg 5 4 0 7 0 7 Cocconeis placentula var. eugylypta (Ehrenburg) Hustedt 137 202 213 390 417 114 Craticula cuspidata (Kützing) D.G.Mann 1 0 0 0 0 0 Craticula halophila (Grunow) D.G.Mann 4 1 0 0 0 4 Cyclotella atomus Hustedt 0 0 1 0 0 0 Cyclotella meneghiniana Kützing 39 0 3 0 6 22 Cymatopleura solea (Brébisson) W.Smith 5 0 0 0 0 2 Cymbella affinis Kützing 0 0 0 0 1 4 Cymbella aspera (Ehrenberg) Cleve 0 0 0 0 0 1 83
Cymbella cistula (Hemprich & Ehrenberg) O.Kirchner 0 0 1 0 0 0 Cymbella delecta A.Sch. 1 0 0 0 0 0 Cymbella tumida (Brébisson in Kützing) van Heurck 1 8 4 3 0 12 Cymbella turgidula Grunow 6 0 0 0 0 0 Cymbopleura amphicephala (Nageli) Krammer 0 0 0 0 0 2 Cymbopleura naviculiformis (Auerswald ex Heiberg) Krammer 0 1 0 2 0 0 Cymbopleura subaequalis (Grunow) Krammer 1 0 0 0 0 2 Denticula tenuis Kützing 0 0 2 1 0 0 Diatoma moniliforme Kützing 0 0 0 0 0 1 Diatoma vulgare Bory 3 1 0 1 0 3 Diploneis parma Cleve 0 0 1 0 0 0 Encyonema caespitosum Kützing 0 0 1 0 0 3 84
Encyonema gracile Rabenhorst 0 0 0 1 0 1 Encyonema minutum (Hilse) D.G.Mann 30 51 0 38 1 36 Encyonema muelleri (Hustedt) D.G.Mann 0 0 2 0 0 0 Encyonema prostratum Kützing 1 0 1 1 0 0 Encyonema silesiacum (Bleisch) D.G.Mann 1 4 1 2 0 9 Eunotia diodon Ehrenberg 0 0 0 0 0 1 Eunotia exigua (Brébisson ex Kützing) Rabenhorst 1 11 34 19 0 6 Eunotia glacialis Meister 0 1 1 0 0 0 Eunotia steineckii Petersen 0 0 2 0 0 0 Fallacia forcipata (Greville) Stickle & Mann 0 0 0 0 0 1 Fallacia subhamulata (Grunow in van Heurck) D.G.Mann 1 0 0 0 0 0 85
Fragilaria biceps (Kützing) Lange-Bertalot 2 31 46 17 1 1 Fragilaria capucina Desmazires 27 27 32 18 22 36 Fragilaria famelica (Kützing) Lange-Bertalot 0 0 12 3 1 3 Fragilaria heidenii Østrup 0 0 0 0 0 1 Fragilaria tenera (W.Smith) Lange-Bertalot 0 0 4 0 0 0 Frustulia rhomboides (Ehrenberg) De Toni 0 0 1 0 0 0 Frustulia rhomboides var. viridula (Brébisson) Cleve 1 0 0 0 0 0 Frustulia vulgaris (Thwaites) De Toni 0 0 1 0 2 0 Geissleria decussis (Østrup) Lange- Bertalot & Metzeltin 5 0 0 0 0 1 Gomphonema angustatum (Kützing) Rabenhorst 0 1 0 0 0 0 Gomphonema helveticum Brun 0 0 3 0 0 0 86
Gomphonema olivaceum (Lyngbye) Desmazires 0 0 0 0 0 3 Gomphonema parvulum (Kützing) H.F.Van Heurck 0 11 8 3 3 4 Gomphonema pseudoaugur Lange-Bertalot 1 2 0 0 0 1 Gomphonema sphaerophorum Ehrenberg 1 0 0 1 1 0 Gomphonema vibrio var. intricatum (Kützing) Playfair 0 0 2 0 0 4 Grunowia tabellaria (Grunow) Rabenhorst 1 0 0 0 1 0 Gyrosigma acuminatum (Kützing) Rabenhorst 13 0 0 0 0 8 Gyrosigma scalproides (Rabenhorst) Cleve 4 0 1 0 0 3 Gyrosigma wansbeckii (Donkin) Cleve 0 0 0 0 1 1 Halamphora normanii (Rabenhorst) Levkov 1 0 0 1 0 0 87
Halamphora veneta (Kützing) Levkov 0 1 1 0 3 0 Hantzschia amphioxys (Ehrenberg) Grunow 0 0 0 0 0 2 Hippodonta capitata (Ehrenberg) Lange-Bertalot, Metzeltin & Witkowski 4 0 0 0 0 1
Melosira lineata (Dillwyn) Agardh 72 5 4 9 12 89 Melosira sp. C.Agardh 37 3 0 2 1 40 Navicella pusilla (Grunow) Krammer 0 0 8 0 2 5 Navicula cari Ehrenberg 6 0 0 0 0 5 Navicula clementis Grunow 3 1 0 0 0 4 Navicula cryptocephala Kützing 101 2 3 1 3 70 Navicula cryptotenella Lange-Bertalot 10 2 0 1 0 7 Navicula detenta Hustedt 0 0 1 1 0 2 Navicula elginensis (W.Gregory) Ralfs 2 0 0 0 0 0 88
Navicula exilis Kützing 3 0 0 0 0 0 Navicula gregaria Donkin 0 0 0 0 1 2 Navicula hustedtii Krasske 0 0 0 1 0 0 Navicula laterostrata Hustedt 9 0 0 0 0 0 Navicula lundii Reichardt 30 6 2 6 2 29 Navicula menisculus Schumann 0 1 0 0 1 0 Navicula pseudoventralis Hustedt 0 1 0 0 0 0 Navicula recens (Lange- Bertalot) Lange- Bertalot 0 0 4 0 0 0 Navicula reichardtiana Lange-Bertalot 0 0 0 0 0 1 Navicula rhynchocephala Kützing 5 1 0 0 0 24 Navicula schroeteri F.Meister 2 0 0 0 0 5 Navicula tripunctata (O.F.Müller) Bory de Saint- Vincent 15 3 2 1 2 2 Navicula trivialis Lange- Bertalot 1 0 0 0 0 0 89
