containers by 1/4 -inch copper tubing. Air pressure 

 in the distribution lines was controlled by an adjust - 

 able diaphragm -type regulator. An outlet, with a 

 brass stopcock, was provided for each test jar. Vinyl 

 plastic tubing in 34 -inch lengths and equipped with 

 stone aerators conveyed air to the test containers. 



The electrical power for the bioassay trailer was 

 furnished by a 5-K. V. A. , 115-volt generator mount- 

 ed on the bed of a 4- by 8 -foot steel trailer (fig. 2). 

 Six 100 -pound tanks of propane carried on the same 

 trailer provided fuel for the generator. The genera- 

 tor trailer also provided transportation for intake and 

 discharge hoses used in the water system and eight 

 10 -gallon milk cans for holding larvae and fish. 



BIOASSAY PROCEDURE 



Groups of four tests were run at each concentra- 

 tion tested. Thus the 44 places provided for bioas- 

 say at 11 different concentrations. Test jars were 

 7. 5-liter polyethylene containers. Use of this type 

 of jar prevented breakage, facilitated storage, and 

 eliminated the problem of keeping glassware clean 

 during field operations. Disposable polyethylene 

 plastic bags were used as liners to prevent chemical 

 contamination of the containers (fig. 3). Five liters 

 of the stream water to be tested were placed in each 

 test jar. The upper portion of the polyethylene bag 

 was then folded over the edge and down the outside. 

 Test concentrations were obtained by adding a 1: 200 

 stock solution of TFM, based on weight of active in- 

 gredient, in distilled water. One milliliter of the 

 stock was added to the 5 liters of stream water for 

 each part per million (p. p. m. ) desired in the test. 



Water circulating through the troughs kept the 

 test solutions near the temperature of the stream. 

 Thus the bioassay information was obtained under 

 the same varying water temperatures that would nor- 

 mally occur during a stream treatment. A single - 

 pen recording thermograph provided a continuous 

 record of water temperatures during a bioassay. 



Test positions were numbered consecutively 

 from 1 to 44; the odd -numbered positions were along 

 the near sides of the troughs and the even -numbered 

 positions along the wall. Each bioassay observation 

 card was stamped with the same identifying number 

 as the test container. 



TEST ANIMALS 



Lamprey larvae, 2 to 4 inches long, and finger - 

 ling rainbow trout of approximately the same size 

 proved to be well suited to the test containers and to 

 the volume of test solution. Normally, two trout and 

 two larvae were placed in each jar; since the tests 

 were in quadruplicate, eight animals of each species 

 were subjected to each level of concentration. Occa- 

 sionally, when supplies of test animals were abundant, 

 three of each species were placed in a container. Con- 

 versely, when test specimens were in short supply, only 

 one of each species was used. 



Lamprey larvae were obtained from nearby streams 

 with a light portable electric shocker (Braem and Ebel, 

 1961). Larvae of the various lamprey species in waters 

 tributary to the U. S. shore of Lake Superior, includ- 

 ing those in process of transformation into the adult 

 stage, were used in bioassays. We have no evidence 

 that species of lamprey exhibit substantially different 

 susceptability to the larvicide. 



Holding facilities for larvae consisted of fine - 

 mesh screen cages with 3 to 6 inches of fine sand or 

 silt in the bottom to permit the larvae to bury them- 

 selves. These holding cages were placed in the stream. 

 Larvae could be collected at one time for four or five 

 bioassays and retained in good condition until needed. 

 Lamprey larvae obtained by shocking were allowed to 

 rest at least 24 hours before they were used as test 

 animals. 



The rainbow trout were provided by the States of 

 Michigan and Wisconsin. They were usually held in 

 the hatchery until needed for bioassay. Small rain- 

 bow trout could be held in a live car in the stream 

 for 4 or 5 days, however, without impairing their val- 

 ue as test animals. The fish were not fed during this 

 time, and they remained vigorous and healthy if not 

 too closely confined. 



Test animals that were transferred from one wa- 

 ter to another were tempered for one -half hour for each 

 5° F. difference in water temperatures. The test 

 specimens and air hoses were placed in each test jar 

 prior to the introduction of the chemical. All jars 

 were covered by a sheet of clear glass to prevent the 

 escape of fish and the accidental pollution of testsolu- 

 tions by foreign substances. Fish and larvae were left 

 undisturbed 1 hour to permit them to adapt to the test 

 environment. 



