The author explains some aspects of sampling phytoplankton blooms and the evaluation of results obtained from different methods. Qualitative and quantitative sampling is covered as well as filtration, freeze-drying and toxin separation

Lindholm, Tore

English

Aquatic Commons

SAMPLING TOXIC PHYTOPLANKTON 97 
THE MEANING OF SOME COMMON TERMS USED IN SAMPLING 
TOXIC PHYTOPLANKTON 
TORE LINDHOLM 
(Dr T. Lindholm, Department of Biology, Abo Akademi University, 
Biocity, SF'-20520 Abo, Finland.) 
Introduction 
Newspapers, radio and television keep us informed about the sudden 
appearance of phytoplankton blooms, consisting of cyanobacteria (blue-
green algae) and other microalgae, emphasising their harmful effects and 
the need for care and vigilance. Biologists who are directly involved in 
the study of these blooms talk about phytoplankters with unfamiliar 
names and refer to toxic samples, toxicity tests and peculiar toxins. For 
those who have to respond to this information, but have little or no 
experience in field studies or work related to these nuisance organisms, it 
is often hard to understand not only the dimensions and distribution of 
blooms but also the kind and significance of the samples and tests being 
used. In this brief note I shall try to explain some aspects of sampling 
phytoplankton blooms and the evaluation of results obtained from 
different methods. However, I shall not attempt to cover the entire field of 
blooms and methods of sampling that are available. Rather, my 
comments reflect some of the problems which I have encountered during 
field work and in the laboratory, when dealing with the public and 
newspapers, and, of course, when discussing matters with colleagues 
who have a background different from mine. Hopefully, some of these 
comments will be of use to the general reader and, perhaps, stimulate 
further discussion. 
Dimensions and distribution of blooms 
Marine and freshwater phytoplankton communities may contain 
hundreds of species of microalgae (often called protists) and 
cyanobacteria. Some of these are abundant and dominant, numbering 
thousands of cells per millilitre of water, while others are relatively rare, 
perhaps less than ten cells per cubic metre of water. Some species tend to 
form surface or subsurface maxima, either actively or passively. 
Remarkable phytoplankton maxima, in which organisms are highly 
concentrated, are usually called blooms, and these are often almost 
monospecific. They may form very local discoloured patches occupying a 
98 T. LINDHOLM 
few square metres of a lake, frequently accumulating in sheltered bays 
along the shore, but they can extend over huge areas of oceans, covering 
hundreds of kilometres. Some blooms, notably the surface scums formed 
by cyanobacteria (formerly called blue-green algae), may suddenly 
appear for brief periods lasting for only a few hours, but others develop 
and persist over long periods of weeks and months. Some bloom events 
are recurrent and predictable, but most are episodic and their appearance 
may even depend on exceptional weather conditions. 
Frequently one or several species in a bloom may produce substances 
or effects which are harmful or toxic to some or most other organisms 
(including other kinds of algae). Examples are given in Table 1 and some 
were mentioned by Reynolds (1991). Examples concerning the 
occurrence of toxic cyanobacteria and toxins in lakes are also found in, 
for example, Lindholm et al. (1989), Watanabe et al. (1992), and Kotak et 
al. (1993). Examples of marine phytoplankton blooms and shellfish 
poisoning events are reviewed by Smayda (1990), Shumway (1990) and 
Todd (1993). Carmichael (1994) summarises toxins and their effects. 
Most phytoplankton species are grazed upon by one or several 
zooplankton animals, which may migrate vertically and often occur in 
swarms or distinct layers. Therefore, when discrete samples of water 
and/or plankton are taken at a depth of about 1 metre, as frequently 
happens, they may or may not be entirely representative of the local 
SAMPLING TOXIC PHYTOPLANKTON 99 
water mass. Occasionally a considerable amount of the total 
phytoplankton biomass and amount of toxin may be contained in the guts 
of fast-swimming zooplankters, which easily escape the sampler. Below I 
will briefly mention some of the different kinds of samples that are often 
encountered in work on toxic phytoplankton. 
Qualitative samples of organisms (dip and net samples) 
Samples taken by "dipping" with a container (e.g. bottle or bucket), or 
caught by sweeping the water with a net, may be highly selective and 
should not be considered as quantitative. A net with a large mesh size 
(mesh size larger than ca. 50 urn) will of course collect the large and 
robust species. A fine net (ca. 25 urn) retains a broader spectrum of 
organisms but fragile ones may be missing. In most cases net sampling 
fails to catch the picoplankton and nanoplankton (smaller than 20 |im by 
definition). Even some filamentous, gliding cyanobacteria like 
Planktothrix agardhii easily pass through the net. However, a net drawn 
through a large volume of water may provide relatively large amounts of 
