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Aqua-Tox, Inc. <br />4880 Robb Street, Suite 8 <br />_ Wheat Ridge, CO 80033 <br />(303) 621-4880 aqua-tox.com <br /> <br />ACUTE TOXICITY REPORT <br />Ceriodaphnia dubia - 3rd Accelerated <br />CLIENT NAME: Twentymile Coal Company SAMPLE I.D.: 110609-1 <br />50 % MORTALITY TEST: Pass TEST RESULT LC50: >100% <br />% SURVIVAL IN 100% DILUTION: 60% <br />SAMPLE DATE: 12-10-09 TIME: 08:30AM CLIENT ID: Site 62 <br />ORGANISM: Ceriodaphnia dubia AGE: < 24 hours old <br />TEST START: 12-11-09 12:15PM TEST FINISH: 12-13-09 12:15PM <br />TYPE OF TEST: LC50, 48 hours, static, renewal, moderately hard fresh synthetic dilution water. <br />MODERATELY HARD FRESH SYNTHETIC DILUTION WATER CONSTITUENT <br />AMMONIA: ND D.O.: 7.29 mg/L <br />HARDNESS: 93.49 mg/L CHLORINE: ND <br />ALKALINITY: 54.04 mg/L TEMPERATURE: 20.4 deg C <br />pH: 7.93 <br />SURVIVAL DATA DILUTIONS (%) <br />DATE (HOURS)CONTROL 6.25 12.5 25 50 75 100 <br />12/11/2009 INITIAL 20 20 20 20 20 20 20 <br />12/12/2009 24 20 20 20 20 20 20 20 <br />12/13/2009 48 20 19 20 19 20 20 12 <br />MORTALITY: 0 1 0 1 0 0 8 <br />% SURVIVAL: 100% 95% 100% 95% 100% 100% 60% <br />DAILY CHEMISTRY - (MIN/MAX) DILUTIONS (%) <br />0 6.25 12.5 25 50 75 100 <br />CONDUCTIVITY (microS) 318/330 565/581 784/788 1129/1245 1855/1874 2630/2650 32803330 <br />D.O. (mg/L) 7.1/7.3 7.2/7.4 7.0/7.4 6.7/7.4 6.6/7.3 6.5/7.3 6.5/7.2 <br />pH 7.9/8.1 7.3/7.9 7.1/7.8 6.8/7.6 6.7/7.4 6.6/7.2 6.5/7.1 <br />TEMPERATURE (deg C) 19.8/20.4 19.8/20.4 19.8/20.4 19.8/20.4 19.8/20.4 19.8/20.4 19.8/20.4 <br />GENERAL CHEMISTRY - SAMPLE <br />AMMONIA: ND D.O.: 6.48 mg/L <br />HARDNESS: 1700.00 mg/L CHLORINE: ND <br />ALKALINITY: 232.47 mg /L TEMPERATURE: 20.4 deg C <br />pH: 6.49 D.O AFTER AERATION: NA <br />ANOEC CALCULATION,METHOD METHOD:REFERENCE <br />75% SPEARMAN-KARBER EPA -821-R-02-012 October 2002 pg.78 <br />ADDITIONAL COMMENTS: <br />Analysis <br />Conducted by: Reviewed by: <br />Date: Date:,/ <br />/ , b