D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 92 Vol. 12, Special Issue 4 Histopathological Analysis of Fish from Acorn Fork Creek, Kentucky, Exposed to Hydraulic Fracturing Fluid Releases Diana M. Papoulias1,* and Anthony L. Velasco2 Abstract - Fracking fluids were released into Acorn Fork, KY, a designated Outstanding State Resource Water, and habitat for the threatened Chrosomus cumberlandensis (Blackside Dace). As a result, stream pH dropped to 5.6 and stream conductivity increased to 35,000 μS/cm, and aquatic invertebrates and fish were killed or distressed. The objective of this study was to describe post-fracking water quality in Acorn Fork and evaluate if the changes in water quality could have extirpated Blackside Dace populations. Semotilus atromaculatus (Creek Chub) and Lepomis cyanellus (Green Sunfish) were collected from Acorn Fork a month after fracking in lieu of unavailable Blackside Dace. Tissues were histologically analyzed for indicators of stress and percent of fish with lesions. Fish exposed to affected Acorn Fork waters showed general signs of stress and had a higher incidence of gill lesions than unexposed reference fish. Gill lesions observed were consistent with exposure to low pH and toxic concentrations of heavy metals. Gill uptake of aluminum and iron was demonstrated at sites with correspondingly high concentrations of these metals. The abrupt and persistent changes in post-fracking water quality resulted in toxic conditions that could have been deleterious to Blackside Dace health and survival. Introduction Development of methods to inject fluids under high pressure to fracture subsurface rock, hydraulic fracturing or “fracking”, has allowed exploitation and recovery of new sources of natural gas and oil. However, chemicals used in fracking have degraded water quality and injured aquatic ecosystems (Kargbo et al. 2010, Wiseman 2009). Mixtures of several different chemicals are used in fracturing fluids, and acids are a key component to inhibit scale and to dissolve rock (Colburn et al. 2011, EPA 2004). Fracking fluids and waste releases may be toxic to fish and wildlife if not contained and disposed of properly (Osborn et al. 2011, Pennsylvania Land and Trust Association 2010, The Academy of Natural Sciences 2010). In 2007, fracking fluids used during the development of four natural gas wells in Knox County, KY were released into Acorn Fork, a second-order tributary of Stinking Creek in the upper Cumberland River basin. Fracking effluent overflowed the retention pits directly into Acorn Fork. As a result, hydrochloric acid, dissolved minerals and metals, and other chemicals entered Acorn Fork, significantly reducing stream pH (from pH 7.5 to 5.6) and increasing stream conductivity (from 200 to 35,000 μS/cm). Subsequently, long reaches of the stream 1US Geological Survey, Columbia Environmental Research Center, 4200 New Haven Road, Columbia, MO 65201. 2US Fish and Wildlife Service - Environmental Contaminants Division, Kentucky Ecological Services Field Office, J.C. Watts Federal Building - Suite 265, 330 West Broadway, Frankfort, KY 40601-1922. *Corresponding author - dpapoulias@usgs.gov. Ecology and Conservation of the Threatened Blackside Dace, Chrosomus cumberlandensis 2013 Southeastern Naturalist 12(Special Issue 4):92–111 93 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 developed a suspended, and later precipitated, orange-red flocculent assumed to be composed of an organo-colloidal complex of iron, aluminum, and other metals. In some places the flocculent was several inches thick. As a result of these releases, fish and aquatic invertebrates were killed or displaced for months in over 2.7 km of the approximate 5 km of af fected waters in Acorn Fork. The federally threatened Chrosomus cumberlandensis (Starnes and Starnes) (Blackside Dace) was among the fishes killed as a result of the sudden and persistent change in water chemistry. Prior to the discharge of the fracking fluids, Acorn Fork had maintained adequate water quality and habitat conditions needed to support a healthy population of Blackside Dace (USFWS 2001). The dace occupies cool headwater streams with slow-moving pools under extensive forest canopy (Starnes and Starnes 1981). Formerly ranging throughout the upper Cumberland River drainage, Blackside Dace have declined and now occupy only a small portion of their historic range (see McAbee et al. 2013[this issue]:supplemental appendix 1). Widespread extirpation of Blackside Dace populations is believed to have been caused by natural resource extraction activities such as surface mining, logging, and natural gas and oil development (Starnes and Starnes 1981, USFWS 1988). It is not known how many dace were killed during the 2007 event because two wells had already been fracked, and peak mortality was likely missed before researchers arrived to document the incident. However, one dead, one moribund, and several living but distressed Blackside Dace, along with three distressed Semotilus atromaculatus (Mitchill) (Creek Chub) were observed. Physico-chemical changes in water quality associated with mining is known to affect the morphology and thereby the function of superficial tissues in fish (Daye and Garside 1976, Henry et al. 2001, Ledy et al. 2003). Therefore, the objective of this opportunistic study was to assess whether fracking-related degradation in Acorn