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Controls on the nature and distribution of an alga in coal mine-waste environments and its potential impact on water quality

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 A dominant non-bacterial microorganism that may strongly impact environmental conditions in acid mine drainage at several Indiana coal mine sites is a single-celled protozoan, Euglena mutabilis. Field data suggest E. mutabilis has high tolerance for
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  Cases and solutions 458 Environmental Geology 40 (4-5) February 2001 7 Q Springer-Verlag Received: 13 January 2000 7 Accepted: 2 May 2000S.S. Brake ( Y )Department of Geography, Geology, and Anthropology,Indiana State University, Terre Haute, IN 47809, USAe-mail: gebrake 6 scifac.indstate.eduTel: 812-237-2257Fax: 812-237-8029H.K. DannellyDepartment of Life Sciences, Indiana State University,Terre Haute, IN 47809, USAK.A. ConnorsWestern Mining Corporation, 8008E. Arapahoe Ct, Suite 110,Englewood, CO 80112, USA Controls on the nature anddistribution of an alga in coalmine-waste environments and itspotential impact on water quality S.S. Brake 7 H.K. Dannelly 7 K.A. Connors Abstract A dominant non-bacterial microorganismthat may strongly impact environmental conditionsin acid mine drainage at several Indiana coal minesites is a single-celled protozoan, Euglena mutabilis .Field data suggest E. mutabilis has high tolerancefor elevated total dissolved solids (TDS), to 18g/l,and acid conditions to pH1.7. Distribution is re-stricted to unmixed effluent pH ~ 4.6, with prolificgrowth between pH3.0 and 3.5. Additional factorsinfluencing E. mutabilis include preference for ar-eas with lower mineral/colloidal precipitation ratesand a stable substrate of iron-rich precipitates. Ini-tial studies indicate that in areas of prolific growthit contributes to oversaturation of dissolved oxygenby up to 200%. The presence of small orange intra-cellular crystalline-like structures, similar in colorto iron oxyhydroxides, suggests that E. mutabilis may be sequestering iron, and possibly other me-tals. Further work is needed to determine if E. mu-tabilis contributes to natural mitigation of poor wa-ter quality at these and other coal mine sites. Keywords Acid mine drainage 7 Algae 7 Euglenamutabilis 7 Water quality  Introduction Coal mining regions are known for producing extremely acidic effluent, commonly referred to as acid mine drai-nage (AMD). AMD is produced during the weatheringprocess from the oxidation of sulfide minerals dissemi-nated in coal and coal waste-rock. As the acidic effluentmoves through the near surface environment, consti-tuents that are soluble under acid, oxidizing conditions,such as Fe, Al, SO 4 , Cu, Pb, Zn, Cd, and Se, become con-centrated in the effluent (Anderson and Youngstrom1976; Davis and Boegly 1981). Some of these constituentsreach concentrations that are toxic to most aquatic or-ganisms. Only a few species of bacteria, protozoa, fungi,and algae have adapted to life under these hostile condi-tions. Some of the bacterial species residing in AMD en-vironments have the ability to increase the rate of acidproduction as well as leach or precipitate metals (Ehrlich1996; Konhauser 1998). The role of non-bacterial micro-organisms in acid generation or in sequestering metals isless well known and poorly documented. Understandingthe influence of non-bacterial microorganisms on waterquality in AMD systems is essential in developing suc-cessful remediation measures, possibly using environ-mental-friendly microorganisms, in an effort to mitigateAMD-related contamination.One of the non-bacterial microorganisms that demon-strates a resistance to acidic environments is Euglenamutabilis (Protista: Euglenophyta), a unicellular proto-zoan commonly referred to as an alga (Lackey 1939; vonDach 1943; Bennett 1969; Hargreaves and others 1975). E.mutabilis has been detected in a wide variety of surficialenvironments (Hargreaves and Whitton 1976), but showsa strong preference for bogs (Pentecost 1982), acidic tun-dra ponds (Sheath and others 1982), and AMD (Lackey 1938; Bennett 1969; Hargreaves and Whitton 1976; Kapfer1998). This species also exhibits an extremely high toler-ance for elevated concentrations of dissolved constituents(Nakatsu and Hutchinson 1988), preferring to live in themetal-laden, low pH ( ~ 3) waters commonly associatedwith coal mining (Lackey 1938).In this study, the temporal and spatial distribution of E.mutabilis