Navicula viridula (Kützing) Ehrenberg 59 5 5 1 3 53 Naviculadicta absoluta (Hustedt) Lange-Bertalot 0 0 1 0 0 0
Neidium dubium (Ehrenberg) Cleve 1 0 0 0 0 0 Nitzschia acicularioides Hustedt 0 1 1 1 0 0 Nitzschia acidoclinata Lange-Bertalot 0 0 5 0 0 0 Nitzschia angustata (W.Smith) Grunow 0 0 0 0 0 4 Nitzschia capitellata Hustedt 5 2 0 0 0 2
Nitzschia clausii Hantzsch 0 3 3 1 0 1 Nitzschia commutata Grunow 0 0 0 0 0 1 Nitzschia dissipata (Kützing) Grunow 13 5 5 0 3 17 Nitzschia fossilis (Grunow) Grunow 0 1 0 0 0 0 90
Nitzschia gracilis Hantzsch 1 0 0 1 0 0 Nitzschia inconspicua Grunow 86 411 306 204 267 43 Nitzschia intermedia Hantzsch ex Cleve & Grunow 11 9 17 6 4 13 Nitzschia linearis (C.Agardh) W.Smith 8 0 0 0 1 3 Nitzschia microcephala Grunow 0 0 0 0 0 4 Nitzschia nana Grunow 0 8 12 6 4 5 Nitzschia palea (Kützing) W.Smith 31 9 17 5 6 23 Nitzschia scalpelliformis Grunow 0 0 0 0 0 1 Nitzschia sigmoidea (Nitzsch) W.Smith 0 0 0 0 0 3 Nitzschia tryblionella Hantzsch 4 0 0 0 0 0 Pinnularia brandelii Cleve 0 0 1 0 0 0 Pinnularia gibba Ehrenberg 0 0 0 0 0 1 Pinnularia interrupta W.Smith 0 1 1 0 0 1 91
Pinnularia microstauron (Ehrenberg) Cleve 0 0 2 0 1 2 Pinnularia subcapitata W.Gregory 0 1 4 0 0 1 Pinnularia viridis (Nitzsch) Ehrenberg 0 0 0 0 0 2 Planothidium delicatulum (Kützing) Round & L.Bukhtiyarova 6 1 2 3 8 0 Planothidium frequentissimum (Lange- Bertalot) Lange- Bertalot 6 4 1 7 3 4 Pleurosira laevis (Ehrenberg) Compère 1 0 0 0 0 2 Psammodictyon constrictum (Gregory) D.G.Mann 6 1 1 0 0 7 Reimeria sinuata (Gregory) Kociolek & Stoermer 6 19 8 6 18 10 Rhoicosphenia abbreviata (C.Agardh) Lange-Bertalot 35 16 4 11 17 25 92
Rossithidium pusillum (Grunow) Round & L.Bukhtiyarova 5 1 0 11 1 14 Sellaphora bacillum (Ehrenberg) D.G.Mann 2 4 0 0 0 2 Sellaphora pupula (Kützing) Mereschkovsky 10 5 0 3 2 6 Sellaphora seminulum (Grunow) D.G.Mann 0 0 0 2 3 0 Stauroneis smithii Grunow 3 2 0 0 0 2 Staurosirella pinnata (Ehrenberg) D.M.Williams & Round 4 30 0 6 0 5 Stenopterobia delicatissima (F.W.Lewis) Brébisson ex van Heurck 0 0 0 0 1 2 Staurosira construens Ehrenberg 0 0 0 0 0 1 Surirella amphioxys W.Smith 5 1 1 0 0 0
Surirella angusta Kützing 0 0 0 0 1 7 Surirella bifrons Ehrenberg 3 0 1 0 3 0 93
Surirella linearis var. helvetica (Brun) Meister 0 0 0 0 0 7 Surirella linearis W.Smith 2 2 8 4 8 21 Surirella minuta Brébisson 0 0 1 0 0 0 Surirella subsalsa W.Smith 12 1 1 1 1 7 Surirella tenera W.Gregory 3 0 2 0 1 7 Thalassiosira baltica (Grunow) Ostenfeld 0 0 0 0 0 16 Tryblionella levidensis W.Smith 2 0 0 0 0 1
94
APPENDIX F: BRAY-CURTIS DISSIMILARITY SCORES FOR EACH SITE AND SAMPLING TIME USING POOLED REPLICATES (N = 3). MEAN IS THE MEAN DISSIMILARITY BETWEEN CLEAR CREEK AND HEWETT FORK SITES.
Site Sampling Time Clear Creek HF039 HF045 HF060 HF075 Week 1 (08/18/10) HF039 0.4647137 - HF045 0.469086 0.3137454 - HF060 0.441601 0.3326739 0.2716378 - HF075 0.4702774 0.3879854 0.230563 0.1753927 - HF090 0.2416063 0.5335759 0.4629259 0.4409654 0.4819435 Mean 0.41745688 Week 3 (09/01/10) HF039 0.5776521 - HF045 0.6346025 0.252411 - HF060 0.6449468 0.3075141 0.2180851 - HF075 0.6394535 0.3125415 0.2462512 0.2623549 - HF090 0.5199203 0.4195041 0.4209827 0.3582287 0.2931434 Mean 0.60331504 Week 6 (09/23/10) HF039 0.5378989 - HF045 0.5324503 0.2773333 - HF060 0.6195832 0.4518815 0.4288557 - HF075 0.7131553 0.6023896 0.5570248 0.3698811 - HF090 0.531733 0.5014896 0.4830201 0.5144928 0.6703874 Mean 0.58696414
APPENDIX G: BRAY-CURTIS DISSIMILARITY SCORES FOR EACH SITE AND SAMPLING TIME, ROUNDED TO TWO DECIMAL PLACES.