Fork water quality could have harmed or resulted in mortality of the federally protected Blackside Dace. Tissues from Creek Chub and Lepomis cyanellus (Rafinesque) (Green Sunfish), proxies for unavailable Blackside Dace, exposed to affected Acorn Fork waters were histologically analyzed for indicators of stress and lesion prevalence, and these results were compared to results for the same measurements on fish from a section of creek where no gas development was taking place. Field Site Description The study was conducted in Acorn Fork mainstem, its west branch, and two unnamed tributaries that join the west branch (Fig. 1). Acorn Fork is located in Knox County approximately 40 km southeast of London, KY. The watershed is mainly undeveloped with few residences. A 2.4-hectare fishing lake is situated immediately below wells #1 and #2 on Unnamed Tributary 1 (Fig. 1B). An earthen dam separates the lake from a short section of stream that flows into a meadow and network of slow-flowing pools backed-up by small beaver dams. Unnamed Tributaries 1 and 2 join below well #3 and flow as the west branch of Acorn Fork for a short segment before entering another small network of beaver pools and streamlets (Fig. 1B). This network again collects into a small stream D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 94 Vol. 12, Special Issue 4 before entering a small culvert downstream of well #3. The confluence of the west branch of Acorn Fork and its mainstem occurs about 75 m downstream of a plunge-pool created by a large culvert that directs the mainstem Acorn Fork under the trail leading up to the four wells. From this point, Acorn Fork continues approximately 2 km to the confluence with Carter-Roark Branch, and flows for another 1 km before emptying into Stinking Creek (Fig. 1A). Methods Fish collection Gas well #2 was fracked on 14 May 2007, and well #1 was fracked on 23 May 2007; well #3 was fracked on 12 June 2007, and well #4 was fracked on 18 June 2007 (John Brumley, Kentucky Division of Water, Lexington, KY, pers. comm.). Water and fish samples were collected opportunistically and as close to the fracking events as possible, at various locations along the mainstem, branch, and tributaries (collectively called Acorn Fork) to best represent affected and unaffected areas of the creek. Creek Chub and Green Sunfish, the only relatively numerous species available, were targeted for collection by seining or dipnetting. Figure 1. Location of gas wells and sample collection sites on Acorn Fork. Panel (A) shows the entire Acorn Fork system to the confluence with Stinking Creek and site 1. Panel (B) shows in detail the spatial relationship among the gas wells and sites 2–4. Yellow circles indicate gas wells, green circles indicate sampling sites, and white circles indicate culverts. 95 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 We first collected fish from Acorn Fork (sites 1 and 2) for this study on 10 July 2007, although limited measurements and observations were made of creek condition and fish presence at these and other locations within the study site back to 23 May 2007. On 24 July 2007, we collected 18 fish from the mainstem Acorn Fork plunge-pool, an unaffected area with no wells, just upstream of the confluence of Acorn Fork with the west branch. Four fish collected at this site (hereafter referred to as the fish reference site) were euthanized to serve as unaffected reference samples (hereafter referred to as reference fish). The remaining 14 fish were transported to site 3 and used for on-site testing. On-site testing involved timed treatments holding unaffected fish in 19-L buckets (n = 8) of the affected west branch stream water, and freely (n = 6) in an isolated slow-moving pool in this same affected area for 3 and 48 hours, respectively, before collecting and preserving the fish for histology. On 9 August 2007 the west branch (site 4), near site 3, was sampled and 3 Creek Chubs, believed to have recently moved downstream to this area (the area was fishless in June and July 2007), were collected for histology. Upon collection, fish were stunned with a blow to the head, and abdomens of all fishes were slit. Fish were preserved whole in 10% neutral buffered formalin, and then later shipped to Columbia Environmental Research Center (CERC) for histological analysis. Fish histology Fish specimens were rinsed in buffer to remove formalin before dissection. Sections of liver, head and trunk kidney, spleen, and gonad were removed and placed in tissue cassettes for processing in a Shanndon Excelsior automatic tissue processor (Thermo Fisher Scientific, Waltham, MA). Tissue processing followed a routine paraffin protocol, and blocks were sectioned at 7 microns, with 3–4 sections per slide for 2 slides. Sections were cut at 3 different depths to ensure microscopic evaluations were representative of the entire tissue. Sections were stained with hematoxylin and eosin (H & E; Luna 1968). Gill tissue was treated similarly but was decalcified for 1 hour (Surgipath® Decalcifier II, Medical Industries, Inc., Richmond, IL) to soften boney structures for cutting, prior to tissue processing. Following Denton and Oughton (2010), gills were also stained with acid solochrome azurine to identify aluminum (blue) and iron (red). Gonad sections were evaluated to assign sex and stage of reproductive maturity. Testes were classified