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Mountain Coal Company CO-0038776 SeaCrest Number: 303251 Test Procedures Upon receipt at the lab, the water samples were analyzed for alkalinity, hardness, conductivity, dissolved oxygen, chlorine, ammonia and pH. Alkalinity and hardness were determined titrimetrically according to methods described in Hach Chemical Co. (2002). Ammonia was measured by a Thermo Orion ion-specific electrode according to procedures in APHA/AWWANVEF (1998). A total of twenty organisms at each concentration were used for the daphnia test. Daphnia magna were exposed in 30 ml plastic disposable beakers. Approximately twenty ml of the effluent mixture was added to each beaker. Fathead minnows were exposed in 260 ml plastic cups containing 200 ml of medium. Forty minnows were used at each concentration, following State of Colorado WET testing guidelines. Exposure medium was replaced after each 24 hour period and organisms surviving at each concentration were counted and recorded. Routine measurements were made of temperature, pH, and dissolved oxygen in each concentration. The acute tests were started on July 16, 2003 (fathead minnows) and July 30, 2003 {Daphnia magna), and were performed according to the procedures outlined in Weber {1993). The Daphnia magna test was conducted over 48 hours, the fathead minnow test for 96 hours. The tests were run with exposure concentrations of 100%, 50%, 25%, 12.5%, and 1.9% effluent. Water used for the control and as the dilution water was moderately-hard laboratory reconstituted freshwater. Data Analysis Data from the tests were analyzed on a Pentium personal computer. Where applicable, LC50's were calculated using EPA's TOXDATMnlti-Method Program according to the binomial, probit, or moving average methods. Statistically significant differences between control and exposed organisms were determined using the TOXSTAT software developed at the University of Wyoming. RESULTS The results of the tests are summarized in Appendix 2. In the Daphnia magna test survival was 80% in the 100% effluent and ranged from 95-100% in the remaining effluent concentrations. Survival was 95% in the control. No LC50 concentration was produced in this test. The fathead minnow test showed 100% survival in the 100% effluent and survival ranging from 98-100% in the remaining effluent concentrations. Control survival was 100%. The SeaCrest Group 3