Clear Rep. Creek HF039 HF045 HF060 HF075 HF090 Wk Rep. 1 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 Clear Creek 2 0.27 - 3 0.22 0.24 - HF039 1 0.48 0.54 0.44 - 2 0.45 0.51 0.42 0.20 - 3 0.49 0.56 0.49 0.17 0.16 - HF045 1 0.45 0.56 0.50 0.32 0.28 0.30 - 2 0.43 0.54 0.51 0.30 0.29 0.32 0.14 - 3 0.44 0.53 0.51 0.55 0.46 0.50 0.41 0.36 - HF060 1 0.44 0.55 0.45 0.44 0.35 0.38 0.33 0.39 0.38 - 2 0.41 0.53 0.44 0.39 0.29 0.31 0.32 0.31 0.33 0.16 - 3 0.41 0.53 0.45 0.42 0.35 0.38 0.30 0.31 0.33 0.21 0.14 - HF075 1 0.43 0.55 0.50 0.41 0.33 0.37 0.20 0.23 0.37 0.23 0.21 0.23 - 2 0.43 0.55 0.50 0.40 0.33 0.36 0.21 0.25 0.37 0.24 0.20 0.22 0.08 - 3 0.41 0.52 0.47 0.51 0.46 0.48 0.41 0.39 0.36 0.34 0.27 0.23 0.26 0.24 - HF090 1 0.31 0.31 0.27 0.53 0.47 0.56 0.54 0.52 0.49 0.51 0.46 0.50 0.54 0.53 0.49 - 2 0.29 0.37 0.33 0.59 0.56 0.60 0.47 0.51 0.49 0.47 0.49 0.48 0.49 0.47 0.51 0.29 - 3 0.30 0.30 0.32 0.58 0.55 0.59 0.51 0.50 0.46 0.51 0.47 0.48 0.51 0.50 0.48 0.26 0.28
Wk 3 Clear Creek 2 0.23 - 3 0.21 0.24 - HF039 1 0.60 0.67 0.58 - 96
2 0.61 0.68 0.58 0.25 - 3 0.64 0.68 0.61 0.45 0.30 - HF045 1 0.65 0.69 0.62 0.27 0.33 0.50 - 2 0.65 0.72 0.64 0.23 0.32 0.48 0.21 - 3 0.65 0.71 0.67 0.46 0.38 0.34 0.49 0.39 - HF060 1 0.66 0.71 0.63 0.30 0.33 0.46 0.22 0.29 0.43 - 2 0.65 0.70 0.61 0.40 0.34 0.36 0.41 0.39 0.26 0.30 - 3 0.66 0.71 0.62 0.30 0.36 0.53 0.23 0.29 0.52 0.17 0.40 - HF075 1 0.64 0.69 0.61 0.39 0.37 0.38 0.39 0.32 0.36 0.32 0.26 0.41 - 2 0.67 0.73 0.64 0.26 0.33 0.47 0.26 0.18 0.41 0.24 0.32 0.25 0.20 - 3 0.62 0.69 0.59 0.46 0.40 0.43 0.44 0.42 0.43 0.38 0.28 0.44 0.24 0.32 - HF090 1 0.78 0.79 0.76 0.68 0.67 0.60 0.74 0.71 0.67 0.71 0.59 0.74 0.61 0.64 0.47 - 2 0.53 0.57 0.45 0.37 0.49 0.56 0.33 0.43 0.61 0.34 0.51 0.33 0.48 0.40 0.52 0.71 - 3 0.56 0.58 0.48 0.44 0.55 0.60 0.39 0.47 0.62 0.37 0.55 0.34 0.51 0.46 0.52 0.76 0.25
Wk 6 Clear Creek 2 0.25 - 3 0.25 0.23 - HF039 1 0.50 0.55 0.52 - 2 0.60 0.61 0.61 0.24 - 3 0.55 0.55 0.57 0.28 0.27 - HF045 1 0.52 0.59 0.56 0.32 0.33 0.35 - 2 0.56 0.59 0.58 0.36 0.39 0.31 0.25 - 3 0.53 0.57 0.56 0.32 0.39 0.40 0.23 0.28 - HF060 1 0.63 0.64 0.63 0.44 0.46 0.40 0.46 0.31 0.49 - 2 0.73 0.75 0.74 0.54 0.55 0.47 0.56 0.44 0.59 0.27 - 3 0.58 0.62 0.59 0.50 0.53 0.46 0.46 0.34 0.50 0.25 0.34 - HF075 1 0.75 0.75 0.76 0.63 0.64 0.57 0.59 0.55 0.62 0.40 0.31 0.52 - 2 0.65 0.69 0.66 0.61 0.65 0.57 0.50 0.49 0.58 0.39 0.47 0.46 0.34 - 3 0.79 0.81 0.81 0.69 0.67 0.63 0.69 0.58 0.72 0.43 0.34 0.55 0.27 0.24 - HF090 1 0.58 0.56 0.57 0.59 0.66 0.60 0.60 0.54 0.61 0.54 0.61 0.45 0.75 0.70 0.79 - 97
2 0.56 0.57 0.57 0.53 0.57 0.52 0.50 0.49 0.53 0.55 0.62 0.52 0.64 0.63 0.72 0.39 - 3 0.66 0.64 0.65 0.52 0.54 0.52 0.57 0.57 0.57 0.59 0.64 0.66 0.69 0.73 0.75 0.44 0.34 98
APPENDIX H: NUMBER OF CUMULATIVE (N = 3) TAXA IDENTIFIED AT EACH SITE FOR WEEK 3 SAMPLING.
Clear Creek HF039 HF045 HF060 HF075 HF090 Achnanthes biasolettiana var. subatomus Lange-Bertalot 0 0 2 0 0 0 Achnanthes childanos Hohn & Hellerman 34 31 31 44 24 37 Achnanthes minutissima var. macrocephala Hustedt 0 0 0 0 2 0 Achnanthidium biasolettianum (Grunow) Round & Bukhtiyarova 2 0 5 0 1 0 Achnanthidium jackii Rabenhorst 0 0 0 0 2 2 Achnanthidium minutissimum (Kützing) Czarnecki 70 140 168 117 185 78 Actinocyclus normanii (Gregory) Hustedt 1 0 0 0 4 0 Amphipleura pellucida (Kützing) Kützing 0 2 4 0 0 1 Amphora aequalis Krammer 31 28 0 31 0 36 99
Amphora delicatissima Krasske 0 5 0 0 0 1 Amphora inariensis Krammer 10 9 3 0 4 12 Amphora ovalis (Kützing) Kützing 10 6 0 5 9 3 Amphora pediculus (Kützing) Grunow ex A.Schmidt 156 223 327 455 296 353 Aulocoseria sp. Thwaites 0 0 0 0 0 1 Brachysira Brébissonii R.Ross 0 0 0 1 0 0 Brachysira microcephala (Grunow) Compère 0 52 51 77 151 152 Brachysira styriaca (Grunow) R.Ross 0 0 0 0 1 1 Caloneis silicula (Ehrenberg) Cleve 0 0 0 0 0 1 Cocconeis pediculus Ehrenburg 32 5 9 4 11 8 Cocconeis placentula Ehrenburg 0 2 0 0 0 0 Cocconeis placentula var. eugylypta (Ehrenburg) Hustedt 49 233 328 365 273 111 100
Craticula accomoda (Hustedt) D.G.Mann 0 1 0 0 0 0 Craticula halophila (Grunow) D.G.Mann 9 0 0 0 0 4 Cyclotella atomus Hustedt 5 0 0 0 0 0 Cyclotella meneghiniana Kützing 41 3 0 3 9 13 Cymatopleura solea (Brébisson) W.Smith 1 0 0 0 0 1 Cymbella affinis Kützing 2 0 0 0 0 1 Cymbella aspera (Ehrenberg) Cleve 0 8 0 0 0 0 Cymbella cistula (Hemprich & Ehrenberg) O.Kirchner 0 14 0 0 3 0 Cymbella tumida (Brébisson in Kützing) van Heurck 3 0 0 0 0 0 Cymbella turgidula Grunow 0 0 0 0 0 1 Cymbopleura naviculiformis (Auerswald ex Heiberg) Krammer 0 3 0 0 2 1 101