as follows: immature—spermatogonia are the primary component of lobules; spermatogenic—lobules contain primarily spermatocytes and spermatids, with limited spermatozoa; mature—spermatozoa are prominent. Ovaries were classified as follows: pre-vitellogenic—contain young oogonia with no vitellogenin deposition; mid-vitellogenic—oogonia undergoing vitellogenesis, and the germinal vesicle has not migrated to the animal pole; mature—oocyte filled with vitellogen, and the germinal vesicle has migrated to the animal pole. Juvenile fish had undifferentiated germ cells and could not be assigned to a sex. Gill, gonad, liver, head and trunk kidney, and spleen were qualitatively evaluated using a light microscope (Nikon Eclipse 90i, Nikon Instruments Inc., Mellville, D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 96 Vol. 12, Special Issue 4 NY) for presence of stress indicators and lesions associated with exposure to low pH and to heavy metals. Gills are vulnerable to environmental stressors due to their exposure to the environment and their delicate, highly vascularized structure (Laurent and Perry 1991, Roberts 1978). Stress in fish may be reflected in the condition of liver and spleen tissues. An increase or decrease in hepatic lipid or glycogen content can indicate exposure to an environmental stressor and result in the disruption of metabolism (Chindah et al. 2008, Ferguson 1989). A calm, unstressed fish will have a large spleen filled with erythrocytes, whereas a fish that is stressed will have a thin spleen and erythrocytes will have been released (Takashima and Hibiya 1995). In this study, histological sections of reference fish were evaluated for the presence and severity of stress indicators and lesions, and then fish exposed to the affected Acorn Fork water were blindly scored. The incidence of fish with lesions from affected sites or of healthy fish exposed to water from affected sites was compared to the number of reference fish with lesions. Lipids and glycogen appear as vacuolar structures in paraffin-embedded, H & E-stained liver sections. Lipids tend to form round structures, and glycogen forms irregularly shaped structures (Takashima and Hibiya 1995). Liver sections (Creek Chub only) were scored as high–moderate or low–none for lipid and glycogen collectively, as an indicator of metabolic condition. As an indicator of stress, spleen sections were scored as high– moderate or low–none for blood cell content. Water quality Conductivity was measured on-site using an Oakton 300 series multimeter (Cole-Palmer, IL) when fish and water samples were collected. Field pH measurements were made 15 June 2007 with Fisher Scientific ALKACID® Test Ribbon on water collected 23 May, 30 May, and 5 June 2007 into Mason® jars and kept at room temperature. Kentucky Division of Water tested water pH, conductivity, and hardness on 25 June 2007 at selected locations on Acorn Fork. Water samples for elemental analysis were collected 22 June 2007 at the approximate time and place dead Blackside Dace were discovered (site 4). Additional water samples were collected above well #4 to serve as an unaffected water reference sample (no water was collected at the fish reference site and no fish were collected at the water reference site), and at sites 1, 2, and 3 on 10 July 2007. No water was collected at site 4 on 9 August 2007 when fish were collected. Water samples were collected directly from the creek into 500-mL I-Chem Certified 300 Series chemically cleaned sample jars (Thermo Fisher Scientific, Waltham, MA). Samples were chilled to 4 °C until overnight shipment on wet ice to Alpha Analytical Laboratories, Inc. (Mansfield, MA) for analysis. Water samples at each site were collected in 2 jars: one sample was filtered using vacuum filtration and 0.45-mm filter paper, and the filtrate was used for ion analyses and metals scan; the other water sample was not filtered and was used for a metals scan only. Samples for metals scans were prepared for ICP-MS (EPA Method 6020A) using a routine acid-digestion procedure (PerkinElmer Corp. 1985). Chloride ions (Cl-) and sulfate ions (SO4 2−) were measured by ion 97 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 chromatography following EPA Method 300.0 (EPA 2003). Blanks, duplicates, and spikes were included. Only filtered results are presented. Hardness values from the water reference site (maximum value) and site 3 (minimum value) were used to calculate hardness-specific water-quality standards for dissolved metals using equations provided in Kentucky Surface Water Standards (State of Kentucky 2011). Water-quality results were evaluated against Kentucky Surface Water Standards criteria when available (State of Kentucky 201 1). Statistics Only one water sample at each site was collected and analyzed; therefore no statistical comparisons among sites could be made using the water-chemistry data. Differences in the odds ratio for incidence of fish with gill and kidney lesions between the reference fish site and sites 1–4, where fish were exposed to affected Acorn Fork water, were tested with a one-sided Fisher’s Exact test due to small sample size. A significant difference between results from the reference site and the exposed sites was set at P ≤ 0.05. Results A small population of Blackside Dace, Green Sunfish, Creek Chub, and other fishes persisted in Unnamed Tributary 1 in the creek below