the less abundant (and presumably less important) organisms which are 
seldom seen in discrete samples. As all net samples are extremely 
concentrated, the organisms caught in them are frequently damaged, and 
they usually deteriorate in a matter of hours. Such samples are usually 
taken for provisional inspection and identification of the main organisms 
present. Extracts of freeze-dried net samples may be used for toxicity tests 
with mouse bioassays and, for example, for the purification of toxins to be 
used as toxin standards. 
Quantitative (discrete) samples of water and plankton in blooms 
Water samples are usually obtained by filling bottles of various shapes 
and sizes, including sampling devices that have been specially developed 
for opening and shutting at various defined depths. A single sample of 
water and the organisms in it is usually called a "quantitative sample" 
because the results of chemical analyses and counts of organisms in the 
sample are expressed as quantities per volume, usually a litre of water. 
Nevertheless, strictly speaking a single sample is only a semi-quantitative 
or relative estimate (orders of magnitude) of a population occupying a 
column of water (Table 2). Fully quantitative sampling (e.g. Heaney 1976; 
Irish 1980) is rarely undertaken, as this involves taking a series of 
replicated samples (of known volume) both across and down through the 
water column containing the bloom (Table 2). 
As stated above, a single "quantitative" sample is unlikely to be truly 
representative of any but the smallest bloom. Even if a toxic species is 
100 T. L1NDHOLM 
present in the water, the amount of toxin collected in a single sample is 
likely to be relatively low, unless the water is very strongly "green" in 
colour, due to very large numbers of algal cells. Therefore it may be 
necessary to collect large volumes of water in order to concentrate the 
toxins for chemical analysis and/or toxicity tests. 
"Clean" water samples can usually be stored for several days for species 
identification if they are kept cool. Cysts (resting stages) can sometimes 
survive for months in cold conditions and may be of use for identifying 
some toxic species. As most phytoplankton samples change or are spoiled 
in a relatively short time, it is preferable and wise to take subsamples 
immediately in the field, and preserve subsamples with a fixative such as 
Lugol's iodine solution (e.g. Wetzel & Likens 1979). 
SAMPtINC TOXIC PHYTOPLANKTON 101 
Although the results from semi-quantitative samples are expressed as 
amounts (concentrations) or numbers of organisms per litre, it should be 
remembered that nevertheless they are really estimates of relative 
abundance (orders of magnitude). This is often sufficient for most 
investigations on phytoplankton blooms, because the organisms and 
chemical components associated with them are present in large amounts 
and these probably fluctuate within quite wide limits in both space 
(especially in windy conditions in lakes) and short periods of time (hours). 
Filtration of samples in the laboratory 
Various filters can be used for semi-quantitative sampling of 
phytoplankton as, for example, in the case of routine analysis of amounts 
or concentrations of chlorophyll-a. Material collected on the filters can 
easily be stored deep-frozen and also be used for various chemical 
analyses. As long as the sampling and subsampling routines are 
appropriate and the filtered volume is known, material on the filters may 
be used to estimate the quantities of toxins present in the biomass of 
phytoplankton (toxins remaining in cells). 
The filtrate, i.e. the water passing through the filter, contains excreted 
and dissolved substances (and fine material from broken cells). Free 
toxins in solution may be of interest, e.g. in drinking water treatment 
plants and in laboratories which use filtered sea-water or lake-water for 
experiments. Usually, rather low concentrations of toxins are likely to 
occur in filtered water samples, and they may have to be concentrated for 
analysis. 
Concentrating samples by freeze-drying 
Samples and/or subsamples may be concentrated in the laboratory by 
freeze-drying. Freeze-dried water samples may be used for toxicity tests. 
Large volumes of water collected for freeze-drying may contain repres-
entatives of all or most of the organisms and debris in the water column, 
but they are rarely "quantitative" samples. Freeze-dried material is, 
however, easy to store and useful for provisional screening for toxins and 
for certain chemical analysis. Freeze-dried samples of phytoplankton 
blooms that have been collected with a net are usually much more select-
ive than freeze-dried samples of water (some toxins may even be absent). 
Samples from algal cultures in the laboratory 
Cultures of algae are usually used for research. Samples taken on different 
days from cultures and filtered, freeze-dried and homogenized are much 
more homogenous in content than natural water or bloom samples, but 
102 T. LINDHOLM 
nevertheless they can still be quite hard to precisely define. They may 
represent one or several strains and be in different growth phases. Axenic 