Cymbopleura subaequalis (Grunow) Krammer 0 0 1 0 0 0 Denticula tenuis Kützing 2 5 1 0 1 1 Diatoma moniliforme Kützing 1 0 0 0 0 0 Diatoma vulgare Bory 22 1 0 0 0 0 Diploneis lineata (Donkin) Cleve 0 1 0 0 0 0 Diploneis parma Cleve 0 10 3 0 0 1 Diploneis subconstricta f. alpina (Meister) Cleve-Euler 0 0 1 0 0 0 Diploneis subovalis Cleve 1 1 1 0 0 0 Encyonema caespitosum Kützing 0 16 2 0 0 0 Encyonema gracile Rabenhorst 1 4 0 0 0 2 Encyonema minutum (Hilse) D.G.Mann 11 22 3 7 2 4 Encyonema muelleri (Hustedt) D.G.Mann 0 1 3 0 0 0 Encyonema obscurum (Krasske) D.G.Mann 0 1 0 0 0 0 Encyonema prostratum Kützing 0 0 3 0 1 0 102
Encyonema silesiacum (Bleisch) D.G.Mann 0 12 7 2 4 1 Eunotia bilunaris (Ehrenberg) Schaarschmidt 15 6 1 8 0 8 Eunotia diodon Ehrenberg 0 1 0 0 0 0 Eunotia exigua (Brébisson ex Kützing) Rabenhorst 0 58 27 70 135 150 Eunotia formica Ehrenberg 0 1 0 0 0 0 Eunotia glacialis Meister 0 3 0 4 0 0 Eunotia monodon Ehrenberg 0 1 2 0 0 0 Eunotia steineckii Petersen 0 5 3 0 1 0 Eunotia vanheurckii R.M.Patrick 0 0 4 0 0 0 Fallacia pygmaea (Kützing) A.J.Stickle & D.G.Mann 1 0 0 0 0 0 Fallacia subhamulata (Grunow in van Heurck) D.G.Mann 3 0 0 1 0 0 Fragilaria biceps (Kützing) Lange-Bertalot 1 56 9 16 34 11 103
Fragilaria capucina Desmazires 37 94 63 28 25 28 Fragilaria famelica (Kützing) Lange- Bertalot 0 6 17 12 2 4 Fragilaria tenera (W.Smith) Lange-Bertalot 0 1 0 0 0 0 Frustulia rhomboides (Ehrenberg) De Toni 2 6 0 1 0 4 Geissleria decussis (Østrup) Lange-Bertalot & Metzeltin 6 0 0 0 0 4 Gomphonema angustatum (Kützing) Rabenhorst 1 0 0 0 1 0 Gomphonema clavatum Ehrenberg 0 0 1 0 0 0 Gomphonema gracile Ehrenberg 0 4 0 0 0 0 Gomphonema hebridense W.Gregory 0 1 0 0 0 0 Gomphonema minutum (C.Agardh) C.Agardh 0 1 0 0 0 0 Gomphonema olivaceum (Lyngbye) Desmazires 1 0 0 0 0 0 104
Gomphonema parvulum (Kützing) H.F.Van Heurck 1 65 36 12 10 0 Gomphonema parvulum var. exilissima Grunow 0 0 0 0 0 15 Gomphonema pseudoaugur Lange-Bertalot 0 0 3 1 0 0 Gomphonema sphaerophorum Ehrenberg 2 1 0 1 0 1 Gomphonema truncatum Ehrenberg 0 0 1 0 0 0 Gomphonitzschia sp. Grunow 0 0 1 0 0 1 Grunowia solgensis (Cleve- Euler) M.Aboal 0 0 1 0 0 1 Gyrosigma acuminatum (Kützing) Rabenhorst 8 2 0 0 0 3 Gyrosigma obscurum (W.Smith) J.W.Griffith & Henfrey 0 1 0 0 0 0 Gyrosigma scalproides (Rabenhorst) Cleve 3 0 0 0 0 1 Gyrosigma wansbeckii (Donkin) Cleve 0 2 1 1 0 1 Halamphora montana (Krasske) 0 0 0 2 0 0 105
Levkov
Halamphora veneta (Kützing) Levkov 2 0 0 0 0 2 Hippodonta capitata (Ehrenberg) Lange-Bertalot, Metzeltin & Witkowski 1 2 0 0 1 1 Melosira lineata (Dillwyn) Agardh 149 4 5 7 27 38 Melosira sp. C.Agardh 55 1 1 0 2 6 Meridion circulare (Greville) C.Agardh 1 0 1 0 0 0 Meridion circulare var. constrictum (Ralfs) Van Heurck 0 0 0 1 0 0 Navicella pusilla (Grunow) Krammer 0 0 13 0 19 0 Navicula angusta Grunow 1 0 0 0 0 0 Navicula capitoradiata Germain 8 0 0 0 0 0 Navicula cari Ehrenberg 14 4 0 1 0 2 Navicula clementis Grunow 0 0 3 0 0 0 106
Navicula cryptocephala Kützing 265 8 10 7 11 20 Navicula cryptotenella Lange-Bertalot 7 8 1 1 0 3 Navicula detenta Hustedt 1 0 0 0 0 2 Navicula elginensis (W.Gregory) Ralfs 0 2 0 0 0 1 Navicula exilis Kützing 0 0 2 0 0 0 Navicula gregaria Donkin 0 0 0 0 0 1 Navicula krasskei Hustedt 0 0 0 0 1 0 Navicula lanceolata Ehrenberg 1 0 0 0 0 0 Navicula laterostrata Hustedt 0 0 0 0 0 8 Navicula leptostriata Jorgensen 2 0 0 0 0 0 Navicula lundii Reichardt 37 3 2 2 3 11 Navicula menisculus Schumann 1 0 0 1 0 0 Navicula modica Hustedt 5 1 0 0 0 0 Navicula porifera var. opportuna (Hustedt) Lang- Bertalot 0 0 0 0 1 0 107
Navicula pseudoventralis Hustedt 0 0 0 0 1 0 Navicula radiosa Kützing 4 0 0 0 0 0 Navicula reichardtiana Lange-Bertalot 0 0 0 4 0 6 Navicula rhynchocephala Kützing 0 1 0 0 0 1 Navicula subtilissima Cleve 0 0 1 0 0 0 Navicula tripunctata (O.F.Müller) Bory de Saint- Vincent 17 0 1 0 3 6 Navicula viridula (Kützing) Ehrenberg 26 12 7 2 9 27 Naviculadicta absoluta (Hustedt) Lange- Bertalot 3 0 0 1 1 0 Neidium dubium (Ehrenberg) Cleve 2 0 0 0 0 0 Nitzschia acicularioides Hustedt 2 0 1 0 2 0 Nitzschia acidoclinata Lange-Bertalot 2 7 10 0 0 3 Nitzschia amphibia Grunow 0 0 1 0 0 0 108
Nitzschia angustata (W.Smith) Grunow 1 0 3 0 0 0 Nitzschia capitellata Hustedt 9 12 0 4 3 16 Nitzschia clausii Hantzsch 0 1 0 1 1 4 Nitzschia constricta var. subconstricta Grunow 1 0 0 0 0 0 Nitzschia dissipata (Kützing) Grunow 16 12 10 5 5 13 Nitzschia gracilis Hantzsch 1 9 0 0 0 0 Nitzschia inconspicua Grunow 52 130 161 135 96 50 Nitzschia intermedia Hantzsch ex Cleve & Grunow 14 17 21 21 35 26 Nitzschia linearis (C.Agardh) W.Smith 5 3 2 0 0 2 Nitzschia lorenziana var. incerta Grunow 0 0 0 0 1 0 Nitzschia nana Grunow 0 16 22 5 27 12 Nitzschia palea (Kützing) W.Smith 39 37 28 7 12 33 109
