toxicants with which certain concentrations per- 

 mitted indefinite survival and normal appearance 

 but blocked spawning completely. Such evidence 

 makes estimates of safe concentrations based on 

 acute lethal test data alone very difficult and fre- 

 quently erroneous. Equally problematical is the 

 near-total lack of information on the sensitivity 

 of the various life stages of organisms. Many or- 

 ganisms are the most sensitive in the larval, 

 nymph, molting, or fry state; some may be the 

 most sensitive in the egg and sperm stage. 



A further difficulty is encountered in recom- 

 mending criteria because continuous acceptable 

 concentrations must be lower than the intermittent 

 concentrations that may be reached occasionally 

 without causing damage. There seems to be only 

 one way in which to resolve this difficulty and 

 that is to use both maximum concentration and a 

 range of concentrations. It is recognized that the 

 extremes do limit organisms, but, within these ex- 

 tremes there is a range in concentration that can 

 be tolerated and is safe for prolonged periods of 

 exposure. 



Average 24-hour concentrations can be deter- 

 mined by using a small water pump to collect 1 to 

 5 ml samples every minute. After 24 hours, the 

 sample is mixed and analyzed. The concentration 

 found represents the average concentration. Sam- 

 ples obtained this way are more reproducible and 

 easier to secure than the maximum instantaneous 

 concentration. Maximum concentrations must be 

 considered in the criteria, however, because an 

 average concentration alone could be met and yet 

 permit a lethal concentration to exist for a critical 

 period. 



Bioassay 



The use of some type of bioassay to determine 

 the toxicity of a material or waste can be the most 

 effective and accurate method of assessing poten- 

 tial danger. With these methods, no assumptions 

 need be made concerning the chemical structure 

 or form of the pollutant, nor does the investigator 

 have to know the constituent substances. The ef- 

 fects of water quality on toxicity also may be 

 measured. Naturally, the more that is known about 

 the chemical and physical behavior of a toxicant 

 in water, the more precise the assay can be. 



While there are many types of assays, two 

 are in general use: ( 1 ) the static bioassay in which 

 the test solution is not changed during the period 

 of exposure, and (2) the flow-through bioassay 

 in which the test solution continually is renewed. 

 It is nearly impossible in a static test to use the 

 introduced test concentration for calculating TLn, 

 values, especially for substances or wastes that are 



toxic at concentrations of 1 mg/1 or less, because 

 the quantity taken into the test organism may be 

 a very large percentage of the amount contained 

 in the test water. A 48-hour TLm based on the 

 introduced concentration could give a value much 

 higher than the true concentration because of this 

 decrease in toxicant concentration. The initial test 

 concentration is usually not measured in static 

 tests because of the changing concentration. 

 Knowledge of the concentration of the toxicant 

 at the end of the test can be of value. 



The static test can give useful relative measures 

 of toxicity for wastes of high toxicity, but for the 

 reason mentioned above, it should not be used 

 for absolute values. Less toxic substances can -be 

 assayed much more accurately and lethal concen- 

 trations can be determined with confidence. The 

 chemical nature of the tested substances has an 

 important effect on the accuracy of the results 

 as well. Substances that are volatile, unstable, or 

 relatively insoluble may not be accurately assayed 

 whUe substances having opposite properties can 

 be assayed more accurately. 



The problem of maintaining oxygen concen- 

 trations suitable for aquatic life in the test chamber 

 can be very difficult. Insufficient oxygen may be 

 present in the test water volume because a BOD 

 or COD may consume much of the available dis- 

 solved oxygen and aeration or oxygenation may 

 degrade or remove the test material. Devices for 

 maintaining satisfactory dissolved oxygen in static 

 tests have been proposed and used with some de- 

 gree of effectiveness. A rather complete account of 

 static assays can be found in Sandard Methods 

 for the Examination of Water , and Wastewater, 

 12th edition (1965), and Doudoroff, et al. 

 (1951). 



In the flow-through type of bioassay a device 

 is used to add toxicant to a flow of water and the 

 mixture is discharged into the test container. This 

 method of testing has few of the problems men- 

 tioned in connection with the static test and has 

 other advantages in addition. Its important dis- 

 advantage is the more complicated work of build- 

 ing the necessary equipment; namely, a water 

 supply system, metering devices, and the provision 

 of a large quantity of the test substance. 



Its important advantages are that a predeter- 

 mined concentration of test material can be main- 

 tained, oxygen concentrations can be kept high 

 or be controlled, metabolites and waste products 

 are removed (animals can be fed), absolute rather 

 than relative TL^ values can be obtained, and 

 volatile, unstable, and sparingly soluble materials 

 can be tested. Additionally, multifactor experi- 

 ments are possible in which several variables can 

 be controlled (pH, dissolved oxygen, carbon diox- 



57 