the lake to the confluence with Unnamed Tributary 2 for the entire period of our assessment (May– September, 2007). However, Acorn Fork appeared to be completely devoid of all fishes, invertebrates, and other biota downstream from this confluence for greater than 2 km. On 22 and 27 June 2007 a dead dace and a moribund dace, respectively, were collected in the west branch below well #3 and above the confluence with Acorn Fork mainstem (site #3). Three living but severely distressed dace were also seen here on 27 June 2007. These fish were observed uncharacteristically at the surface, rostrum pointed downward, slowly rocking back and forth. The Blackside Dace in this condition were easily caught by dipnet, whereas healthy Blackside Dace would typically avoid capture and quickly swim away. On 9 August 2007, only 3 Creek Chub were observed and collected in a shallow, isolated pool near this same reach (site 4). Their swimming was atypically erratic and agitated— swimming erratically and dashing their sides against the substrate. However, once collected, they appeared to be in good physical conditio n. Downstream, small groups of fishes were also occasionally collected from Carter-Roark Branch near its confluence with the affected portions of the mainstem Acorn Fork. Further downstream to the confluence with Stinking Creek, larger fishes of each species were sometimes collected. A total of 38 Creek Chubs and 7 Green Sunfish were captured and evaluated for this study. A few small tissues were lost in processing for some individuals, such that a complete set of tissues was not available for 16 fish. Mean (± SD) length and weight of Creek Chubs were 86 ± 20 mm and 7.74 ± 4.87 g, respectively. Mean (± SD) length and weight of Green Sunfish were 78 ± 15 mm and 9.75 ± 5.16 g, respectively. The collection consisted of 18 females, 12 males, 14 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 98 Vol. 12, Special Issue 4 immature, and 1 unsexed fish. No lesions were observed in the gonads of any fish. Creek Chubs were either immature or non/post-reproductive, with the exception of 1 female which appeared to have recently spawned and was used for on-site testing. Green Sunfish were either mature or immature, with the exception of 1 female which appeared to have recently spawned and was used for on-site testing. Figure 2. Histological sections of gill tissue stained with hemotoxylin and eosin (A, B, C) from Creek Chub taken from locations in Acorn Fork unaffected by fracking fluids (A) and where fracking fluids had contaminated the stream (B and C). Panel (B) is an example of extensive lamellar hyperplasia (asterisk) and touching of gill filament tips (arrow). Panel (C) shows examples of epithelial lifting (arrow) on secondary lamellae, curling (arrowhead), and clubbing (open triangle). Scale bar is 100 microns. 99 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 Histological observations Gills of reference fish. Gills of Creek Chubs (n = 2) and Green Sunfish (n = 2) from the fish reference site (Fig. 2A) had focal areas of slight epithelial hyperplasia of the primary and secondary lamellae, and occasional touching, fusion, and swelling of secondary lamellae. Gills of exposed fish, sites 1 and 2. All Creek Chubs (nsite 1 = 9, nsite 2 = 11) and Green Sunfish (nsite 1 = 2, nsite 2 = 1) had gills with epithelial hyperplasia of primary and secondary lamellae, and the condition was diffuse and pervasive in 78% of fish from site 1. The hyperplasia was diffuse, pervasive, and severe in 83% of site 2 fish (Fig. 2B). Concomitantly with the hyperplasia, touching of secondary lamellae often occurred, sometimes fusing, as did extensive and severe swelling of secondary lamellae. One Creek Chub (site 2) had extensive areas of hemangioendothelio sarcoma (vascular tumor). Curling or clubbing of the tips of the secondary lamellae was not observed in the reference fish but was apparent in 44% of fish from site 1 and 9% from site 2 (Psite 1 = 0.27, Psite 2 = 0.56, Figs. 2C, 3A). Lifting of epithelium away from the secondary lamellae Figure 3. Percent number of fish with gills showing clubbing or curling at ends of gill filaments (A) and separation of the epithelium from the secondary lamella (epithelial lifting; B). Asterisks indicate a signficant difference between results at the fish reference site (Reference) and the affected site (Fisher Exact Test, P < 0.05). D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 100 Vol. 12, Special Issue 4 was observed in fish from sites 1 (55%) and 2 (83%), but not reference fish (Psite 1 = 0.12, Psite 2 = 0.01; Figs. 2C, 3B). Gills of reference fish exposed on-site, site 3. Reference fish (Creek Chubs, n = 8) exposed for 3 hours developed hyperplasia of the epithelium on the primary and secondary lamellae, and the condition was diffuse, severe, and pervasive for 50% of the fish. All fish (Creek Chubs, n = 4; Green Sunfish, n = 2) exposed for 48 hours had epithelial hyperplasia on primary and secondary lamellae, and the condition was severe on the primary lamellae for 50% of the fish. Epithelial lifting was apparent in 88% of the fish exposed for 3 hours; the condition was severe in 50% of these, and some swelling of secondary lamellae was observed (P = 0.01, Fig. 3B;). All the fish exposed for 48 hours had swollen secondary lamellae, and 50% had epithelial lifting (P = 0.17). Curling of the secondary