Basic HTML Version
International Journal of Marine Science 2014, Vol.4, No.52, 1-9
http://ijms.biopublisher.ca
3
1.2 Bioassay
In the present study, a continuous flow-through
bioassay test system was used to find out the acute
toxic (LC
50
)
(USEPA, 2002) and cytotoxic effects of
heavy metals to
M. philippinarum
. The toxicant
concentrations selected for continuous flow-through
bioassay tests were similar concentrations that were
already used for conducting the static renewal
bioassay tests (Table 2) and the LC
50
values for
M.
philippinarum
derived through the definitive toxicity
tests were 0.023, 0.221, 2.876, 2.337 and 0.007 mg.
L
-1
in Cu, Cd, Pb, Zn and Hg, respectively
(Ramakritinan et al.,
2012).
Table 2 Concentrations of metals selected for conducting acute
continuous flow through bioassay tests for
M. philippinarum
Toxicant Used
Acute metal concentrations
(mg.L
-1
)
Copper
0.01, 0.02, 0.04, 0.08, 0.16
Lead
0.929, 1.858, 3.716, 7.432, 14.864
Cadmium
0.06, 0.12, 0.24, 0.48, 0.96
Zinc
1.015, 2.030, 4.061, 8.122, 16.444
Mercury
0.002, 0.004, 0.008, 0.017, 0.034
Copper sulphate [CuSO
4
.5H
2
O], lead nitrate [Pb(NO
3
)
2
],
cadmium chloride [CdCl
2
.5H
2
O], zinc chloride [ZnCl
2
]
and mercury chloride [HgCl
2
] (Merck India Ltd,
Mumbai) were used for conducting the heavy metal
toxicity studies. Stock solutions of heavy metals i.e.,
1000 mg.L
-1
were prepared by dissolving 3.9322 g of
CuSO
4
.5H
2
O, 1.598 g of Pb(NO
3
)
2
, 2.032 g of
CdCl
2
.5H
2
O, 2.641 g of ZnCl
2
and 1.354 g of HgCl
2
in 1000 ml Milli-Q-water in 1000 ml glass volumetric
flasks separately. This was treated as main stock
solutions (MSS). They were stored in a refrigerator
until use. From the MSS, sub-stock solutions (SSS) of
each metal was prepared daily and used for
preparation of required concentrations of toxicant.
Glassware used for storage stock of solutions had
previously been washed using 10% nitric acid and
well rinsed with distilled water.
A continuous flow-through bioassay test system was
constructed and the flow of seawater and toxicant
media were automatically controlled using
Microprocessor based ISMATEC Peristaltic Pumps
(Model 1256; USA). The operation of the flow
through system was as follows: a final flow rate of
20ml.min
-1
in each test tank (12 Channel pumps) was
maintained by an outflow of 40ml.min
-1
from the
mixing chamber (Palanikumar et al.,
2013). The
toxicant inflow of 4ml.min
-1
(8 channels pumps) and
seawater inflow of 36ml.min
-1
(8 channels pumps)
were maintained. If a test concentration of 1.0 mg.L
-1
was desired in a test tank, ten times higher
concentration was placed at top of the main toxicant
reservoir. This flow rate would ensure a 90%
replacement of water contained in a five litres test
tank in about ten hour. A five litres capacity toxicant
reservoir can be left to run automatically for 12-14h.
For toxicity study, the well-acclimated animals
were
introduced in five selected test concentrations of each
metal including control (Table 2). Two replicates were
maintained for each test concentration and also for
seawater control. Ten animals were exposed in each
test tank. During the period of bioassay, the organisms
were starved and the dead animals were removed
immediately from the medium (ASTM, 2007). The
mortality was observed during 24, 48, 72 and 96h and
the LC
50
values were analyzed at respective hours of
exposure (USEPA, 1994). Safe concentrations of the
metals were determined by following the application
factor 1/100
th
of the 96h LC
50
values (Kameswara Rao,
1974; Miller and Miller, 1986).
1.3 Micronucleus Assay
Similar to
acute toxicity studies, the well-acclimated
animals were introduced in five selected test
concentrations of each metal including seawater
control also for comparison (Table 2) (Ramakritinan et
al.,
2012). Two replicates were maintained for each
test concentration and also for seawater control. Ten
animals were exposed in each test tank under
continuous flow through bioassay test method for
micronuclei (MN) and binuclei (BN) analysis, The
medium was not aerated throughout the study and the
test animals were not offered food (Sprague, 1973).
After 96 h exposure, live mussels in each metal
concentration including control were collected and the
whole body tissues were removed and placed in a big
drop of 3:1 ethanol and acetic acid solution separately
on two clean microscopic slides. Since the length of
the test animal was 9-11 mm in size with 5-10 mg of
body tissues, the whole body was used for cytotoxicity
assay. They were gently nipped with cover slip for 2-3
min (until cells spread within a drop). Then the cell
suspensions were softly smeared on both slides. Dried