Biological field and laboratory methods for measuring the quality of ...

The maximum standing crop in any flask is
defined as the maximum algal biomass achieved
during incubation. For practical purposes, the
maximum standing crop is assumed to have been
achieved when the rate of increase in biomass
has declined to less than 5 percent per day.
2.8 Additions (Spikes)
The quantity of cells produced in a given
medium is limited by the nutrient present in the
lowest relative quantity with respect to the
needs of the organism. If a quantity of the
limiting substance were added to the test flasks,
cell production would increase until this additional
supply was depleted or until some other
substance became limiting to the organism.
Adding substances other than the limiting substance
would not increase algal growth. Nutrient
additions may be made singly or in combination,
and the growth response can be compared with
that of unspiked controls to identify those substances
that limit growth rate or cell production.
In all instances, the volume of a spike should
be as small as possible. The concentration of
spikes will vary and must be matched to the
waters being tested. When selecting the spike
concentration, keep in mind that (l) the concentration
should be kept small to minimize
alterations of the sample, but at the same time,
be sufficiently large to yield a potentially
measureable response; and (2) the concentration
should be related to the fertility of the sample.
2.9 Data Analysis and Interpretation
Present the results of spiking assays together
with the results from two types of reference
samples: the assay reference medium and unspiked
samples of the water under consideration.
Preferably, the entire growth curves should be
presented for each of the two types of reference
samples. Present the results of individual assays
in the form of the maximum specific growth
rate (with time of occurrence) and maximum
standing crop (with time at which it was
reached), both with the confidence interval
indicated.
Growth rate limiting nutrients can be determined
by spiking a number of replicate flasks
with single nutrients, determining the maximum
5
ALGAL ASSAY
specific growth rate for each flask, and comparing
the averages by a Students' t-test or other
appropriate statistical tests.
Data analysis for multiple nutrient spiking can
be performed by analysis of variance calculations.
In multiple nutrient spiking, accounting
for the possible interaction between different
nutrients is important and can readily be done
by factorial analysis. The same methods described
above can be used to determine the
nutrient limiting growth of the maximum
standing crop.
2.10 Assays to Determine Toxicity
As previously pointed out, the assay may be
used to determine whether or not various compounds
or water samples are either toxic or
inhibitory to algal growth. In this case the substance
to be tested for toxicity is added to the
standard algal culture medium in varying concentrations,
the algal test species is added, and
either the maximum standing crop or maximum
specific growth rate (or both) determined. These
are then compared to those obtained in the
standard culture medium without the additions
(controls). The LCSO, or that concentration at
which either 50% of the maximum standing crop
or maximum specific growth rate is obtained, as
compared with the controls, is then calculated.
3.0 PERIPHYTON
Uniform methods for conducting bioassays
with periphyton have not been developed, and
their environmental requirements and toxicology
are still relatively unknown. Many of the
common species have not been successfully
cultured, and the bioassays that have been
carried out with the algae and other microorganisms
occurring in this community were
conducted principally to screen potential
algicides, fungicides, and other control agents.
Two kinds of tests can be conducted with
periphyton: static and continuous flow.
3.1 Static
Because the techniques currently employed in
the Algal Assay Procedure: Bottle Test (USEPA,
1971) have been more rigorously tested than
any procedure previously used for periphyton,