lamellae occurred in 75% and 50% or the fish exposed for 3 and 48 hours, respectively (P3h = 0.03, P48h = 0.17, Fig. 3A). Gills of exposed fish, site 4. The Creek Chubs collected at site 4 (n = 3) all had slight epithelial hyperplasia of the secondary lamellae but only focal or none on the primary lamellae. Swollen secondary lamellae were observed in all fish but it was not extensive, and only 1 fish had curled lamellae (P = 0.43). None of the fish from site 4 was observed to have epithelial lifting or edem a. Gill uptake of Al and Fe. Distinct differences in gill sections among fish collected from the 5 sites were observed after staining with acid solochrome azurine. Gills of fish from the fish reference site had little Al or Fe uptake, whereas a gradient of these metals was seen in gill tissues of fish collected from affected waters at sites below the wells (sites 1 and 2) and those exposed on-site (site 3) to contaminated creek water (Fig. 4). Gill tissues of Creek Chub from site 1 were a Figure 4. Gill sections from fish collected from the fish reference site and 4 sites in Acorn Fork below fracked wells. Sections were stained with acid solochrome azurine for aluminum (blue color) or iron (red color). 101 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 reddish color, indicating Fe had passed across the epithelial membrane, whereas gills of Creek Chub from sites 2, 3, and 4 were stained blue to varying degrees, indicating that Al had been taken-up. Creek Chub that were collected at sites 2 and 4 had much less Al than those fish continuously exposed for 3 and 48 hours to affected water at site 3. Liver. Creek Chubs (n = 2) collected from the fish reference site and from site 4 (n = 3) were scored as having moderate to high liver glycogen/lipid. Fifty-percent of the Creek Chubs from site 1 (n = 10) and 64% from site 2 (n = 18) had low to no glycogen/lipid. Thirty-eight percent of Creek Chubs (n = 8) exposed for 3 hours had low to no glycogen/lipid, whereas 50% of those (n = 4) exposed for 48 hours had low to no glycogen/lipid. No lesions were observed in the liver. Spleen. Spleens of all Creek Chubs (n = 2) collected from the fish reference site were moderately to highly perfused with red blood cells. In contrast, spleens of 37% of Creek Chubs from site 1 (n = 11) and 50% from site 2 (n = 10) were moderately to highly perfused with red blood cells. Spleens of 63% of Creek Chubs treated for 3 hours (n = 8) at site 3 were moderately to highly perfused with red blood cells, whereas 25% of those exposed for 48 hours (n = 4) scored similarly. No Creek Chubs at site 4 (n = 1) had moderate to high amounts of red blood cells. No lesions were observed in the spleen. Kidney. Reference fish (n = 3) and those collected from site 4 (n = 3) were without head or trunk kidney abnormalities. Varying numbers of fish exposed to affected Acorn Fork water had granulomatous inflammation, but only at site 2 was the incidence significantly greater than at the fish reference site (Psite 1 = 0.80, Psite 2 = 0.02, Psite 3 = 0.06, Psite 4 = 0.18, Fig. 5). Water quality Conductivity measured at the water reference site above well #4 between 15 June and 9 August 2007 ranged from 430–639 μS/cm. At the fish reference site, conductivity was 190 μS/cm and 244 μS/cm on 15 June and 24 July 2007, respectively. At the time of fish sampling (10 July 2007), conductivity at site 1 was 1917 μS/cm having decreased from a high of 2690 μS/cm on 22 June 2007; by 24 July 2007, conductivity at site 1 was still elevated at 1265 μS/cm (Table 1). Conductivity measured at site 2 ranged between 279 and 308 μS/cm from 22 June to 9 August 2007 and was 291 μS/cm on 10 July 2007 when fish were collected Table 1. Conductivity measured between 22 June 2007 and 9 August 2007 in water at four fishcollection sites in Acorn Fork and its unnamed tributaries. Conductivity (μS/cm) Date Site 1 Site 2 Site 3 Site 4 June 22 2690 279 8190 35,900 July 10 1917 291 8380 29,200 July 24 1265 293 7650 25,200 July 26 5500 August 9 308 4620 12,610 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 102 Vol. 12, Special Issue 4 Figure 5. Percent number of fish with kidney tisue showing granulomatous inflammation. Asterisk indicates a signficant difference between results at the fish reference site (Reference) and the affected site (Fisher’s Exact Test, P < 0.05). for this study (Table 1). Conductivities at sites 3 and 4 were the highest on 22 June 2007 (greater than 8000 and 35,000 μS/cm, respectively) and were still up to 50 times greater than reference sites when reference fish were collected and on-site testing occurred 24 July 2007 (Table 1). On 9 August 2007, nearly 7 weeks after wells #3 and #4 were fracked, conductivity at site 4 remained elevated at 12,610 μS/cm. Water from two locations uninfluenced by wells (i.e., above wells #1 and #4) measured pH 7.1 and 7.8 on 25 June. Creek water had a pH of 5.5 on 23 May and 5 June 2007 just below the confluence of the Acorn Fork mainstem with the west branch. On 25 June 2007, creek water below well #3 and above site 4 was pH 6.5; pH was 7.31 at Site 2; 5.61 at site 4; and 7.25 at site 1. No other pH measurements were made. On 25 June 2007, hardness measured 41.7 mg/L CaCO3 at the water reference site, 30.1 mg/L CaCO3 at site 1; 45.5 mg/L CaCO3 at site 2, and 10.9 mg/L CaCO3 at site 3. On 22 June 2007, approximately 1 week after wells #3 and #4 were fracked, when conductivity at site 4 was over 35,000 μS/cm (the highest measured), chloride ions, sulfate ions, and all the metals except Al and Fe were also at the highest levels measured at any site in this study (Table 2). With the exception of sulfate, water concentrations of the other analytes were greatest at sites 3 and 4 and greater at site 1 when compared to site 2 (Table 2). Sulfates were highest (450 mg/L) at the water reference site, followed by sites 2 and 1 103 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 (Table 2). The water reference site was similar to sites 1 or 2 for most elements except chloride, Fe, and Mn (Table 2). Chloride at site 1 was elevated over concentrations at site 2 and the water reference site (Table 2). Chloride concentrations exceeded Kentucky water-quality standards only at sites 3 and 4 (Table 2). Iron and Mn were slightly higher at sites 1 and 2 compared to the water reference site (Table 2). At least 3 heavy metals exceeded Kentucky Table 2. Concentrations of selected elements measured in unfiltered water samples collected from Acorn Fork and tributaries in 2007. Acute and chronic surface water standard values are from State of Kentucky (2011) unless otherwise noted. NC = not collected. < = below detection. Dashes indicate no aquatic-life standard available. Symbol (§ or *) indicates an exceedance above corresponding standard value for CaCO3 (acute or chronic, respectively). Cond. = conductivity. Element (mg/L) Date Cond. (2007) Site (μS/cm) Al Cd Cl- Cr2 B Cu Fe 24-Jul Ref (fish) 244 NC NC NC NC NC NC 10-Jul Ref (water) 430 0.196* less than 0.0001 less than 1 0.0006 0.0023§ 1 10-Jul 1 1917 0.075 less than 0.0001 480 0.0013 0.0041§ 2** 10-Jul 2 291 0.108* less than 0.0001 17 0.0008 0.0024§ 1** 10-Jul 3 8380 1.670§§ 0.0003§ 2900§§ 0.0018 0.0114§§ 251§§ 22-Jun 4 35,900 0.355* 0.0005§ 8500§§ 0.0048 0.0349§§ 41§§ Standard values as mg/L CaCO3 10.9 mg/L = § - 0.0002 - - 0.0017 - 41.7 mg/L = §§ 0.750A 0.0009 1200 0.0061 4 10.9 mg/L = * 0.0870A 0.0001 - - 0.0014 - 41.7 mg/L = ** - 0.0001 600 0.0044 1 Element (mg/L) Date (2007) Site Mg Mn Ni Pb SO4 2 – Sr Zn 24-Jul Ref (fish) NC NC NC NC NC NC NC 10-Jul Ref (water) 31 0.1 0.002 less than 0.0005 450 0.2 0.006 10-Jul 1 31 0.1 0.005 less than 0.0005 150 1.8 0.061§§ 10-Jul 2 15 1.8 0.002 less than 0.0005 200 0.1 less than 0.005 10-Jul 3 133 12.0 0.047** 0.0028** 8 4.6 0.053§ 22-Jun 4 330 19.6 0.071** 0.0038** 35 44.6 0.064§§ Standard values as mg/L CaCO3 10.9 mg/L = § - - 0.010 0.0050 - - 0.018 41.7 mg/L = §§ - - 0.224 0.0270 - - 0.057 10.9 mg/L = * - - 0.008 0.0002 - - 0.018 41.7 mg/L = ** - - 0.025 0.0010 - - 0.057 AValues (CMC and CCC for aquatic life) from EPA (2009) are not hardness specific. BTotal chromium. There are no Kentucky surface water standards for aquatic habitats for total chromium. Values for chromium VI are not hardness specific and are 0.016 and 0.011 for acute and chronic values, respectively; chromium III acute and chronic at 41.7 mg/L CaCO3 are 0.881 and 0.294 mg/L, respectively; chromium III acute and chronic at 10.9 mg/L CaCO3 are 0.042 and 0.014 mg/L, respectively. D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 104 Vol. 12, Special Issue 4 water-quality standards at all sites (Table 2). Kentucky water-quality standards for Al were exceeded at sites 2, 3, and 4 and for Zn at sites 1, 3, and 4 (Table 2). Chromium was the only metal of the 11 measured to not exceed the Kentucky water criterion at sites 3 and 4 (Table 2). Discussion Distress and tissue injury can occur in freshwater fishes with o smoregulatory systems not suited to rapid changes in pH or conductivity such as that which occurred during the spill incident in Acorn Fork. Fishes have a defined range of tolerance to aquatic pH and dissolved solids measured as specific conductivity. A pH between 6.5 and 9.0 is required by most freshwater fishes (Fromm 1980). Although technical problems with a field pH meter did not permit measurements on all dates, the present study shows that creek waters of the Acorn Fork system unaffected by fracking normally have a pH 7.0 or higher, conductivities less than 500 μS/cm, low to moderate hardness, and are low in dissolved elements. Recently, the EPA (2011) established a chronic field-based aquatic-life benchmark for conductivity of 300 μS/cm specifically for the Appalachian region including eastern Kentucky, where the pH-neutral waters are dominated by salts of Ca2+, Mg2+, SO4 2−, and HCO3−. This benchmark is consistent with the upper limit of 240 μS/cm identified by Black et al. (2013[this issue]) as a good predictor of streams occupied by Blackside Dace populations. Acorn Fork water downstream of fracked wells was observed to be as low as 5.6 pH and as high as 35,900 μS/cm conductivity. Overall, the water elemental and ionic laboratory analyses were consistent with field-measured conductivity, such that elements and ions tended to be elevated in water samples from sites where conductivity also was elevated. Conductivity, elements, and ions were higher nearer to wells #3 and #4 than further downstream. However, well fracking activity effects on water quality were still notable at site 1, a distance of approximately 3 km downstream of the wells. Elevated sulfates, at the water reference site above well #4, suspected to be due to historic coal mining activity, may explain the higher conductivity values