Sampling, Biomass Estimation and Counts of Freshwater Algae - Freshwater Algae: Identification, Enumeration and Use as Bioindicators - page 60

(1975). Total carotenoids can be estimated by mea-
suringtheextinctioncoeficientat480nm(Strickland
and Parsons, 1972).

contribution of each algal group is particularly use-
ful since it partitions the known total chlorophyll-
a (overall phytoplankton biomass) into the separate
phytoplankton groups (individual biomass determi-
nations).
Although there are some quantitative limitations
with this method - including variation in carotenoid
ratios within groups, lack of absolute phylum-
specificity and co-elution of some pigments (e.g.
lutein and zeaxanthin) during HPLC separation -
HPLC has been widely used in phytoplankton deter-
minations. Various studies, using corroborative stan-
dard algal count procedures, have demonstrated the
validity of using CHEMTAX ® in assessing major
taxonomic groups with mixed phytoplankton sam-
ples (Paerl et al ., 2005).
Numerous studies have also shown that examin-
ing phytoplankton community dynamics and succes-
sional changes at the phylum (rather than species)
level often provides excellent insight into environ-
mental changes. HPLC studies on the effects of
estuarine eutrophication, for example, have shown
clear alterations in taxonomic composition during
the major phase of aquatic productivity and also in
changes to seasonal succession (see Section 3.5.2).
In a eutrophication-related study on marine phyto-
plankton, Goericke (1998) demonstrated that algal
biomass in the Sargasso Sea varied over the annual
cycle - probably in relation to nutrient availability.
These overall biomass changes did not relate to any
alteration in the composition of the eukaryote algal
community or to the relative biomass of blue-green
algae, as determined by HPLC analysis.

High-performance liquid chromatography
HPLC analysis of phytoplankton samples provides
a useful supplement to the use of algal counts. The
main limitation of HPLC is that it cannot determine
individual algal species, but instead classifies the
phytoplankton sample in terms of major taxonomic
groups (phyla). The main advantage is that it can be
used to analyse a large number of samples over an
extended aquatic area, and has been employed par-
ticularly with marine (Goericke, 1998) and estuarine
investigations. Paerl et al . (2005), for example, used
this approach to monitor freshwater algae as bioindi-
cators of eutrophication in various estuary systems of
the Southern United States (see Section 3.5.2).

Taxonomiccarotenoids HPLC,coupledwithphoto-
diode array spectrophotometry (PDAS), can be used
for chlorophyll and carotenoid analysis. The identity
of pigments within the sample is established by com-
parison with elution times of pigments isolated from
reference material. Taxonomic-specific carotenoids
(alloxanthin, fucoxanthin, peridinin and zeaxanthin)
are particularly useful as diagnostic biomarkers for
determining the relative abundance of different algal
groups (Fig. 2.11). Of these carotenoids, only peri-
dinin and alloxanthin are specific to a single phylum
(plus violaxanthin - used by some workers as a spe-
cific marker for green algae).

Quantitation Absolute and relative abundances of
major algal groups can be calculated using a matrix
factorisation programme such as CHEMTAX ® (Paerl
et al ., 2005). Using this programme, input consists of
a raw data matrix of phytopigment concentrations
obtained from HPLC analyses, plus an initial pig-
ment ratio file (standard ratios of secondary pigments
to chlorophyll- a in different algal groups). The data
matrix is subjected to a factor minimisation algorithm
that calculated a best-fit pigment ratio matrix and a
final phytoplankton group composition matrix. Rela-
tively large errors in the initial estimates of pigment
ratios have little influence on the final determination
of algal group abundances. The relative chlorophyll- a

2.4 Flow cytometry: automated
analysis of phytoplankton
populations

Analysis of total algal biomass or biomass of major
taxonomic groups gives little detailed information
about the composition of algal populations in rela-
tiontotaxonomiccomposition(speciesanalysis),size
range or numbers of individual algal units (cells or
colonies).