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2.0 METHODS 2.1 TEST PROTOCOLS Maintenance of Pimephales and their testing procedures are described in general form by California Department of Fish and Game,Water Pollution Control Laboratory (1987). The test was conducted as a static test with no renewal of the test solutions. 2.2 TEST SOLUTION PREPARATION Dilution water for the Pimephales test solutions consisted of Nanopure grade water adjusted to soft(40-48 mg/L CaCO3) using spring water. The test concentrations used were 250 and 750 mg/L. The proper amounts of well-mixed sample were weighed out and added to 250-m1 flasks with 200 ml of Nanopure grade water. Duplicates of each concentration were prepared separately. The containers were covered and shaken for 24 hours on a three-dimensional shaker. Each aliquot was then added to 9.8 L of dilution water in a 5-gallon tank. 2.3 SUMMARY OF BIOASSAY TEST PROCEDURES Juvenile fathead minnows were used in the bioassay. Test animals were obtained from Thomas Fish Co., Anderson, California and held for at least 10 days prior to test initiation. The test was carried out in 5-gallon aquaria using 10 L of test solution and ten fish per tank. Duplicates were run for each treatment(concentration)and the control. Water quality measurements, consisting of pH, dissolved oxygen, and temperature, as well as mortality, were taken daily. Alkalinity, hardness, and conductivity of the the control and the highest concentration tested were measured at the beginning and end of the testing period. Testing was conducted at 20 ± 2°C under a 16 hours light : 8 hours dark photoperiod for the 96-hour duration of the test. The fish were not fed during testing. Upon termination of the test, fifteen fish, chosen randomly, were weighed and measured. 2.4 STATISTICAL ANALYSIS At the conclusion of the test, the LC50 was determined. 2