at this site. Conductivity was consistently lowest at site 2. Site 2 is below a 2.4-ha lake into which fracking effluent flowed and was diluted. State and federal standards established to protect aquatic life were exceeded for heavy metals and chloride ions at some or all affected sites (EPA 2009, State of Kentucky 2011). No measurements of the reference water exceeded standards. Three of the 11 metals exceeded standards at sites 1 and 2, whereas 7 of the metals and chloride ions exceeded levels protective of aquatic life at sites 3 and 4. The toxicity of many metals is increased in soft waters low in calcium and magnesium ions (Allen and Janssen 2006). Moreover, the toxicity of waters containing heavy metals tends to increase under reduced pH because the elements are maintained in the water column where they are bioavailable (Manahan 1972). Creek Chubs, and to a lesser extent Green Sunfish, exposed to Acorn Fork water contaminated with fracking effluent showed more tissue damage and stress than 105 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 fish from an unaffected reference site. The most severely affected fish were those collected at site 2, and the reference site fish exposed on-site to contaminated water at site 3. Fewer signs of tissue damage were observed in fish collected in August at site 4, despite higher conductivities here than at any other site. However, behavioral observations and spleen condition of these fish indicated they were experiencing stress. It is likely these Creek Chubs had only recently moved downstream into this area where previously all fish had been killed by a large pulse of wastewater. Fish at site 1, 3 km downstream from the wells, showed signs of tissue damage and stress, but generally fewer individuals were affected than at sites 2 and 3. The magnitude of effects of aquatic acidification on fish will vary dependent on the source of the hydrogen ions, water calcium ion concentration, the presence of heavy metals, and their speciation (Henry et al. 2001). Aquatic acidification changes the biochemical properties of the gill tissue and disrupts the flow of ions (e.g., sodium and chloride) at the gill-water interface, subsequently changing blood ion concentrations (Evans 1987). Respiratory distress also can occur from exposure to low environmental pH as a result of excess epithelial hyperplasia and mucus accumulation on gills (Daye and Garside 1976, Evans 1987). The same gill lesions observed on Creek Chubs and Green Sunfish exposed to Acorn Fork waters (epithelial lifting and edema, epithelial cel l hyperplasia and swelling, curling of secondary lamellae, touching and fusion of lamellae) have been reported for many fish species exposed to low environmental pH, dissolved heavy metals, or both (Chevalier et al. 1985, Evans et al. 1988, Figueiredo-Fernandes et al. 2007, Gill et al. 1988, Karan et al. 1988, Visoottiviseth et al. 1999). Granulomatous inflammation is commonly observed in fish organs due to disease and parasites (Hedrick et al. 1993, Rahimian 1998). Chronic exposure to very low pH has been reported to affect fish kidney morphology (Saenphet et al. 2009), but no reports were found of renal granulomas as a result of exposure to acidic conditions or from exposure to heavy metals. Although granulomatous inflammation was only observed in fish exposed to affected Acorn Fork water, its association with the degraded water quality may be a secondary response in chronically stressed fish. Metals can affect gill tissue by adsorbing to the surface and by active or passive transport across the gill epithelia. Metal ions will vary in their ability to form ionic bonds at the surface of gill tissue or to move across epithelia to form covalent bonds in cytosol (Wepener et al. 2001). Modes of action of these metals are variable but generally involve interference with enzymes, resulting in adverse consequences for osmoregulation, respiration, and reproduction (Goyer and Clarkson 2001). Metals detected in this study also have been shown to affect behaviors such as avoidance, coughing, and changes in ventilation rate at or below guidance levels (Atchinson et al. 1987). Two of the metals, Al and Fe, were elevated in water samples and were also detected by histochemistry only in gill tissue of fish collected from the D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist 106 Vol. 12, Special Issue 4 affected areas of Acorn Fork. The toxicity of Al to fishes has been wellstudied (Sparling and Lowe 1996). National aquatic life guidance criteria for Al (i.e., for chronic exposure, 87 μg/L at pH 6.5–9.0; EPA 2009) were exceeded at sites 2, 3, and 4. However, site-specific water quality, particularly pH, calcium ion concentration, and presence of organic or inorganic complexing agents, together with the species and life-stage of the fish determine the degree of Al toxicity (see references in DeLonay et al. 1993). Observed accumulation of Al in gill tissue of fish collected in this study reflected the concentration of Al measured in water samples (i.e., site 1 < site 2 < site 4 < site 3). Although Al was not measured at the fish reference site, the absence of Al in gill tissue is consistent with expected low concentrations of Al at the conductivity measured, based on historical records of unaffected stream waters of eastern Kentucky (Dyer and Curtis 1977). Unlike Al, Fe toxicity to fish has not