Nitzschia recta Hantzsch ex Rabenhorst 2 0 0 0 0 0 Nitzschia sigmoidea (Nitzsch) W.Smith 1 0 0 0 0 0 Nitzschia tryblionella Hantzsch 1 0 0 0 0 0 Pinnularia appendiculata (C.Agardh) Cleve 0 0 0 0 0 3 Pinnularia gibba Ehrenberg 0 0 0 0 0 1 Pinnularia macilenta Ehrenberg 0 0 0 0 1 0 Pinnularia microstauron (Ehrenberg) Cleve 0 0 0 0 6 0 Pinnularia subcapitata W.Gregory 0 0 1 0 1 3 Plagiotropis sp. Pfitzer 0 1 0 0 0 0 Planothidium delicatulum (Kützing) Round & L.Bukhtiyarova 15 12 8 3 3 4 Planothidium frequentissimum (Lange-Bertalot) Lange-Bertalot 5 0 1 3 6 5 Pleurosira laevis (Ehrenberg) Compère 0 0 0 0 0 1 110
Psammodictyon constrictum (Gregory) D.G.Mann 10 2 0 0 0 7 Reimeria sinuata (Gregory) Kociolek & Stoermer 5 13 20 8 6 5 Rhoicosphenia abbreviata (C.Agardh) Lange-Bertalot 30 6 19 11 8 7 Rhopalodia Brébissonii Krammer 0 0 0 0 0 1 Rossithidium pusillum (Grunow) Round & L.Bukhtiyarova 6 13 5 0 0 2 Sellaphora bacillum (Ehrenberg) D.G.Mann 5 0 0 0 0 3 Sellaphora pupula (Kützing) Mereschkovsky 12 1 3 5 4 4 Sellaphora seminulum (Grunow) D.G.Mann 2 2 0 0 1 0 Stauroneis smithii Grunow 0 0 1 1 0 1 Staurosirella pinnata (Ehrenberg) D.M.Williams & Round 1 3 0 0 0 0 111
Stenopterobia delicatissima (F.W.Lewis) Brébisson ex van Heurck 0 0 1 0 1 8 Staurosira construens Ehrenberg 1 0 0 0 0 0 Surirella amphioxys W.Smith 1 1 0 0 2 0 Surirella angusta Kützing 1 0 3 0 0 1 Surirella bifrons Ehrenberg 0 0 0 0 1 2 Surirella capronii Brébisson ex F.Kitton 0 0 0 0 0 5 Surirella linearis W.Smith 1 7 4 3 7 87 Surirella minuta Brébisson 1 0 0 0 0 2 Surirella subsalsa W.Smith 30 2 0 0 1 1 Surirella tenera W.Gregory 15 2 1 2 2 4 Tabellaria flocculosa (Roth) Kützing 0 0 1 2 0 0 Thalassiosira baltica (Grunow) Ostenfeld 29 0 0 0 0 2 Tryblionella levidensis W.Smith 4 0 0 0 0 3 Tryblionella littoralis (Grunow) D.G.Mann 0 1 0 0 0 0 112
APPENDIX I: NUMBER OF CUMULATIVE (N = 3) TAXA IDENTIFIED AT EACH SITE FOR WEEK 6 SAMPLING
Clear Creek HF039 HF045 HF060 HF075 HF090
Achnanthes biasolettiana var. subatomus Lange-Bertalot 56 0 0 0 0 0
Achnanthes childanos Hohn & Hellerman 83 47 45 27 30 1 Achnanthes laevis Østrup 0 0 0 0 0 1
Achnanthes minutissima var. gracillima (Meister) Lange- Bertalot 0 0 2 0 0 0
Achnanthidium biasolettianum (Grunow) Round & Bukhtiyarova 0 7 6 0 0 0
Achnanthidium jackii Rabenhorst 0 3 0 0 0 0
Achnanthidium minutissimum (Kützing) Czarnecki 39 227 231 131 44 128 113
Actinocyclus normanii (Gregory) Hustedt 37 0 0 0 1 0 Amphipleura pellucida (Kützing) Kützing 0 17 5 0 0 0 Amphora aequalis Krammer 42 8 0 20 0 22 Amphora commutata Grunow 1 1 0 0 0 0 Amphora inariensis Krammer 37 17 4 17 6 275
Amphora ovalis (Kützing) Kützing 7 0 3 1 1 5 Amphora pediculus (Kützing) Grunow ex A.Schmidt 209 95 228 176 130 233 Bacillaria paradoxa J.F.Gmelin 0 1 0 0 0 4 Brachysira Brébissonii R.Ross 0 1 0 0 0 0 Brachysira microcephala (Grunow) Compère 0 96 103 319 485 40
Brachysira styriaca (Grunow) R.Ross 0 0 0 1 7 0 114
Caloneis tenuis (W.Gregory) Krammer 1 0 0 0 0 0
Capartogramma crucicula (Grunow) R.Ross 0 1 0 0 0 0 Cavinula variostriata (Krasske) D.G.Mann & A.J.Stickle 2 0 0 0 0 0 Cocconeis pediculus Ehrenburg 13 6 4 2 2 1 Cocconeis placentula Ehrenburg 2 4 0 2 0 19 Cocconeis placentula var. eugylypta (Ehrenburg) Hustedt 103 156 122 331 93 164 Craticula cuspidata (Kützing) D.G.Mann 0 0 0 0 0 1 Craticula halophila (Grunow) D.G.Mann 3 3 0 0 0 0
Cyclotella atomus Hustedt 0 1 0 0 0 0 Cyclotella meneghiniana Kützing 16 11 0 2 2 4 115
Cymatopleura elliptica (Brébisson) W.Smith 1 0 0 0 0 0
Cymatopleura solea (Brébisson) W.Smith 3 0 0 0 0 2
Cymbella affinis Kützing 0 1 0 0 0 1
Cymbella aspera (Ehrenberg) Cleve 0 9 0 0 0 0
Cymbella cistula (Hemprich & Ehrenberg) O.Kirchner 0 0 0 1 0 0
Cymbella descripta (Hustedt) Krammer & Lange-Bertalot 2 0 0 1 0 3 Cymbella hustedtii (Krasske) 0 0 0 0 0 5 Cymbella leptoceros (Ehrenberg) Kützing 0 0 0 1 0 0
Cymbella tumida (Brébisson in Kützing) van Heurck 0 0 0 0 0 5 Cymbella turgidula Grunow 1 1 0 0 0 0 116
Cymbopleura amphicephala (Nageli) Krammer 0 2 0 0 0 0 Cymbopleura hybrida (Grunow) Krammer 0 0 0 0 0 5 Cymbopleura naviculiformis (Auerswald ex Heiberg) Krammer 0 8 13 0 0 2 Cymbopleura subaequalis (Grunow) Krammer 0 1 0 0 0 0 Delicata delicatula (Kützing) Krammer 2 0 0 0 0 0
Denticula tenuis Kützing 1 3 6 0 0 1 Diatoma vulgare Bory 6 0 0 0 0 0 Diploneis parma Cleve 0 24 4 0 1 0
