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Figure 1: AMD sample site map. This map shows each of the four sampling sites as well as the healthy and impacted flows. Acid mine drainage occurs in areas where water comes in contact with exposed rocks that have a high concentration of sulfide minerals. Pyrite, also known as fool’s gold, is a common mineral found with coal. The oxidation of pyrite and other sulfide-rich minerals causes the release of sulfuric acid and metal ions. If the stream has a limited buffer capacity, the pH will continue to decrease, thus increasing the oxidation reactions and increasing the precipitation of metals (5).

Figure 2: Healthy Water 40 m upstream of AMD Site (40.40833,-78.65473)Before passing Hughes Borehole, the Little Conemaugh River is a healthy stream. We sample roughly 40 meters upstream of the introduction AMD. In this location, we have found a wide variety of diatom species and macroinvertebrates. The water at this sampling site has an average pH of 6.9 and shows no signs of being impacted by AMD.

Figure 3: 40 m upstream Navicula lanceolata. Navicula lanceolata collected from a healthy stream in Pennsylvania, 40 m upstream from Acid Mine Drainage. Diatoms were washed with nitric acid and mounted with Naphrax. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 4: 40 m upstream Navicula lanceolata (Labeled view)Identification of the diatom was determined by the terminal raphe fissures hooking to the secondary side, the irregular oval central area, and the number of striae in 10 microns. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining the diatoms taxonomy. The raphe is a slit that forms during the diatoms development and runs along the apical axis. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 5: 40 m upstream Surirella brebissonii. Surirella brebissonii collected from a healthy stream in Pennsylvania, 40 m upstream from Acid Mine Drainage.  Diatoms were washed with nitric acid and mounted with Naphrax. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 6: 40 m upstream Surirella brebissonii (Labeled view)Identification of the diatom was determined by its heteropolar shape and the number of fibula in 10 microns. Fibulae provide support around the raphe which is a slit that runs through the axis of the diatom and can be used in identification.

Figure 7: 40 m upstream Staurosirella leptostauron. Staurosirella leptostauron collected from healthy stream in Pennsylvania, 40 m upstream from Acid Mine Drainage.  Diatoms were washed with nitric acid and mounted with Naphrax. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 8: 40 m upstream Staurosirella leptostauron (Labeled view)Identification of the diatom was determined by the number of striae in 10 microns and a key identifier of S. leptostauron is that the striae are wider than the costae. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining the diatoms taxonomy. Costae are unornamented and thickened silica areas of the valve. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 9: 40 m upstream Eunotia spp. Eunotia spp. collected from a healthy stream in Pennsylvania, 40 m upstream from Acid Mine Drainage. Diatoms were washed with nitric acid and mounted with Naphrax. The infrequent presence of this diatom in the healthy stream is not an indicator of a healthy or poor water quality. Eunotia can tolerate a wide range of water qualities, but in our studies, was found infrequently in the healthy stream, likely due to the ability of other diatoms to thrive there. At the AMD impacted sites, Eunotia was the only diatom observed in some samples.

Figure 10: 40 m upstream Eunotia spp. (Labeled view)Identification was determined by a slightly concave ventral margin, slightly convex dorsal margin, and the capitated apices which are the rounded ends of the valve. The infrequent presence of this diatom in the healthy stream is not an indicator of a healthy or poor water quality. Eunotia can tolerate a wide range of water qualities, but in our studies was found infrequently in the healthy stream, likely due to the ability of other diatoms to thrive there. At the AMD impacted sites, Eunotia was the only diatom observed in some samples.

Figure 11: Hughes Borehole (discharge). Hughes Borehole is an Acid Mine Drainage (AMD) site near Portage, Pennsylvania. Polluted water from underground mines in the area flows out of the borehole at a rate of 800-3,500 gallons per minute. The drainage contains a high concentration of dissolved metals, mostly iron, and has an average pH of 3.4. Over time, this source has flooded an area 6 acres in size, leaving it covered with a thick layer of brownish-red, iron-rich soilless precipitate which kills surrounding vegetation. Eventually this water flows into and pollutes the Little Conemaugh River which is flowing parallel to the AMD site.

Figure 12: 5 m downstream (40.40884,-78.65477)The sampling for the borehole is about 5 meters away from where the water emerges at the surface because a fence surrounds the site. The water here is extremely acidic, with an average pH of 3.4, and saturated with dissolved metals, making it the lowest quality water we sampled from. To date, we have only found one genus of diatom living in this water, Eunotia spp. This diatom shows considerable variation in size and shape due to this harsh environment.

Figure 13: 5 m downstream Eunotia spp. Eunotia spp. collected from the outflow of the Acid Mine Drainage 5 m from the origin. The environmental conditions limit the variety of diatoms to only Eunotia spp. Diatoms were washed with nitric acid and mounted with Naphrax.

Figure 14: 5 m downstream Eunotia spp. (Labeled view)Identification of Eunotia was determined by a slightly concave ventral margin, slightly convex dorsal margin, and the capitate apices which are the reflexed ends of the valve.

Figure 15: 5 m downstream Eunotia spp. girdle. Eunotia spp. collected from the outflow of the Acid Mine Drainage 5 m from the origin.  The environmental conditions limit the biodiversity of diatoms to only Eunotia spp.  Diatoms were washed with nitric acid and mounted with Naphrax.