(sterile) cultures are monospecific and are not contaminated with bacteria. 
Toxins 
Certain organisms manufacture organic substances that are toxic to others 
(Table 1). The toxins are either excreted into the surrounding water or 
stored within the cell and released when it dies and decomposes. It is 
very difficult to distinguish toxins that are already present in the water 
from those held in the cells, because of difficulties in separating water 
from cells without damaging the latter. Usually the toxins present in a 
natural bloom are studied by extracting them from the algal cells with 
organic solvents such as methanol. Crude extracts are sufficiently pure for 
certain purposes (tests). The extract may contain either a fraction of a 
particular toxin or, in some cases, most of the toxin(s) present in, for 
example, a freeze-dried algal sample. 
What is a "pure" toxin? 
Separation of a certain toxin, which is present in a crude extract, may be 
done with various organic solvents and, for example, HPLC methods 
(high performance liquid chromatography). Pure toxins are useful tools for 
studying the chemical structures of toxins and their physiological effects 
at the cellular and organismic levels. For pure toxins it is relevant to talk 
about toxicity, if properly tested and the solvent used does not influence 
toxicity. Many of the toxins which are produced by phytoplankton are in 
fact supertoxic, with toxicities of about 100 u.g per kg body weight in 
mouse assays, i.e. a dose of a few micrograms is enough to kill a mouse 
(usually intraperitoneal injection or i.p. is applied). 
Final comments 
A sample from a toxic bloom may, in fact, consist of anything between a 
bucket of water containing a large diversity of organisms, and a white 
powder of purified toxin. As different kinds of samples can have very 
different properties, be more or less concentrated, and be more or less 
representative, it is urgent that adequate descriptions of sampling pro-
cedures and treatments are provided. Some readers of a report may have 
to judge whether the toxic sample originated from a trivial, extremely 
local bloom or whether there is a real threat to the health of people or 
domestic animals. A single "positive" sample (perhaps even "amplified" 
during weeks in a culture medium), does not necessarily mean that a large 
lake must be closed! On the other hand, it is urgent to detect, respond to 
and inform others about toxic algal blooms that may be harmful. 
SAMPLING TOXIC PHYTOPLANKTON 103 
The testing of toxicity and the units and vocabulary involved require a 
separate article to cover them adequately. This also applies to the 
complex topic of exposure to toxins in the field, which is difficult to 
evaluate and quantify. For example, a water-bird may swim in the middle 
of a toxic bloom without being lethally exposed to toxins in the water, but 
it may receive a lethal dose of toxins via food items like fish - even miles 
away from the toxic bloom which originally contaminated the fish. 
Acknowledgements 
I wish to thank a referee and Dr D. W. Sutcliffe for kind assistance in 
preparing this article. 
References 
Carmichael, W. W. (1994). The toxins of cyanobacteria. Scientific American, 
(January), 64 -74. 
Heaney, S. I. (1976). Temporal and spatial distribution of the dinoflagellate 
Ceratium hirunclinella O. F. Miiller within a small productive lake. Freshwater 
Biology, 6, 531-542. 
Irish, A. E. (1980). A modified one-metre Friedinger sampler: a description and 
some selected results. Freshwater Biology, 10, 135-139. 
Kotak, B. C, Kenefick, S. L, Fritz, D. L, Rousseaux, C. C, Prepas, E. E. & Hrudey, 
S. E. (1993). Occurrence and toxicological evaluation of cyanobacterial toxins in 
Alberta lakes and farm dugouts. Water Research, 27, 495-506. 
Lindholm, T., Eriksson, J. E. & Meriluoto, J. A. O. (1989). Toxic cyanobacteria and 
water quality problems - examples from a eutrophic lake on Aland, south west 
Finland. Water Research, 23, 481 -486. 
Reynolds, C. S. (1991). Toxic blue-green algae: the "problem" in perspective. 
Freshwater Forum, 1, 29-38. 
Shumway, S. E. (1990). A review of effects of algal blooms on shellfish and 
aquacukure. Journal of the WorldAquaculture Society, 21, 65-104. 
Smayda, T. J. (1990). Novel and nuisance phytoplankton blooms in the seas: 
evidence for a global epidemic. In Proceedings of the Fourth International 
Conference on Toxic Marine Phytoplankton, held June 26-30, 1989, in Lund, 
Sweden (eds E. Graneli, B. Sundstrom, L. Edler & D. M. Anderson), pp. 29-40. 
Elsevier, New York. 
Todd, E. C. D. (1993). Domoic acid and amnesic shellfish poisoning- a review. 
journal of Food Protection, 56, 69-83. 
Watanabe, M. M., Kaya, K. & Takamura, N. (1992). Fate of toxic cyclic 
heptapeptides, the microcystins, from blooms of Microcystis (cyanobacteria) in a 
hypertrophic lake. Journal of Phycology, 28, 761 -767. 
Wetzel, R. G. & Likens, G. E. (1979). Limnological Analyses. 357 pp. Saunders, 
Philadelphia. 


The meaning of some common terms used in sampling toxic phytoplankton

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The meaning of some common terms used in sampling toxic phytoplankton

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