Page 29 - Volume 4, Issue 2, July 2011

Emmanouil

et al.

58

tection Agency “thiram is expected to be

sufficiently mobile and persistent in some

cases to reach surface waters in concentra-

tions high enough to impact aquatic life”

(20). Moreover, it may not be readily catab-

olised and it may persist in soil for several

weeks.

A series of

in vivo

experiments have

shown that thiram may affect certain bio-

logical or biochemical aspects in aquatic

organisms namely cladocerans (31), fish (6,

42) and mussels (15). The aim of the present

project was to test for bioaccumulation and

effects of thiram, which is

in vivo

non-clas-

togenic for mammals (19) in the model ma-

rine mussel of the Mediterranean Sea

Myti-

lus galloprovincialis

, in relation to tissue and

dose. Following positive results, mechanistic

aspects of this interaction have been sought

(oxidative DNA damage and early apoptotic

DNA damage). These pathways are implicat-

ed in thiram toxicity in mammals but they

have not yet been examined in bivalve spe-

cies.

Materials and Methods

Test animals

Commercially available

Mytilus gallopro-

vincialis

from a mussel farm in Evia, Greece

were purchased. Mussels of similar size (8-9

cm) were kept in continuously aerated glass

aquaria of 15 L saltwater (salinity 33‰) at

ambient temperature of 25°C, under natu-

ral light. The mussels were fed with approxi-

mately 0.05 g powdered

Spirulina

(M.Rohrer,

Netherlands)

every day. Nitrate and nitrite

levels were periodically checked (API phar-

maceuticals, USA) and they did not exceed

0.5 and 0.25 μg mL

-1

respectively. The mus-

sels were acclimatized in laboratory condi-

tions for at least 5 days before the beginning

of the experiments. These species absorb

quickly contaminants from their environ-

ment but they depurate in an equally rapid

manner when found in clean water (2).

Fungicide and Solvents

A stock solution of 10 mg thiram/L was

prepared fresh in distilled water from com-

mercially available thiram 80% w/w and was

diluted to nominal concentrations of 0.1, 1.0

and 10.0 mg thiram/L in saltwater. Thiram an-

alytical standard was purchased from Fluka

(Buchs, Switzerland) and a stock solution of

100 μg mL

-1

was prepared in methanol. The

working solutions were prepared from this

stock solution in methanol. Methanol and ac-

etonitrile were purchased fromMerck (Darm-

stadt, Germany) and were LC-MS grade. Eth-

yl acetate (pro analysi) was also purchased

from Merck (Darmstadt, Germany).

Experimental set up

Healthy mussels from the aquaria were

exposed in 2 L glass beakers (Simax, Czech

Republic) at a density of 4 mussels/beaker

to final concentrations of the agrochemi-

cal stated in 2.2. The exposures lasted for

48 h after which the animals were sacrificed.

The water was changed every 12 h and was

spiked with thiram after each renewal. Dur-

ing the exposure the animals were not fed.

The general condition of the animals and

the mortalities were recorded daily.

Chemical analysis

Soxhlet Extraction

Lyophilized mussels (7 g) were placed

with equal amount of sodium sulfate at the

bottom of the Soxhlet apparatus covered

with glass wool. The solvent of choice was a

mixture of ethyl acetate/hexane (1:1 respec-

tively, 200 mL) and the extraction time was

4 h. After the extraction the solvent was re-

moved in vacuum and the mixture was re-

constituted with ethyl acetate (2 mL).

Removal of sulfur compounds and Solid

Phase Extraction (SPE)

The removal procedure was the follow-

ing: 1 g of activated copper was added to

the reconstituted mixture (2 mL) and stirred

vigorously for 2 min. Centrifugation (Her-

aeus Labofuge 400R Thermo Electron Cor-

poration) of the mixture was followed by

the separation of the organic phase. Then

the organic phase was passed from Flori-