Figure 16: 5 m downstream Eunotia spp. 40x. Eunotia spp. collected from the outflow of the Acid Mine Drainage 5 m from the origin.  The environmental conditions limit the biodiversity of diatoms to only Eunotia spp. Diatoms were washed with nitric acid and mounted with Naphrax.

Figure 17: 5 m downstream Eunotia spp. 40x. (Labeled view)There is a dramatic variation in length among Eunotia spp. found in the AMD outflow.

Figure 18: 50 m downstream (40.40945,-78.65468) The settling pond sampling site is about 50 meters downstream from the Hughes Borehole. The water that is expelled from the borehole flows across a soilless iron precipitate formed following many years of pollution. This water then collects in a naturally formed settling pond which also gathers rainwater runoff and continues to move slowly towards the Little Conemaugh River. The samples taken at this site exhibit a great diversity of diatom species despite the very low pH (2.7) of the water in this location.

Figure 19: 50 m downstream Encyonema latum. Encyonema latum was collected from AMD outflow that was 50 m downstream from the origin in a naturally formed settling pond. Identification was determined by the strongly arched dorsal margin, rounded apicies, and 9 striae in 10 microns. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining diatom taxonomy.

Figure 20: 50 m downstream Encyonema latum (Labeled view). Encyonema latum was collected from AMD outflow that was 50 m downstream from the origin in a naturally formed settling pond. Identification was determined by the strongly arched dorsal margin, rounded apicies, and 9 striae in 10 microns. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining diatom taxonomy.

Figure 21: 50 m downstream Pinnularia divergentissima. Pinnularia divergentissima was collected 50 m downstream from AMD outflow in a naturally formed settling pond. Identification was determined by the closely spaced raphe ends, the central area reaches both margins, and 13 striae in 10 microns. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining diatom taxonomy.

Figure 22: 50 m downstream Pinnularia divergentissima (Labeled view). Pinnularia divergentissima was collected 50 m downstream from AMD outflow in a naturally formed settling pond. Identification was determined by the closely spaced raphe ends, the central area reaches both margins, and 13 striae in 10 microns. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining diatom taxonomy.

Figure 23: 50 m downstream Pseudostaurosira brevistriata. Pseudostaurosira brevistriata was collected 50 m downstream from AMD outflow in a naturally formed settling pond.

Figure 24: 50 m downstream Pseudostaurosira brevistriata (Labeled view). Identification was determined by the location of striae near the valve mantle. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining diatom taxonomy.

Figure 25: 600 m downstream (40.41199,-78.65948). The bridge sampling site is about 600 meters downstream from Hughes Borehole. The water in this section of the stream has an average pH of 5.3, lower than our healthy sampling site upstream of the borehole, pH of 6.9, but still considerably higher than the water coming out of the borehole itself, pH of 3.4. The streambed here exhibits less diatom and macroinvertebrate species diversity as compared to the healthy stream sampling site, a result of AMD pollution.

Figure 26: 600 m downstream Diatoma vulgaris. Diatoma vulgaris was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification was determined by rounded subrostrate ends and by 8 transapical costae in 10 microns.

Figure 27: 600 m downstream Diatoma vulgaris (Labeled view). Diatoma vulgaris was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification was determined by rounded subrostrate ends and by 8 transapical costae in 10 microns.

Figure 28: 600 m downstream Navicula lanceolata. Navicula lanceolata was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification of the diatom was determined by the terminal raphe fissures hooking to the secondary side, the irregular oval central area, and the number of striae in 10 microns. Striae are rows of pores in the diatoms valve that are oriented along the axis and are used in determining diatom taxonomy. The raphe is a slit that forms during the diatoms development and runs along the apical axis. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 29: 600 m downstream Navicula lanceolata (Labeled view). Navicula lanceolata was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification of the diatom was determined by the terminal raphe fissures hooking to the secondary side, the irregular oval central area, and the number of striae in 10 microns. Striae are rows of pores in the diatom valve that are oriented along the axis and are used in determining diatom taxonomy. The raphe is a slit that forms during the diatoms development and runs along the apical axis. This diatom is typically found in streams that are around a neutral pH and can be used as an identifier of a healthy water source.

Figure 30: 600 m downstream Cocconeis placentula. Cocconeis placentulas was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification of the diatom was determined by a small oval central area, striae interrupted by hyaline ring, and by 20 striae in 10 microns. The hyaline ring is an unornamented area the interrupts the striae on the diatom.

Figure 31: 600 m downstream Cocconeis placentula (Labeled view). Cocconeis placentulas was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification of the diatom was determined by a small oval central area, striae interrupted by hyaline ring, and by 20 striae in 10 microns. The hyaline ring is an unornamented area the interrupts the striae on the diatom.

Figure 32: 600 m downstream Diatoma tenuis. Diatoma tenuis was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification of the diatom was determined by 6 transapical costae in 10 microns and capitate ends.

Figure 33: 600 m downstream Diatoma tenuis (Labeled view). Diatoma tenuis was collected 600 m downstream where the healthy stream and AMD outflow mixes. Identification of the diatom was determined by 6 transapical costae in 10 microns and capitate ends.