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Mountain Coal Company CO-0038776 SeaCrest Number: 303369, 303385 <br />reference toxicant test, conducted during the effluent test or as soon as possible afterwards. <br />Test Procedures <br />Upon receipt at the lab, the water samples were analyzed for alkalinity, hardness, <br />conductivity, dissolved oxygen, chlorine, ammonia and pH. Alkalinity and hardness were <br />determined titrimetrically according to methods described in Hach Chemical Co. (2002). <br />Ammonia was measured by a Thermo Orion ion-specific electrode according to procedures in <br />APHA/AWWA/WEF (1998). <br />A total of twenty organisms at each concentration were used for the daphnia test. <br />Daphnia magna were exposed in 30 ml plastic disposable beakers. Approximately twenty ml <br />of the effluent mixture was added to each beaker. Fathead minnows were exposed in 260 ml <br />plastic cups containing 200 mi of medium. Forty minnows were used at each concentration, <br />following State of Colorado WET testing guidelines. Exposure medium was replaced after each <br />24 hour period and organisms surviving at each concentration were counted and recorded. <br />Routine measurements were made of temperature, pH, and dissolved oxygen in each <br />concentration. <br />The acute tests were started on October 17, 2003 (Daphnia magna) and November 4, <br />2003 (fathead minnows), and were performed according to the procedures outlined in Weber <br />(1993). The Daphnia magna test was conducted over 48 hours, the fathead minnow test for <br />96 hours. The tests were run with exposure concentrations of 100%, 50%, 25%, 12.5%, and <br />1.9% effluent. Water used for the control and as the dilution water was moderately-hard <br />laboratory reconstituted freshwater. <br />Data Analysis <br />Data from the tests were analyzed on a Pentium personal computer. Where applicable, <br />LC50's were calculated using EPA's TOXDATMnlti-Method Program according to the binomial, <br />probit, or moving average methods. Statistically significant differences between control and <br />exposed organisms were determined using the TOXSTAT software developed at the University <br />of Wyoming. <br />RESULTS <br />The results of the tests are summarized in Appendix 2. In the Daphnia magna test <br />survival was 15% in the 100% effluent and ranged from 70-100% in the remaining effluent <br />concentrations. Survival was 100% in the control. An LC50 of 63.7% was created for this <br />sample. <br />Sq°~sr.t%-./"i..,.J-SJ i...~eL.icc. riP~:+'r_Rt.. CL.....''.. _...... ..... ~..^'.,... ..:.~.,...,, ..u."i fv, v`tiv _ __ _ <br />The SeaCrest Groups <br />