Diploneis subconstricta f. alpina (Meister) Cleve-Euler 0 1 0 0 0 0 Encyonema caespitosum Kützing 0 0 2 0 0 0 Encyonema gracile Rabenhorst 0 4 1 0 0 0 117
Encyonema minutum (Hilse) D.G.Mann 23 23 10 18 5 1 Encyonema muelleri (Hustedt) D.G.Mann 0 0 1 0 0 0 Encyonema prostratum Kützing 0 1 0 0 0 0 Encyonema silesiacum (Bleisch) D.G.Mann 1 26 13 1 0 2
Eolimna minima (Grunow) Lange- Bertalot 0 0 0 0 0 1
Eunotia bilunaris (Ehrenberg) Schaarschmidt 0 0 5 0 2 0
Eunotia circumborealis Lange-Bertalot & Norpel 0 1 0 0 0 0
Eunotia exigua (Brébisson ex Kützing) Rabenhorst 0 52 76 186 475 52
Eunotia glacialis Meister 2 3 0 3 2 0 118
Eunotia monodon var. bidens (W.Gregory) Hustedt 0 1 0 0 0 0 Eunotia steineckii Petersen 0 1 0 0 0 1 Eunotia vanheurckii R.M.Patrick 0 0 2 0 1 0 Fallacia subhamulata (Grunow in van Heurck) D.G.Mann 6 0 0 0 0 0
Fragilaria biceps (Kützing) Lange- Bertalot 0 6 13 10 23 1 Fragilaria capucina Desmazires 18 76 45 16 0 33
Fragilaria famelica (Kützing) Lange- Bertalot 2 13 11 8 5 5 Fragilaria nitzschioides Grunow 1 0 0 0 0 0 Frustulia rhomboides (Ehrenberg) De Toni 0 6 1 2 0 2 Frustulia vulgaris (Thwaites) De Toni 0 8 0 2 1 0 119
Geissleria decussis (Østrup) Lange-Bertalot & Metzeltin 0 0 0 0 0 2
Geissleria similis (Krasske) Lange- Bertalot & Metzeltin 0 0 0 0 0 2
Gomphonema amoenum Lange- Bertalot 0 0 0 0 0 1 Gomphonema angustatum (Kützing) Rabenhorst 1 0 0 0 0 0 Gomphonema clavatum Ehrenberg 0 0 0 0 1 1 Gomphonema gracile Ehrenberg 0 1 0 0 0 0 Gomphonema olivaceum (Lyngbye) Desmazires 0 0 0 0 0 1
Gomphonema parvulum (Kützing) H.F.Van Heurck 0 17 19 8 2 3
Gomphonema pseudoaugur Lange-Bertalot 0 0 3 0 0 0 120
Gomphonema vibrio var. intricatum (Kützing) Playfair 0 6 1 0 0 0
Gomphosphenia grovei (M.Schmidt) Lange-Bertalot 2 0 0 0 0 0 Gyrosigma acuminatum (Kützing) Rabenhorst 9 0 0 0 0 6 Gyrosigma scalproides (Rabenhorst) Cleve 3 0 0 0 0 0
Gyrosigma wansbeckii (Donkin) Cleve 0 11 0 0 2 0 Halamphora montana (Krasske) Levkov 1 0 0 0 0 0
Halamphora veneta (Kützing) Levkov 1 1 2 0 0 2
Hippodonta capitata (Ehrenberg) Lange-Bertalot, Metzeltin & Witkowski 3 0 0 0 0 0 121
Karayevia clevei (Grunow) Round & Bukhtiyarova 0 1 0 0 0 0
Melosira lineata (Dillwyn) Agardh 25 1 0 1 2 20 Melosira sp. C.Agardh 18 5 0 1 0 24
Navicella pusilla (Grunow) Krammer 0 0 5 1 0 0 Navicula capitoradiata Germain 0 0 0 0 0 7 Navicula cari Ehrenberg 5 0 3 2 0 0 Navicula cryptocephala Kützing 136 33 15 1 0 20
Navicula cryptofallax Lange-Bertalot & Hofmann 0 0 0 3 0 0
Navicula cryptotenella Lange-Bertalot 18 1 1 0 0 10 Navicula denudata Pantocsek 0 0 0 0 0 1
Navicula detenta Hustedt 7 0 0 0 2 1 Navicula diluviana Krasske 1 0 0 0 0 1 122
Navicula elginensis (W.Gregory) Ralfs 5 0 0 0 0 0
Navicula gregaria Donkin 0 0 0 1 0 0 Navicula longicephala Hustedt 0 4 0 0 1 0
Navicula lundii Reichardt 62 9 5 2 4 6 Navicula menisculus Schumann 0 1 0 0 1 1
Navicula recens (Lange-Bertalot) Lange-Bertalot 0 0 0 0 0 1
Navicula reichardtiana Lange-Bertalot 1 0 0 0 0 0 Navicula rhynchocephala Kützing 4 2 0 0 0 26 Navicula schroeteri F.Meister 0 5 0 0 0 0 Navicula tridentula Krasske 0 1 0 0 0 0 Navicula tripunctata (O.F.Müller) Bory de Saint- Vincent 19 2 1 1 0 0 123
Navicula viridula (Kützing) Ehrenberg 65 19 4 2 5 29
Naviculadicta absoluta (Hustedt) Lange- Bertalot 25 0 0 0 0 0 Nitzschia acicularioides Hustedt 0 7 7 1 0 5 Nitzschia acicularis (Kützing) W.Smith 0 0 0 0 0 1
Nitzschia acidoclinata Lange-Bertalot 0 0 21 1 0 0 Nitzschia amphibia Grunow 1 0 0 0 0 0 Nitzschia angustata (W.Smith) Grunow 1 0 0 0 0 1 Nitzschia capitellata Hustedt 11 7 12 2 0 1
Nitzschia clausii Hantzsch 0 1 6 3 0 0
Nitzschia constricta var. subconstricta Grunow 0 2 0 0 0 0 124
Nitzschia dissipata (Kützing) Grunow 19 15 5 2 3 7 Nitzschia fonticola (Grunow) Grunow 0 1 0 0 0 6
Nitzschia gracilis Hantzsch 3 24 0 5 0 0 Nitzschia inconspicua Grunow 130 129 247 67 42 45
Nitzschia intermedia Hantzsch ex Cleve & Grunow 11 37 29 37 33 15
Nitzschia linearis (C.Agardh) W.Smith 8 3 1 1 0 1
Nitzschia lorenziana var. incerta Grunow 0 10 5 4 1 0 Nitzschia nana Grunow 0 12 22 6 24 2
Nitzschia palea (Kützing) W.Smith 38 70 53 9 33 32 Nitzschia paleacea Grunow 1 0 0 0 0 0 Nitzschia perminuta (Grunow) M.Peragallo 0 0 16 0 0 2 125
Nitzschia sigmoidea (Nitzsch) W.Smith 0 0 0 1 0 0
Pinnularia brandelii Cleve 3 0 0 0 0 0 Pinnularia interrupta W.Smith 0 1 0 0 0 4
Pinnularia maior (Kützing) Cleve 0 1 1 0 0 0 Pinnularia microstauron (Ehrenberg) Cleve 1 2 0 0 0 0 Pinnularia subcapitata W.Gregory 2 1 3 0 0 1
