Biology Reference
In-Depth Information
L
.
majuscula
has been possible by the application of such physicochemical techniques. The availability
of a number of molecular markers has enabled the identifi cation of not only the toxic bloom algae
from laboratory cultures but also from natural bloom samples to the species level.
1) Biological methods
:
(i)
Bioassays
:
The most widely used conventional assay for determining the
toxicity of cyanobacterial toxins is the mouse bioassay. The toxins are administered i.p. or orally to
the inbred lines of mouse and the concentration of the toxin lethal to 50% of the population (LD
50
) is
calculated. In general, the toxicity of bloom material or extracts is tested and by inferring the toxic
response, the identity of the class of toxin can be determined. Though this method is the offi cially
approved method by the Association of Offi cial Analytical Chemists (under EC Law) for determining
marine STXs, it has certain limitations. The foremost is it is unethical and secondly this bioassay
is only a qualitative test for detecting the presence or absence of a toxin but does not help in the
chemical identity of the toxin. The MCs and nodularins cause death within 4 h with symptoms of
liver haemorrhage. The neurotoxins cause death more quickly without any apparent tissue damage,
i.e. within 15 minutes (Falconer, 1993). There are a number of limitations, i.e. (i) since this assay can
potentially be calibrated against MC-LR, the toxicity of any toxin can only be expressed in terms
of MC-LR equivalents, (ii) this test does not have the sensitivity to be applicable to water samples
and (iii) water samples with 1-2 µg L
-1
MC-LR cannot be used directly as it requires a considerable
concentration of the sample. This is exemplifi ed by the fact that for MC-LR with LD
50
of 50 µg
kg
-1
, the lethal dose is about 1 µg for a 20 gm mouse which requires 1 L sample to be administered
intraperitoneously which is not practicable.
A number of other bioassays have been proposed from time to time. Though some of these
proved to be sensitive, these are neither specifi c to MCs nor applicable for measuring the levels
of toxins in waters. Some of these are Microtox test involving luminescent bacteria (Lawton
et al
.,
1990; Volterra
et al
., 1992),
Artemia
bioassay (Kiviranta
et al
., 1991; Campbell
et al
., 1994; Lahti
et al
.,
1995), assay with plant growth of
Sinapis alba
L. (Kos
et al
., 1995), assay with African locust (Hiripi
et al
., 1998), assay with larvae of crustacean
Thamnocephalus platyurus
(Törökné
et al
., 2000) and
toxicity test with crustacean
Daphnia
(Kyselkova and Marsalek, 2000). Cyanobacterial toxicity was
assessed, using the crude extracts obtained from bloom samples dominated by
Microcystis
spp., by
comparing the sensitivity of 17 acute bioassays. The bioassay employing the crustacean
T
.
platyurus
was highly sensitive followed by those of
Drosophila melanogaster
, protozoans
Spirostomum ambiguum
and
Tetrahymena termophyla
and the crustacean
Daphnia pulex
(Marsalek and Blaha, 2004).
As more sophisticated alternative
in vitro
biochemical methods have been developed, these have
gained more prominence over mouse bioassay. For example, the inhibition of protein phosphatases
(for the assay of MCs and nodularins), acetylcholinesterase [for the assay of anatoxin-a (s)] and
protein synthesis (for the assay of CYN) have been developed. For the detection and quantifi cation
of STXs, STX-binding assays have been put to use. These methods proved to be highly sensitive,
reproducible and can be performed at ease.
ii) Immunoassays
:
Another important
in vitro
test system is immunoassay. The cyanobacterial
toxins do not possess antigencity, i.e. these do not act as antigens to evoke an immune response for
generation of antibodies specifi c against them. Such molecules that do not show antigenicity are
known as heptens. That is why these are linked to other larger molecules to make a complex antigen
that can generate the formation of specifi c antibodies. The immunoassay provides two advantages,
i.e. this method is highly specifi c, sensitive and can detect the presence of toxin as low as 1 µg l
-1
of
MC-LR (a concentration considered as a guideline value according to WHO). However, the cross-
