SECT. 2] BIOASSAY OF TRACE SUBSTANCES 221 



parameters of two areas, and found them almost identical. Yet, the invertebrate 

 populations with which he worked could distinguish between them. If the sand 

 from these two areas was cleaned with hot sulfuric acid to remove organic 

 materials, the organisms could no longer distinguish between them. He attri- 

 buted the ability of the invertebrate larvae to distinguish between the two 

 areas to an organic film (non-living) which coated the sand in one area, while 

 the other area was relatively clean. Clearly, then, living organisms are able to 

 distinguish differences in environments which our tests do not show. It follows 

 that, if we can find out what substances the organisms respond to, we can then 

 use the organisms to test for the presence of specific materials. This is the basis 

 for the bioassay technique. 



The bioassay technique for organic materials in sea-water enjoys certain 

 advantages over conventional chemical tests. This latter approach has been 

 used by Johnston (1955) with some success, but there are certain basic problems 

 involved which make this approach impractical. Many of the standard methods 

 require processing of large quantities of sea-water to recover the trace sub- 

 stances in sufficient concentration for analysis. One commonly used method 

 for the extraction of organic materials from sea-water involves adsorption on 

 activated charcoal which selectively removes 65 to 100% of the total soluble 

 carbon-containing materials from the water (Jeffrey and Hood, 1960). The 

 removal of the organic matter may be accomplished by eluting with a variety 

 of organic solvents. Either cold or hot extractions may alter some of the organic 

 compounds ; if a recycled hot-extraction method is used, to obtain quantitative 

 recovery, one might reasonably expect considerable alteration of many of the 

 trace organic materials. Following the elution step, the organic solvents are 

 generally removed by cold evaporation under vacuum. This probably introduces 

 no further changes in the organic products, but quite a bit of inorganic salt 

 may remain after evaporation is complete. Depending upon the analytical tests 

 to be run and the complexity of the mixture of organic materials, it may be 

 desirable to remove these salts. This can be accomplished by dissolving the 

 extract in distilled water and dialyzing against flowing distilled water. The in- 

 organic salts migrate through the dialysis tubing while the organic materials 

 are retained. If such a step is not carried out, chromotography and electro- 

 phoresis, two common methods for resolving individual compounds from a 

 complex mixture, yield often indeterminate results. 



As an example of the quantitative aspect of this problem of isolation and 

 identification, Daisley and Fisher (1958) have reported samples of sea -water to 

 contain 0.03 \iy.g vitamin Bi 2 per ml. For the extraction technique, if one 

 processed 50 1. of such a water sample with 1C0% recovery, the yield of vitamin 

 B12 would be about 15 m[xg. This is a small quantity indeed to attempt to 

 characterize chemically. Yet Euglena gracilis, the bioassay organism, is capable 

 of detecting one-fiftieth of that quantity, with no extraction or concentration 

 required. 



As has been previously stated, the "reagent" for detecting chemical 

 substances in the bioassay is a living organism. An example of a typical bioassay 