Pinnularia viridis (Nitzsch) Ehrenberg 0 1 0 0 0 0
Planothidium delicatulum (Kützing) Round & L.Bukhtiyarova 18 0 8 4 1 0
Planothidium frequentissimum (Lange-Bertalot) Lange-Bertalot 9 1 3 9 2 0
Pleurosira laevis (Ehrenberg) Compère 2 0 0 0 0 4 126
Psammodictyon constrictum (Gregory) D.G.Mann 14 1 1 0 2 1
Pseudostaurosira elliptica (Schumann) Edlund, Morales & Spaulding 0 0 0 0 0 4
Pseudostaurosira parasitica (W.Smith) Morales 1 0 0 0 0 0
Reimeria sinuata (Gregory) Kociolek & Stoermer 5 8 25 11 5 6
Rhoicosphenia abbreviata (C.Agardh) Lange-Bertalot 35 4 5 2 2 19 Rhopalodia Brébissonii Krammer 0 0 2 0 0 0
Rossithidium pusillum (Grunow) Round & L.Bukhtiyarova 10 28 9 5 5 0 Sellaphora bacillum (Ehrenberg) D.G.Mann 1 0 0 1 0 1 127
Sellaphora laevissima (Kützing) D.G.Mann 1 0 0 0 0 0
Sellaphora pupula (Kützing) Mereschkovsky 21 6 3 4 0 10 Stauroneis anceps Ehrenberg 0 1 0 0 0 0 Stauroneis gracillima Hustedt 0 1 0 0 0 0 Stauroneis phoenicenteron (Nitzsch) Ehrenberg 0 0 1 0 0 0
Stauroneis smithii Grunow 0 4 0 0 0 0 Staurosirella pinnata (Ehrenberg) D.M.Williams & Round 6 1 0 16 0 0 Stenopterobia delicatissima (F.W.Lewis) Brébisson ex van Heurck 0 0 0 0 0 32 Staurosira construens Ehrenberg 0 0 0 0 22 0 Surirella amphioxys W.Smith 0 4 0 0 0 0
Surirella angusta Kützing 1 1 1 0 0 0 128
Surirella capronii Brébisson ex F.Kitton 1 0 1 0 0 7
Surirella elegans Ehrenberg 1 0 0 0 0 0
Surirella linearis var. helvetica (Brun) Meister 1 0 0 0 0 0
Surirella linearis W.Smith 2 4 3 5 6 96
Surirella minuta Brébisson 0 5 0 1 0 32 Surirella subsalsa W.Smith 18 0 1 0 1 5
Surirella tenera W.Gregory 5 2 0 1 0 1
Tabellaria flocculosa (Roth) Kützing 0 0 0 7 0 0
Thalassiosira baltica (Grunow) Ostenfeld 0 0 0 0 0 1 Tryblionella levidensis W.Smith 0 5 0 0 1 0 Tryblionella littoralis (Grunow) D.G.Mann 2 1 0 3 0 0
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APPENDIX J: NUMBER OF CUMULATIVE (N = 3) TAXA IDENTIFIED AT EACH SITE FROM THE COMPARATIVE STUDY OF CLEAR CREEK, HF039 AND HF090
Clear Creek HF039 HF090 Achnanthes childanos Hohn & Hellerman 26 0 1 Achnanthes laevis Østrup 0 0 1 Achnanthes rosenstockii Lange-Bertalot 0 6 0 Achnanthidium biasolettianum (Grunow) Round & Bukhtiyarova 2 0 1 Achnanthidium jackii Rabenhorst 0 23 5 Achnanthidium minutissimum (Kützing) Czarnecki 67 720 47 Actinocyclus normanii (Gregory) Hustedt 20 0 0 Amphipleura pellucida (Kützing) Kützing 0 20 1 Amphora aequalis Krammer 2 0 0 Amphora inariensis Krammer 9 1 4 130
Amphora ovalis (Kützing) Kützing 25 0 0 Amphora pediculus (Kützing) Grunow ex A.Schmidt 324 7 3 Bacillaria paradoxa J.F.Gmelin 0 7 0 Brachysira microcephala (Grunow) Compère 1 159 362 Brachysira vitrea (Grunow) R.Ross 0 0 4 Caloneis hyalina Hustedt 1 0 1 Capartogramma crucicula (Grunow) R.Ross 0 2 0 Cavinula cocconeiformis (Gregory ex Greville) D.G.Mann & A.J.Stickle 2 0 0 Cocconeis pediculus Ehrenburg 17 0 0 Cocconeis placentula Ehrenburg 0 5 0 Cocconeis placentula var. eugylypta (Ehrenburg) Hustedt 133 11 2 131
Craticula cuspidata (Kützing) D.G.Mann 1 0 0 Craticula halophila (Grunow) D.G.Mann 0 2 0 Cyclotella atomus Hustedt 3 1 0 Cyclotella glabriuscula (Grunow) Håkansson 6 1 0 Cyclotella meneghiniana Kützing 30 9 0 Cymatopleura elliptica (Brébisson) W.Smith 6 0 0 Cymatopleura solea (Brébisson) W.Smith 2 0 0 Cymbella affinis Kützing 0 1 0 Cymbella aspera (Ehrenberg) Cleve 0 7 0 Cymbella descripta (Hustedt) Krammer & Lange-Bertalot 0 2 0 Cymbella turgidula Grunow 6 1 0 132
Cymbopleura hybrida (Grunow) Krammer 0 1 0 Cymbopleura lata (Grunow) Krammer 0 1 0 Cymbopleura naviculiformis (Auerswald ex Heiberg) Krammer 0 9 0 Cymbopleura subaequalis (Grunow) Krammer 2 3 0 Denticula tenuis Kützing 2 2 0 Diatoma moniliforme Kützing 0 0 1 Diatoma vulgare Bory 4 0 0 Diploneis elliptica (Kützing) Cleve 0 1 0 Diploneis parma Cleve 0 1 0 Diploneis puella (Schumann) Cleve 0 1 0 Diploneis subconstricta f. alpina (Meister) Cleve-Euler 1 0 0 Encyonema caespitosum Kützing 0 15 0 Encyonema gracile Rabenhorst 13 1 0 133
Encyonema minutum (Hilse) D.G.Mann 3 0 2 Encyonema muelleri (Hustedt) D.G.Mann 1 0 0 Encyonema silesiacum (Bleisch) D.G.Mann 1 15 1 Eunotia diodon Ehrenberg 0 1 0 Eunotia elegans Østrup 0 0 1 Eunotia exigua (Brébisson ex Kützing) Rabenhorst 7 68 506 Eunotia formica Ehrenberg 0 1 0 Eunotia glacialis Meister 0 0 2 Eunotia steineckii Petersen 0 1 5 Eunotia vanheurckii R.M.Patrick 0 4 0 Fragilaria biceps (Kützing) Lange-Bertalot 1 9 4 Fragilaria capucina Desmazires 24 39 48 Fragilaria famelica (Kützing) Lange-Bertalot 3 4 4 134
