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Client: Colowyo Coal Company C00045161 SCG Project No.: 419340.13 <br /> Site: 010A Outfall <br /> INTRODUCTION <br /> Biomonitoring provides an effective means by which the toxicity of discharges from <br /> municipal, industrial and mining operations can be tested. Among the advantages of <br /> biornonitoring is the ability to test complex effluents containing a broad range of contaminants. <br /> Blomonitoring, when used in conjunction with chemical analyses, can generate data capable of <br /> identifying a much wider range of contaminants. <br /> The Colorado Water Quality Control Division requires certain NPDF.S permittees to <br /> perform acute and/or chronic bionionitoring tests. The chronic test measures significant <br /> differences in lethality and in reproduction (Ceriodaphnia dubia) or growth (Fathead minnow — <br /> Pimephales promelas) between control and exposed organisms. <br /> The present report discusses the results of chronic biomonitoring tests conducted on <br /> effluent from the Colowyo Coal Company on the 0 1 OA discharge. These tests were conducted in <br /> June 2019. <br /> MATERIALS AND METHODS <br /> Sample Collection <br /> Three or Two gallons of the effluent were collected on three separate dates. Samples <br /> were delivered chilled to the SeaCrest lab where they were held at 0-60C. Chain of custody forms <br /> showing sample collection and lab arrival times are 'included in Appendix 1. <br /> Dilution Water <br /> Laboratory reconstituted water was used as both the dilution water source and the control <br /> for the tests. Reconstituted water for the Cer iodaphnia du is test was produced by adding <br /> sodium bicarbonate, calcium Sulfate,, magnesium sulfate, potassium chloride, and sodium <br /> selenate to delonized water. Reconstituted water for the Fathead minnow test was produced by <br /> adding sodium bicarbonate, calcium sulfate,, magnesium sulfate, and 'potassium chloride to <br /> delonized water. <br /> Test Organisms <br /> The tests were conducted with Cerlodaphnia dubia and Fathead miiiLnows. Cerlodaphnia <br /> dub is cultured in the SeaCrest laboratory. Stock cultures are maintained in 5-gallon aquaria. <br /> Brood females are cultured in individual plastic beakers on brood boards for a period of up to 14- <br /> days. Neonates less than 24-hours old released from third or subsequent broods of eight or more <br /> within an 8-hour period are removed from the brood chambers and used in tests. Brood and <br /> stock organisms are fed daily with a mixture of Yeast, Cereal Leaves, and Trout Chow (YCT). <br /> This is supplemented with an equal volume of green algae (SelenaSlrum capricornufuln). <br /> Less than one-day-old Fathead minnow, cultured In Aquatic BloSysterns, Inc., were also <br /> used in the test. The larval fish are fed newly hatched Brine shrimp (Aftemia sp.) at least twice <br /> per day. <br /> In-house organisms are tested at least monthly in a reference toxicant test using sodillm <br /> chloride to monitor overall health and test reproducibility. <br /> SeaCrest Group 5 <br />