Page 32 - Volume 4, Issue 2, July 2011
Basic HTML Version
© Benaki Phytopathological Institute
Thiram Bioaccumulation in
Mytilus galloprovincialis
61
Results
Validation results
The analytical method applied produced
good response of linearity in the range of
0.20-7.88 μg mL
-1
with correlation coefficient
value r
2
= 0.998 (equation, y=241932x-4695).
LOD and LOQ were determined from the
calibration plot and were 0.32 μg mL
-1
and
0.96 μg mL
-1
respectively. Satisfactory re-
sults were obtained for all levels with recov-
eries for low and high concentrations, well
above the cut off value of 70%. RSDs % for
both low and high concentrations were <
5% therefore acceptable. RSDs % for repeat-
ability and reproducibility were 3.69% and
4.49% respectively. Finally no matrix effect
was observed.
Identification of thiram, accumulation
in mussel soft tissues and concentration
levels in aqueous solutions
The identification of thiram (6.61 min, re-
tention time) was achieved by the LC-ESI-MS
system functioning on the positive mode.
Selected Ion Monitoring mode (SIM mode)
was applied and the characteristic sodi-
um adduct [M+Na]
+
ion of thiram was ob-
served (Figure 1). Accumulation of thiram in
mussel soft tissues for exposure of 0.1 mg/L
(100 μg/kg) was below the LOD and thus
not quantitated. Accumulation for the 1.0
and 10.0 mg/L (1000 and 10000 μg/kg cor-
respondingly) exposure group was 375 μg/
kg d.w. and 3115 μg/kg d.w respectively (Ta-
ble 1). Next step was the determination of
the actual concentrations of thiram in aque-
ous solutions. Thus three different concen-
tration levels were investigated same with
those presented in Table 1. Surprisingly for
0.1 and 1 μg mL
-1
the only peak which ap-
peared (3.55 min, retention time) and was
identified was the peak of another sodium
adduct [C
3
H
7
NS
2
+Na]
+
(47). It seems that in
more dilute aqueous solutions of thiram
the peak of [C
3
H
7
NS
2
+Na]
+
predominates.
One possible fragmentation pathway is de-
picted in Figure 2. The aqueous solution of
10 μg mL
-1
contained both sodium adducts
[M+Na]
+
and [C
3
H
7
NS
2
+Na]
+
as it is shown in
Figure 3. For the sample of 10 μg mL
-1
, thiram
was determined as the sum of areas of peaks
which correspond to thiram and its metab-
olite as equivalent of thiram. The sum con-
centration measured was 8.19 μg mL
-1
.
Mussel mortality
The general condition of the animals (i.e.
reaction to stimuli, excretion of mucus, at-
tachment to glass-walls) was recorded daily
and dead animals were discarded as soon as
possible. Profuse mucus secretion was de-
tected only in the high dose groups. Mortal-
ities were low and did not exceed 12.5 % in
all exposed groups.
SSB in relation to dose group and tissue
type
SSB values were affected by both tissue
and dose, while there was significant inter-
action between dose and tissue (Table 2).
Since significant interaction was observed,
multiple comparisons were conducted at
each tissue for all doses and at each dose for
all tissues (Figure 4).
For gill, exposure to 0.1 mg/L thiram
caused a statistically significant increase in
SSB (see also Figure 5A,B). Higher doses (1.0
and 10.0 mg/L) also caused an increase in
relation to control but these were indistin-
guishable fromeach other. For haemolymph,
exposure to the high dose only (10.00 mg/L)
caused an increase in relation to control
whereas the low and medium dose did not
produce a statistically significant increase
in SSB. For digestive gland there was also a
dose response with the low dose group be-
ing indistinguishable from the control group
and the medium and high dose groups be-
ing different from the control group but not
different from each other.
Oxidative DNA damage in relation to
dose group and tissue type
For gill, exposure to the medium dose of
1.0 mg/L caused a very significant increase
(P<0.001) in Fpg-sensitive sites which corre-
spond to oxidative DNA damage (see also
Figure 5C). For haemolymph and digestive
gland, exposure to the medium dose of 1.0