Fragilaria tenera (W.Smith) Lange-Bertalot 1 0 1 Frustulia rhomboides (Ehrenberg) De Toni 0 6 20 Frustulia vulgaris (Thwaites) De Toni 2 0 0 Geissleria decussis (Østrup) Lange- Bertalot & Metzeltin 6 0 0 Gomphonema affine Kützing 0 4 0 Gomphonema angustatum (Kützing) Rabenhorst 0 1 1 Gomphonema clavatum Ehrenberg 0 3 0 Gomphonema insigne Gregory 0 0 1 Gomphonema minutum (C.Agardh) C.Agardh 0 2 0 Gomphonema parvulum (Kützing) H.F.Van Heurck 2 40 6 Gomphonema pseudoaugur Lange-Bertalot 2 0 0 135
Gyrosigma acuminatum (Kützing) Rabenhorst 0 1 0 Gyrosigma scalproides (Rabenhorst) Cleve 5 0 0 Gyrosigma wansbeckii (Donkin) Cleve 11 5 0 Halamphora normanii (Rabenhorst) Levkov 1 0 0 Halamphora veneta (Kützing) Levkov 4 0 1 Luticola saxophila (Bock ex Hustedt) D.G.Mann 0 1 0 Melosira lineata (Dillwyn) Agardh 106 0 0 Melosira sp. C.Agardh 5 2 3 Meridion circulare (Greville) C.Agardh 0 1 1 Navicella pusilla (Grunow) Krammer 14 1 0 Navicula cryptocephala Kützing 156 19 1 136
Navicula cryptotenella Lange-Bertalot 16 14 0 Navicula detenta Hustedt 3 0 0 Navicula elginensis (W.Gregory) Ralfs 4 1 0 Navicula exilis Kützing 1 0 0 Navicula lapidosa Ehrenberg 0 0 1 Navicula longicephala Hustedt 7 0 0 Navicula lundii Reichardt 35 0 0 Navicula recens (Lange- Bertalot) Lange- Bertalot 31 0 0 Navicula rhynchocephala Kützing 0 6 0 Navicula striolata (Grunow) Lange-Bertalot 0 1 0 Navicula tripunctata (O.F.Müller) Bory de Saint- Vincent 10 0 0 Navicula viridula (Kützing) Ehrenberg 36 18 4 137
Naviculadicta absoluta (Hustedt) Lange-Bertalot 1 1 0 Nitzschia acicularioides Hustedt 1 3 1 Nitzschia acicularis (Kützing) W.Smith 0 5 0 Nitzschia acidoclinata Lange-Bertalot 0 0 2 Nitzschia amphibia Grunow 1 0 0 Nitzschia capitellata Hustedt 9 0 1 Nitzschia clausii Hantzsch 0 3 0 Nitzschia commutata Grunow 1 0 0 Nitzschia constricta var. subconstricta Grunow 0 1 0 Nitzschia dissipata (Kützing) Grunow 15 7 0 Nitzschia filiformis (W.Smith) Hustedt 0 0 11 Nitzschia fonticola (Grunow) Grunow 0 2 0 138
Nitzschia gracilis Hantzsch 0 13 0 Nitzschia inconspicua Grunow 66 33 3 Nitzschia intermedia Hantzsch ex Cleve & Grunow 6 24 40 Nitzschia linearis (C.Agardh) W.Smith 6 4 0 Nitzschia lorenziana var. incerta Grunow 0 2 0 Nitzschia microcephala Grunow 0 1 0 Nitzschia nana Grunow 0 9 18 Nitzschia palea (Kützing) W.Smith 38 85 19 Nitzschia paleacea Grunow 0 0 10 Nitzschia pellucida Grunow 0 0 1 Nitzschia perminuta (Grunow) M.Peragallo 0 14 3 Nitzschia pusilla Grunow 0 0 3 139
Nitzschia sigmoidea (Nitzsch) W.Smith 0 4 0 Pinnularia interrupta W.Smith 1 1 2 Pinnularia microstauron (Ehrenberg) Cleve 1 1 1 Pinnularia obscura Krasske 0 0 1 Pinnularia subcapitata W.Gregory 0 0 7 Plagiotropis sp. Pfitzer 0 1 0 Planothidium delicatulum (Kützing) Round & L.Bukhtiyarova 17 0 0 Planothidium frequentissimum (Lange- Bertalot) Lange- Bertalot 5 0 5 Pleurosira laevis (Ehrenberg) Compère 2 0 0 Psammodictyon constrictum (Gregory) D.G.Mann 13 1 0 Reimeria sinuata (Gregory) Kociolek & Stoermer 15 1 0 140
Rhoicosphenia abbreviata (C.Agardh) Lange-Bertalot 33 11 1 Rossithidium pusillum (Grunow) Round & L.Bukhtiyarova 4 14 2 Sellaphora bacillum (Ehrenberg) D.G.Mann 0 2 0 Sellaphora pupula (Kützing) Mereschkovsky 15 1 4 Stauroneis smithii Grunow 0 6 0 Staurosirella pinnata (Ehrenberg) D.M.Williams & Round 2 0 1 Stenopterobia delicatissima (F.W.Lewis) Brébisson ex van Heurck 0 0 47 Surirella amphioxys W.Smith 4 2 2 Surirella angusta Kützing 1 0 0 Surirella linearis W.Smith 4 5 131 Surirella minuta Brébisson 3 0 100 141
Surirella subsalsa W.Smith 18 3 8 Surirella tenera W.Gregory 2 0 43 Synedra amphicephala Kützing 0 1 0 Tabellaria flocculosa (Roth) Kützing 0 0 4 Tryblionella coarctata (Grunow) D.G.Mann 0 1 0 Tryblionella levidensis W.Smith 3 2 1 Tryblionella littoralis (Grunow) D.G.Mann 0 4 0
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APPENDIX K: BRAY-CURTIS DISSIMILARITY SCORES FOR CLEAR CREEK AND THE MOST DOWNSTREAM (HF039) AND UPSTREAM (HF090) SITES ON HEWETT FORK AFTER A 6-WEEK COLONIZATION PERIOD USING POOLED REPLICATES (N = 3). MEAN IS THE MEAN DISSIMILARITY BETWEEN CLEAR CREEK AND HEWETT FORK SITES.
Clear Creek HF039 HF039 0.7879186 - HF090 0.8836436 0.718933 Mean 0.8357811
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2 APPENDIX L: THE χ 0.05,1 VALUES FROM COMPARISONS OF GROWTH FORMS BETWEEN CLEAR CREEK AND HF039, AND CLEAR CREEK AND HF090 AFTER A 6-WEEK COLONIZATION PERIOD.
2 Comparison χ 0.05,1 P-value Clear Creek HF039 3743.42459 P ≤ 0.001
Clear Creek HF090 4413.48056 P ≤ 0.001
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