is evaluated in AMD environments associatedwith five Indiana coal mine reclamation sites located insouth-western and west-central Indiana on the easternedge of the Illinois Basin, USA (Fig.1). The nature of E.mutabilis and its distribution are investigated with re-spect to environmental variables such as temperature,pH, Eh, total dissolved solids (TDS), and dissolved oxy-gen. The potential impact of this microorganism on wa-ter quality in AMD environments is also considered, par-  Cases and solutions Environmental Geology 40 (4-5) February 2001 7 Q Springer-Verlag 459 Fig.1 Location of Indiana coal mine sites evaluated in this study.Mine sites include G Green Valley, C Chinook, F Friar Tuck, S Shasta,  A American. Shaded area on lower map representsapproximate extent of Illinois basin ticularly with regard to introducing dissolved constituentsand/or sequestering metals. Site description Study areas, which include the Chinook, American, Shas-ta, Friar Tuck, and Green Valley mines, were selected torepresent the broad array of conditions in AMD environ-ments associated with coal mining. Sample sites withineach study area were chosen to characterize the physico-chemical conditions controlling the distribution of E. mu-tabilis. Of the five sites, Green Valley was studied ingreatest detail. Samples were collected during the sum-mer of 1999; however, field observations of E. mutabilis distribution were also made at Green Valley during thewinter months of December 1998 and January 1999.Waste materials generated during coal mining and proc-essing activities are stored on the surface at each of themine sites. Dumping areas srcinally consisted of numer-ous steeply sloping “gob piles” comprised of coarse-grained coal waste rock, along with tailings of fine-grained waste deposited in slurry ponds. Waste materialshave been reclaimed to varying degrees. However, AMDis a continuing problem at all of the investigated sites,and the associated yellowish-brown to orangish-browniron oxyhydroxide precipitates that occur in some of theimpacted drainage areas attest to the poor water quality. Chinook mine At the Chinook strip mine, coal waste-rock was coveredwith imported soil and revegetated with trees. Today, thearea consists of a series of undulating hills supporting amature growth of vegetation. Slightly acidic AMD cur-rently discharges from several seeps located in waste pilesadjacent to a paved highway. This effluent flows througha road ditch, coated with iron precipitates, and eventually discharges into a nearby natural stream. Seven samplesites were selected along a 170-m stretch of the roadditch. American mine Coal waste-rock extracted from underground workings atthe American mine was reshaped into a large, low relief pile covering F 0.3km 2 . The pile was subsequently cap-ped by soil and revegetated with grasses (B. Stevens, per-sonal communication 1999). Effluent from a small pondlocated near the center of the waste pile drains into aconstructed ditch lined with crushed rock. The rip-rapchannel, designed to prevent erosion and divert flow, iscomposed of carbonate rock that provides a local bufferfor acidity. Unlike at the Chinook site, iron-rich AMDprecipitates do not coat the channel bottom. Effluent inthe channel varies from pH4–5 and is diverted into acarbonate rip-rap-lined road ditch located on the north-western edge of the waste pile. Effluent also flows fromseveral horizontal drainage pipes placed on the north-western side of the waste pile. This effluent is considera-bly more acidic (pH2–3). One sample location was se-lected in the rip-rap ditch draining the small pond, andfive were located in the road ditch containing the moreacidic effluent. Drainage from the pipes is divertedthrough the road ditch and eventually combines with ef-fluent draining from the small pond. Shasta mine Coal waste-rock from strip mining operations at theShasta mine was reclaimed by placing fly ash over thewaste pile and revegetating the area with grasses (B. Stev-ens, personal communication 1999). Results of these ef-forts are mixed; effluent is confined to three shallow ponds, one near neutral and the other two acidic(pH2–3). Iron-rich AMD precipitates were not observedin the two acidic ponds, but precipitates were present in  Cases and solutions 460 Environmental Geology 40 (4-5) February 2001 7 Q Springer-Verlag the drainage channels between the ponds. A sample wastaken from each of the two acidic ponds. Friar Tuck mine Coal was extracted by both underground and