should be taken to insure that the test will be 

 conducted under the least favorable environmental 

 conditions that are allowable in the natural en- 

 vironment. Tests should be conducted at water 

 temperatures typical of the mean of maximum 

 daily temperatures during critical periods at the 

 proposed effluent discharge site. 



Test organisms should be selected either on the 

 basis of their economic importance in the area 

 receiving the discharge and their sensitivity or on 

 the basis of their importance in the food web of 

 economically important animals. In the event that 

 organisms meeting these criteria are not suitable 

 or available for the confined conditions of the tests, 

 substitute forms endemic to the area may be uti- 

 lized. Appropriate tests must be undertaken to 

 demonstrate the relative sensitivity of economi- 

 cally important species and substitute species to the 

 test material so that meaningful interpretations of 

 the data can be made. 



Application Factor: It is recognized that the 

 most obviously deleterious effect of toxic sub- 

 stances is increased mortality. More subtle changes 

 such as reduced growth, lowered fecundity, altered 

 physiology, and induced abnormal behavior pat- 

 terns may have more disastrous effects on the 

 continued existence of the species. Evaluations of 

 such sublethal effects generally will provide more 

 meaningful guidelines. 



It is recognized that there should be an applica- 

 tion factor for each waste or material and that 

 these factors may vary widely for these different 

 wastes and materials. The concept and use of ap- 

 plication factors is defined and discussed at length 

 in the toxicity portion of the section on water 

 quality requirements for fresh water organisms. 

 Due to a lack of knowledge of application factors 

 for specific wastes and materials, a single applica- 

 tion factor to be applied to all wastes is being sug- 

 gested at this time. This application factor may 

 require a lower concentration than is necessary in 

 some instances, particularly for those materials 

 that are subject to biological degradation, but it is 

 known that it is not restrictive enough for some 

 materials. Ideally, the determination of application 

 factors should be the result of studies for the de- 

 termination of safe levels of potential toxicants 

 under long-term or continuous exposure. The ap- 

 plication factor is the concentration of a material 

 or waste that is not harmful, divided by the 96- 

 hour TLn, value for that material. A few applica- 

 tion factors have been so determined at the Bureau 

 of Commercial Fisheries Laboratory at Gulf 

 Breeze, Fla. (unpublished data). In the future, as 

 application factors are determined for specific sub- 

 stances, they will replace the recommendation for 



the generalized application factor for these particu- 

 lar materials or wastes. It is clearly understood that 

 as additional data become available recommenda- 

 tions on water quality requirements will be 

 changed so that they conform with the new 

 knowledge. 



Biological Magnification: Biological magnifi- 

 cation is an additional chronic effect of toxic 

 pollutants (such as heavy metals, pesticides, radio- 

 nuclides, bacteria, and viruses) which must be rec- 

 ognized and examined before clearance can be 

 given for the disposal of a waste product into na- 

 tural waters. Many animals, and especially shellfish 

 such as the oyster, have the ability to remove from 

 the environment and store in their tissues sub- 

 stances present at nontoxic levels in the surround- 

 ing water. This process may continue in the oyster 

 or fish, for example, until the body burden of the 

 toxicant reaches such levels that the animal's 

 death would result if the pollutant were released 

 into the bloodstream by physiological activity. This 

 may occur, as in the case of chlorinated hydrocar- 

 bon pesticides (such as DDT and endrin) stored 

 in fat depots, when the animals food supply is re- 

 stricted and the body fat is mobilized. The appear- 

 ance of the toxicant in the bloodstream causes the 

 death of the animal. Equally disastrous is the 

 mobilization of body fat to form sex products 

 which may contain sufficiently high levels of the 

 pollutant so that normal development of the young 

 is impossible. 



The biological magnification and storage of 

 toxic residues of polluting substances and micro- 

 organisms may have another serious after effect. 

 Herbivorous and carnivorous fish at lower trophic 

 stages may gradually build up DDT residues of 

 1 5 to 20 mg/1 without apparent ill effect. Carniv- 

 orous fish, mammals, and birds preying on these 

 contaminated fish may be killed immediately or 

 suffer irreparable damage because of the pesticide 

 residue or infectious agent. 



In the final analysis, laboratory tests alone are 

 not sufficient to assess completely the toxic effects 

 of a substance. These data must be interpreted in 

 combination with field observations. Criteria es- 

 tablished under the artificial conditions of labora- 

 tory tests will probably require adjustment in 

 the light of later and more prolonged field 

 observations. 



Recommendation : In the absence of toxicity data other 

 than the 96-hour TL„,, an arbitrary application factor 

 of Vioo of this amount shall be used as the criterion of 

 permissible levels. 



Additional chronic exposure tests will be conducted 

 within a reasonable period to demonstrate that the 

 estimated maximum safe levels as indicated by the 



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