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Mountain Coal Company SeaCrest Number 399127 <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 of <br />the effluent mixture was added to each beaker. Fathead minnows were exposed in 260 ml plastic <br />cups containing 200 ml of medium. Forty minnows were used at each concentration, following <br />the revised 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 />Acute tests were perfonned according to the procedures outlined in Weber (1993). The <br />Daphnia magna test was started at 12:15 on April 9, 1999 and was conducted over 48 hours. <br />The fathead minnow test was begun at 10:30 on April 2, 1999 and ran for 96 hours. The tests <br />were run with exposure concentrations of 100%, 75%, 50%, 25%, and 12.5% effluent. Water <br />used for the control and as the dilution water was laboratory reconstituted freshwater. <br />Data Analysis <br />Data from the tests were analyzed on a Computer Source 386 personal computer. Where <br />applicable, LC50's were calculated using EPA's TOXDATMulli-Method Program according to the <br />binomial, probit, or moving average methods. Statistically significant differences between control <br />and exposed organisms were determined using the TOXSTAT software developed at the <br />University of Wyoming. <br />LABORATORY ERROR <br />The acute tests with the first effluent sample, collected April 1, 1999, were run with the <br />fathead minnows and the Ceriodaphnia dubia. The Ceriodaphnia were not the correct <br />invertebrate species for this site permit and a second sample had to be collected on April 8, 1999, <br />to run the correct species, Daphnia magna. We apologize for the error in performing a <br />Ceriodaphnia test with the first sample instead of the Daphnia magna test that was required. We <br />will try to prevent this type of error from occurring in the future so that additional samples will not <br />have to be taken. <br />RESULTS <br />The results of the tests are summarized in Appendix 2. No mortality was seen in the <br />Daphnia magna test where survival was 100% in the 100% effluent and ranged from 95-100% <br />in every remaining effluent concentration. Survival was 100% in the control. The technician who <br />terminated the test noted that the animals in the two highest test concentrations were immobile <br />but still alive. <br />The fathead minnow test showed no toxicity, with 100% survival in the 100% effluent and <br />survival ranging from 98-100% in the remaining effluent concentrations. Control survival was <br />100%. <br />The SeaCrest Group 3 <br />