surfacemethods at the Friar Tuck mine, and waste material wasstored on the surface. A preparation plant and powerplant operated at the site (Branam and Harper 1994), andwaste products from these facilities were also stored onthe surface. Three sample locations were located at see-page discharge points near the bottom of an unvegetated,steeply sloping ridge of waste dissected by a series of gul-lies. Drainage from the seeps is routed into a small, shal-low stream channel that runs parallel to the bottom of the ridge. One sample point is located within this streamchannel slightly downgradient of the ridge. Iron-richAMD precipitates are absent in the seepage areas on theside of the ridge and in the stream channel. Green Valley mine Approximately 5million tons of waste materials fromcoal mining and processing operations at the Green Val-ley mine were initially dumped over a nearly 25-ha area(Eggert and others 1981). The waste material was subse-quently reclaimed by recontouring waste piles and back-filling slurry ponds to create a single, low relief moundcovering F 67ha (L. Ladislas and M. Stacy, personalcommunication 1998). A 20-cm layer of lime was placedover the waste to neutralize acidity generated by oxida-tion of contained sulfide minerals. The waste pile wasthen capped by 1m of soil and revegetated with grassesand legumes to prevent erosion. Crushed carbonate rip-rap ditches were also constructed at various locations onthe waste pile and were intended to divert surface-waterrunoff into West Little Sugar Creek, located along thewestern margin of the waste pile. This would have, intheory, decreased penetration of water into the pile,thereby reducing acid generation. However, AMD, be-tween pH2–4.5, is currently flowing in two of the rip-rapchannels; the longest channel, and the one sampled dur-ing this study, is hereafter referred to as the main ef-fluent channel. Rip-rap in this channel is covered by iron-rich, AMD-related precipitates rendering the carbon-ate ineffective as a buffer and, contrary to intentions, therip-rap channel is now providing direct access for AMDto leave the site and to drain into West Little SugarCreek.AMD precipitates in the upper part of the main rip-rapchannel at the Green Valley site form a 3- to 4-cm coat-ing on the rip-rap, whereas in the central portion of thechannel, AMD precipitates almost completely bury therip-rap, forming a series of small terraces. The slopes be-tween the terraces are composed of a yellowish-brownprecipitate (Fig.2), similar in color to the mineral jaro-site, a hydrous iron-potassium sulfate. Effluent discharg-ing from the rip-rap channels into West Little SugarCreek mixes with the stream water resulting in rapiddownstream precipitation of colloidal iron oxyhydroxidesand aluminum hydroxides. Colloidal aggregation of the Fig.2 Terraced section in central portion of main effluent channel atGreen Valley.  Arrows indicate probable jarosite, or amorphousFe–K sulfate, precipitate on terrace slopes precipitants results in sedimentation during periods of low flow, forming an unstable, fluid-rich layer up to20cm thick. During sampling, flow in West Little SugarCreek remained low ( ~ 0.015m/s), leaving the colloidallayer relatively undisturbed. Sample points at the GreenValley site are located primarily in the main effluentchannel and in West Little Sugar Creek, both up- anddowngradient of the waste pile (Fig.3). Methods Temperature, dissolved oxygen, Eh, pH, and conductivity of effluent were measured at each sample site using a YSI600XL Multi-Parameter Water Quality Monitor to charac-terize the physical properties associated with the distribu-tion of E. mutabilis. This instrument has an accuracy of  B 0.15 7 C for temperature, B 20mV for Eh, B 0.5% of the conductivity measurement plus 0.001mS/cm,  Cases and solutions Environmental Geology 40 (4-5) February 2001 7 Q Springer-Verlag 461 Fig.3 a Sample locations along West Little Sugar Creek at the GreenValley Mine. Shaded area represents approximate extent of coalwaste pile. Dashed and dotted lines approximate the location of constructed effluent channels. Map coordinates are meters,UTM zone 16. b Sample locations in main effluent channel atGreen Valley trending north-east–south-west. The north–southbranch is dry. Dashed line indicates continuation of channel,and shaded areas represent seepage points B 0.2mg/l for dissolved oxygen, and B 0.2 units for pH.Conductivity was used to internally calculate TDS withinthe YSI software. In areas where water