Chronic tests are always definitive (multi-concentration) tests. Typically 6
different treatments are specified -- a series of five dilutions of the effluent, and a
dilution water control. For example, the dilutions specified in a permit may be
100%, 50%, 25%, 12.5%, and 6.25%. Normally, the required age of the organisms at the
beginning of chronic tests is usually less than 24-hours old. However, if fecundity of
mysid shrimp is to be observed, the required age for mysid shrimp at test initiation is 7
days.
For chronic tests involving cladocerans (Ceriodaphnia dubia), a minimum of 10
replicates are required for each treatment. Each replicate contains one test
organism, and its survival and the total number of neonates it produces are monitored
daily over the duration of the test. The minimum test volume of each replicate in a
cladoceran test is 15 ml. At least 80% of the organisms in the controls must
survive and produce three broods for the test to be considered valid.
For tests involving fish or mysid shrimp, a minimum
of 4 or 5 replicates are required for each treatment. Each replicate must contain either
8 or 10 organisms for a total of 40 organisms per treatment. The minimum
volume of each replicate is usually 250 ml. Growth is measured at the end of the 7-day
period by carefully drying and measuring the total weight of the organisms in each
replicate. At least 80% of the organisms must survive in the controls and attain a
specified weight in order for a chronic test to be considered valid.
