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Enviro -Chem Analytical, Inc. <br />2493 Hwy 6850 Unit 7 <br />Grand Junction, CO 81505 <br />Chronic Toxicity Testing <br />OXBOW — July 2013 <br />2 Species <br />test chamber. Organisms were obtained from Aquatic Biosystems Inc. Fort <br />Collins, Colorado and were <24 hours old at test initiation. Fish were impartially <br />distributed to each test chamber.Test chamber consisted of 500 mL disposable <br />plastic cups containing 250 mL of test solution. Fathead minnows were fed twice <br />daily, 0.15 mL once at the beginning of the day and 0.15 mL again in the late <br />afternoon, newly hatched brine shrimp (Artemia). Brine shrimp cysts were <br />obtained from Aquatic Biosystems Inc, Fort Collins, Colorado and were hatched <br />in salt water overnight. Brine shrimp were thoroughly rinsed prior to feeding to <br />remove any residual salt. Test solution was renewed daily at approximately 24- <br />hour intervals from the original initiation time of the test. <br />Each day test chambers were observed for survival. At test termination, the fish <br />were euthanized, placed in pre- weighed drying dishes and dried for 23 hours at <br />96 -99 C. The dried fish were then weighed to determine growth measured as <br />mean dry weight. Per USEPA guidelines, mean dry weight is calculated as <br />pooled replicate weight divided by the original number of organisms exposed. <br />Ceriodaphnia dubia - <br />Ten replicates per exposure concentration were tested with one organisms per <br />test chamber. Organisms were obtained from Aquatic Biosystems Inc. Fort <br />Collins, Colorado with neonates that were <24 hours old. Test chamber <br />consisted of 30 mL disposable plastic condiment cups containing 15 mL of test <br />solution. Ce►iodaphnia dubia were fed daily 0.1 mL of a suspension of algae <br />(Selenastrum capricomutum) obtained from Aquatic Biosystems Inc, Fort Collins, <br />Colorado. No additional micro - nutrients were added to MHW as algal <br />suspensions have already been fed the proper amounts of nutrients per USEPA <br />guidelines. Feeding took place immediately after test solution renewal. Test <br />solution was renewed daily at approximately 24 -hour intervals from the original <br />test initiation time. <br />Each day test chambers were observed for survival and presence of new brood <br />neonates. Whenever brooding appeared to have occurred during, or relatively <br />closed to test renewal, the neonates were counted and considered 1 brood. <br />4 <br />