was too shallow for the YSI monitor, a hand-held pH meter, with an ac-curacy of B 0.01 was used.The pH and conductivity meters on the YSI monitor werecalibrated prior to each site visit using pH4 and 7 buffer-ing solutions and a calibration standard of 0.990mS/cmrespectively. The dissolved oxygen probe was calibratedby measuring water saturation and adjusting for barom-etric pressure. Prior to data collection, the YSI monitorwas triple rinsed with reverse osmosis water. Duringsampling events, the probe was allowed to stabilize foreach parameter before recording data.Biological samples were collected at some of the watersample points following determination of field paramet-ers. E. mutabilis communities were easily recognized inthe field due to their bright green color and were readily distinguished from the olive-green to brownish-green fil-amentous algae that also grow in some of the effluentchannels. E. mutabilis was collected in sterile plastictubes by scraping benthic mats attached to substrate sur-faces with the edge of a tube to loosen the coating andby dragging a tube across the water surface to collectfloating communities. In some areas where E. mutabilis was not evident, water and substrate samples were col-lected to confirm their absence. All samples were storedon ice for transport to the laboratory, where they wereimmediately refrigerated and examined within 24h of collection. Wet mounts of each sample were preparedand analyzed using dark field and phase contrast micros-copy. Internal structures of E. mutabilis were examinedby staining fixed smears with methylene blue. Results Field determinations of temperature, dissolved oxygen,Eh, and pH obtained at the various sample sites, as wellas TDS, calculated from conductivity data, are presentedin Table1. Communities of E. mutabilis were observed togrow over a wide range of temperatures, from 0 7 C, basedon their presence below a thin layer of ice, up to 37 7 C.The organism has a high tolerance for elevated TDS, oc-curring in effluent containing from 3.5 to 18.6g/l TDS(Fig.4a). In areas where E. mutabilis is densely popu-  Cases and solutions 462 Environmental Geology 40 (4-5) February 2001 7 Q Springer-Verlag Table1 Analysis of water samplesfrom five Indiana coal minesites. TDS Total dissolvedsolids; SpC specificconductance; DO dissolvedoxygen; GV Green Valley;  AM  American; SHA Shasta; FT  Friar Tuck; CH ChinookSampleDateT ( 7 C)TDS (g/l)SpC (mS/cm)DO (mg/l)Eh (volts)pHAreas with no E. mutabilis GV-B1007/12/99259.0313.911.60.3823.7GV-B1107/12/993113.6721.08.40.3683.5GV-B1207/12/993115.5824.01.30.3913.1GV-B1407/28/99260.761.27.00.1835.9GV-007I07/12/992914.5822.47.10.3763.4GV-W106/22/99220.390.611.5–8.4GV-W206/22/99250.370.614.00.2138.6GV-W306/22/99250.370.613.90.2258.6GV-W406/22/99260.400.614.30.0878.0GV-W1907/27/99389.3614.44.60.2873.1GV-W2307/28/99281.352.16.50.4103.9GV-W2507/28/99240.460.76.70.2397.6GV-W2608/02/99210.030.16.00.4283.5GV-W2708/02/99231.382.16.50.4623.3AM-306/04/99290.160.58.80.2424.7CH-B108/16/99193.465.33.20.2786.2CH-B208/16/99193.515.46.30.2966.2CH-B308/16/99163.485.44.30.3185.8CH-B408/16/99173.385.26.40.3126.0CH-B508/16/99183.455.36.60.3096.1CH-B808/16/99193.445.37.30.2926.2CH-B908/16/99241.342.18.60.2996.4Areas with dead E. mutabilis cellsGV-B507/08/993712.6019.44.10.2813.3GV-B707/08/992317.2926.61.30.3023.3AM-B307/15/99279.8415.15.20.4692.6AM-B407/15/992912.2918.92.30.4242.2Areas with E. mutabilis in trace amountGV-B607/08/992913.1720.318.50.2853.6GV-B1307/12/993616.3125.14.50.3013.3GV-013B07/12/993415.8723.57.00.4233.3GV-W2007/28/992414.0721.74.30.4552.2GV-306/15/993710.2615.83.30.3113.2AM-206/04/99268.4812.90.30.4362.3SHA 106/04/99–––––2.7Areas with abundant E. mutabilis GV-OB112/01/987 a –––––GV-OB201/15/99–2 a –––––GV-B107/08/99203.545.55.50.1434.6GV-B307/08/99223.695.76.70.1854.6GV-B407/08/992614.6822.616.80.2613.5GV-B807/12/992514.6122.514.50.4713.5GV-B907/12/992314.6922.68.80.3873.5GV-007B07/12/992214.7822.720.60.4993.5GV-007C07/12/992614.7422.717.20.4753.5GV-007F07/12/992814.7822.715.90.4183.5GV-007G07/12/992814.8122.813.50.4303.5GV-007H07/12/992914.8222.814.40.4363.5GV-W1607/27/992018.2628.12.80.3013.2GV-W1807/27/993618.1027.810.30.2943.1GV-206/15/992813.4120.617.30.2773.4AM-106/04/99298.3312.70.60.4382.2AM-B107/15/992510.5016.20.50.5082.5SHA 206/04/99–––––2.5FT-106/04/99–––––2.5FT-206/04/99–––––2.8FT-306/04/99244.997.75.60.3962.7FT-406/04/99258.2412.72.80.3741.7 a Represents atmospheric temperature
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