The damaged umbonal area was measured and was recorded as
a percentage of the outer surface of the left valve using an image
analysis system (Leica Application Suite v3) connected to a stereo
microscope (Leica S8APO). Finally, to measure the shell thickness of
both species, the right valves of 30 shells per tank were sectioned
along the growth axis, following embedding in epoxy resin (Araldite
2020, Huntsman) and grinding. Shell thickness was measured
at various specific points (the umbonal area and shell margin and
three points in-between) in these sections using a stereo microscope
and the above-mentioned image analysis system. In
C. gallina, at intermediate points in the sectioned shells, thickness
was measured at both the rib and ridge levels (Fig. 2). In the
sectioned valves from both the control and treated bivalves, the
mean thicknesswas calculated for each specific point on the shell in
the 3rd and 6th month.
For scanning electron microscopy (SEM) analysis, after embedding
the bivalve shells in epoxy resin, they were cut, fixed on
aluminium stubs and gold coated using an Edwards S 150B Sputter
Coater. The specimens were observed with a Stereoscan 260
(Cambridge Instruments) scanning electron microscope, operating
at 12 kV.