20 In Vitro Toxicity and Histopathological Effects Induced in the Mantle. . .
231
During each experiment, the mussels were maintained under constant aeration and
temperature (20
ı C), with natural photoperiod. They were fed a daily microalgae
diet (0.0224 g microalgae/mussel) comprised of Isochrysis galbana, Tetraselmis
suecica and Chatoceros gracilis in a 50:30:20 proportions.
After 15 days acclimatization to the described conditions, the mussels from each
tank were exposed to different concentrations of the tar mixture. Previous studies
by our research team regarding total hydrocarbon accumulation in mussels from the
R´ ıa of Vigo, had shown mean PAH concentrations of 400 g/kg (Ruiz et al. 2011).
This value was the lowest concentration measured in mussel tissues after laboratory
exposure to 10 ppm of the tar mixture, applied for 17 days. We also employed three
higher concentrations: 40, 60 and 80 ppm. Control mussels from the fifth tank were
not treated with tar. The tar mixture was heated to 200
ı C and added in 5 L of
seawater, together with the microalgal diet, at a 2 mL/min flow rate. Treatment was
continued for 17 days and samplings made at days 0, 10 and 17.
Tissue Preparation for Histology
In species of the genus Mytilus, the proximity of the genital papilla to the mantle
tissue is used for the expansion of gonad and the gametogenic development taking
place mainly in this tissue. For each sampling, the mantles from 10 individuals from
each assay tank were dissected, fixed in 10 % neutral buffered formalin, dehydrated
and processed in paraffin. Sections of 5 m thickness were mounted on microscope
slides and stained with Carazzi haematoxilin and eosin. Slides were examined by
light microscopy (Nikon 90i Eclipse). The images were captured by an 8Mpx high
resolution Nikon camera using Nis- Elements BR 2.30 version software.
The gametogenic stage of each mussel was determined according to Lubet (1959)
and Su´ arez et al. (2005). Histological preparations were examined comparatively
with those of the control mussels as a reference.
Statistical Analysis
A statistical survival analysis with a PROBIT regression test was performed
(statistical package SPSS 17.0, Microsoft Co) in order to calculate the median lethal
concentration (LC 50 ) of the tar mixture.
Results
The concentration of total hydrocarbons analyzed in the tar mixture (†13 parent
PAH species) was 40,364 mg/kg. Therefore, the dilutions of 10, 40, 60 and 80 ppm
employed in this experiment represent exposure concentrations of 400, 1,600, 2,400
231
During each experiment, the mussels were maintained under constant aeration and
temperature (20
ı C), with natural photoperiod. They were fed a daily microalgae
diet (0.0224 g microalgae/mussel) comprised of Isochrysis galbana, Tetraselmis
suecica and Chatoceros gracilis in a 50:30:20 proportions.
After 15 days acclimatization to the described conditions, the mussels from each
tank were exposed to different concentrations of the tar mixture. Previous studies
by our research team regarding total hydrocarbon accumulation in mussels from the
R´ ıa of Vigo, had shown mean PAH concentrations of 400 g/kg (Ruiz et al. 2011).
This value was the lowest concentration measured in mussel tissues after laboratory
exposure to 10 ppm of the tar mixture, applied for 17 days. We also employed three
higher concentrations: 40, 60 and 80 ppm. Control mussels from the fifth tank were
not treated with tar. The tar mixture was heated to 200
ı C and added in 5 L of
seawater, together with the microalgal diet, at a 2 mL/min flow rate. Treatment was
continued for 17 days and samplings made at days 0, 10 and 17.
Tissue Preparation for Histology
In species of the genus Mytilus, the proximity of the genital papilla to the mantle
tissue is used for the expansion of gonad and the gametogenic development taking
place mainly in this tissue. For each sampling, the mantles from 10 individuals from
each assay tank were dissected, fixed in 10 % neutral buffered formalin, dehydrated
and processed in paraffin. Sections of 5 m thickness were mounted on microscope
slides and stained with Carazzi haematoxilin and eosin. Slides were examined by
light microscopy (Nikon 90i Eclipse). The images were captured by an 8Mpx high
resolution Nikon camera using Nis- Elements BR 2.30 version software.
The gametogenic stage of each mussel was determined according to Lubet (1959)
and Su´ arez et al. (2005). Histological preparations were examined comparatively
with those of the control mussels as a reference.
Statistical Analysis
A statistical survival analysis with a PROBIT regression test was performed
(statistical package SPSS 17.0, Microsoft Co) in order to calculate the median lethal
concentration (LC 50 ) of the tar mixture.
Results
The concentration of total hydrocarbons analyzed in the tar mixture (†13 parent
PAH species) was 40,364 mg/kg. Therefore, the dilutions of 10, 40, 60 and 80 ppm
employed in this experiment represent exposure concentrations of 400, 1,600, 2,400