been as thoroughly tested. Guidance levels for chronic exposure to iron have been suggested at 1000 μg/L total Fe (Buchman 2008), but toxicity is highly dependent on the ionic form of Fe present (Teien et al. 2008). Total Fe was 40–250 times this level at sites 3 and 4, but only slightly higher than 1000 μg/L at sites 1 and 2. Iron uptake, however, was evident only in gills of fish from site 1. Although Fe concentrations were greater at sites 3 and 4, site-specific water quality, increased transepithelial movement of Al over Fe, or both, may explain why no Fe was detected in gills of fish from these sites by histochemistry. The results of this histological evaluation of Creek Chub and Green Sunfish exposed to affected Acorn Fork water provides evidence that the releases of fracking fluids degraded water quality sufficiently to cause lesions or exacerbate general stress-indicators in these and likely other fish species present, including Blackside Dace. Differences were observed in type and severity of lesions when compared to fish from a reference site, despite uncertainties regarding where collected fish originated, and for how long they were exposed to affected Acorn Fork water at sites 1 and 2. Furthermore, fish collected from sites with elevated Al and Fe concentrations bioaccumulated these metals. Laboratory studies that remove the additional environmental stressors present in the stream environment could aid in identifying specific causes of the lesions and stress. The major loss of fish habitat due to decreased environmental pH and increased conductivity has been attributable to drainage from active and abandoned mine operations (Sams and Beer 2000), as well as activities associated with land-based gas and oil exploration (Sidhu and Mitsch 1987). The water quality of natural mountain stream waters of eastern Kentucky is typified by low conductivity (<100 μS/cm), near neutral pH, moderate buffering (Ca+ <6.5 mg/L, Mg+ ≤3.5 mg/L, HCO3 - <25 mg/L, SO4 2−<30, Cl- <5 mg/L), and low concentrations of dissolved iron (<0.25 mg/L) and aluminum (<0.07 mg/L) (Dyer and Curtis 1977). Fishes of the Appalachian mountain region, such as Blackside Dace, evolved under and adapted to these conditions (Jones 2005). The abrupt and persistent 107 D.M. Papoulias and A.L. Velasco 2013 Southeastern Naturalist Vol. 12, Special Issue 4 post-fracking changes in water quality within the Blackside Dace habitat that resulted in very high conductivity, lowered pH, and lowered alkalinity, coupled with toxic concentrations of metals, could be expected to be deleterious to Blackside Dace health and survival. The timing of this incident was especially injurious to the species because it occurred during the spawning season. Moreover, the adverse effects of this incident in the Acorn Fork headwater extended over several months, likely causing long-term local and downstream disruption of ecosystem function (Freeman et al. 2007). As efforts accelerate to unleash new energy sources, application of technologies such as hydraulic fracturing can, if not carefully developed, compound the effects of ecosystem degradation caused by past resource extraction (Groat and Grimshaw 2012). This is clearly the case in the Appalachian Highlands, where mining, logging, agriculture, and development have cumulatively degraded aquatic ecosystems, fragmenting freshwater fish populations to the extent that population mixing and gene flow have become restricted (Freund 2004, Pond 2012, Warren et al. 2000). The findings of this study will be useful in assessing injury to the threatened Blackside Dace and its habitat, and demonstrate the utility of on-site field exposures, histology, and histochemistry when investigating impacts of hydraulic fracturing on small streams. Acknowledgments The authors are grateful to Bob Snow, Mindi Lawson, Michael Floyd, and Mike Armstrong (US Fish and Wildlife Service) for their many long days of field support; also to John Brumley (Kentucky Division of Water) and John Williams (Kentucky Department of Fish and Wildlife Resources), and their crews, with post-release field assessments. Special thanks to Ryder Velasco for his assistance during the in situ treatment, and in particular to Valerie Hudson (former Deputy Commissioner of the Energy and Environment Cabinet), whose support, coordination, vision, and spirit helped maintain steady progress. The authors thank Mandy Annis and Vanessa Veléz for preparation of fish samples for histopathology. Susan Finger assisted with initial study design, supplies, and advice. Julia Towns-Campbell provided invaluable library support. Dr. Jeffrey Wolf (Experimental Pathology Laboratories) reviewed some of the histology slides. Marcia Nelson, CERC Outreach Coordinator, assisted with graphics and publication outreach. Finally, we are grateful for the determination of the Reverend Ova Grubb, a life-long resident of Acorn Fork, who called agencies and experts until he could resolve the environmental damage occurring to the aquatic ecosystem near his residence. This work was partially supported with funding from US Fish and Wildlife Service, under contract agreement No. 401818N502. Disclaimer Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US Government. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the US Fish and Wildlife Service. D.M. Papoulias and A.L. 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