Total oxyradical scavenging capacity responses in Mytilus galloprovincialis transplanted into the Venice lagoon (Italy) to measure the biological impact of anthropogenic activities

Marine Pollution Bulletin 49 (2004) 801–808 www.elsevier.com/locate/marpolbul

Total oxyradical scavenging capacity responses in Mytilus galloprovincialis transplanted into the Venice lagoon (Italy) to measure the biological impact of anthropogenic activities Lionel Camus

a,b,*

a

c

, Daniela M. Pampanin c, Elisa Volpato c, Eugenia Delaney d, Steinar Sanni b, Cristina Nasci c The University Centre on Svalbard, PB 156, 9170 Longyearbyen, Norway b Akvamiljø as, Mekjarvik 12, 4070 Randaberg, Norway Institute of Marine Science, ISMAR-CNR, Castello 1363/a, Venice, Italy d Thetis Spa, Castello 2737-F, 30122 Venice, Italy

Abstract Oxidative stress related investigations to monitor the impact of the pollutant discharges into the Venice lagoon (Italy) originating from anthropogenic activities (raw sewage water, agricultural and industrial effluents, oil tanker traffic), on marine organisms have classically been carried out by analyzing specific, single antioxidants (i.e. catalase, superoxide dismutase). In this paper, two studies are reported where the total oxyradical scavenging capacity assay (TOSC) was selected and measured toward peroxyl, and hydroxyl free radicals, and peroxynitrite in mussels (Mytilus galloprovincialis) transplanted into the Venice city and throughout the lagoon to measure the biological effects of anthropogenic activities. In the first experiment, mussels from a clean site (farm) were transplanted to the urban area of Venice for 0, 1, 2 and 4 weeks; cytosolic TOSC toward peroxyl and hydroxyl free radicals, and peroxynitrite revealed that the transplantation process caused a stress (handling stress, anoxia, oxidative burst) resulting in a reduction of TOSC in both control and urban sites, therefore, preventing clear interpretation of the data after one week. At week 2, a significant TOSC reduction (P < 0:05) toward peroxyl and hydroxyl radicals in the urban site revealed that mussels experienced oxidative pressure exerted by pollutants. Most TOSC values returned to initial levels at week 4; however, TOSC induction was noticed in the control group toward peroxyl and hydroxyl radicals while in the exposed group it was not indicating an inhibition of the oxidative metabolism. In the second experiment, mussels were deployed at seven different sites throughout the lagoon. After five weeks of exposure, significant TOSC reduction was measured (P < 0:05) toward peroxyl for Palude della Rosa, Chioggia and Valle Millecampi, toward hydroxyl radicals for Valle Millecampi and Campalto and toward peroxynitrite for Valle Millecampi. Although these data indicate a depletion of the low molecular weight scavengers, additional biomarkers are needed to draw a conclusion on the health of the mussels. TOSC was proved to be an interesting health index parameter to measure pollution impact in a transplantation study provided that the mussels are exposed for two weeks and a control is run in parallel.
2004 Published by Elsevier Ltd. Keywords: Mytilus galloprovincialis; Transplantation; Venice lagoon; Oxidative stress; TOSC

1. Introduction In the last few decades, the use of a biomarker integrated approach helped bring about better comprehension of the mechanistic mode of action of environmental *

Corresponding author. Address: Akvaplan-niva, Polar Environmental Centre, 9296 Tromsø, Norway. Tel.: +47-777-50-300; fax: +4777-75-03-01. E-mail address: [email protected] (L. Camus). 0025-326X/$ - see front matter
2004 Published by Elsevier Ltd. doi:10.1016/j.marpolbul.2004.06.009

pollutants on organisms. However, the complexity and high variability of the marine ecosystem pose numerous problems. The physiological response of marine ectothermic organisms is strongly dependent on fluctuations of biotic and abiotic factors such as salinity, oxygen concentration, temperature, food availability, resulting in difficulties in interpreting the biological effects exerted by xenobiotics. In addition, the genetic differences in susceptibility to pollution render comparison between animals originating from different populations difficult

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due to the variability in biomarker responses (Forbes and Depledge, 1996; Astley et al., 1999). With the objective of reducing the physiological variability between populations of indigenous organisms, biomonitoring was carried out with animals originating from the same population (i.e. farmed organisms) grown in a clean site and transplanted into the area of environmental concern (Regoli and Principato, 1995; Nasci et al., 1999, 2000, 2002; Regoli, 2000; Da Ros et al., 2002; Gowland et al., 2002; Shaw et al., 2002; Romeo et al., 2003). Following a large research program funded by UNESCO in the 90s to study the functions of the Venice lagoon ecosystem, numerous studies have focused on the measurements of the background levels of pollutants originating from growing anthropogenic activities and their effects on the indigenous fauna (Lowe et al., 1995; Livingstone et al., 1995; Sol
e et al., 2000). Among biomarkers deployed in field studies, single antioxidant parameters that give an indication of the prooxidant forces associated to reactive oxygen species (ROS) generated during metabolism of contaminants have been thoroughly employed (Livingstone et al., 1995; Nasci et al., 2000, 2002). The main ROS produced during these cellular processes include superoxide anion (O
 2 ), hydrogen peroxide (H2 O2 ), hydroxyl radicals ( OH), peroxyl radicals (ROO ), alkoxyl radicals (RO ) and peroxynitrite (HOONO). Although ROS naturally originate from the partial reduction of molecular oxygen during aerobic metabolism (see DiGiulio et al., 1989 for review), a state of imbalance between ROS production and the antioxidant defence system in a cell or tissue, in favour of the former, leads to oxidative stress (Sies, 1985). The balance between prooxidant forces and antioxidant defences is essential in preventing oxyradical cellular toxicity as oxidative damage will appear when the overall antioxidant capacity is exceeded by ROS generation. The defences consist of various low molecular weight free radical scavengers, such as glutathione, ß-carotene and vitamins A, E and C and a number of specific enzymes, superoxide dismutase (SOD) which reduces the superoxide anion into peroxide water (H2 O2 ), catalase which reduces H2 O2 produced by SOD to produce water and oxygen, and glutathione peroxidase which reduces peroxides (H2 O2 or hydroperoxides) by oxidizing two molecules of glutathione. Cytotoxic effects of ROS comprise lipid peroxidation, DNA damage, protein degradation, metabolic malfunctions and cell death (Winston and DiGiulio, 1991). The drawback of measuring single antioxidant parameters such as enzyme activities (i.e. catalase) or the concentration of low molecular weight compounds (i.e. glutathione) is that their large numbers render the assessment of the overall antioxidant capacity of an organism complex. In addition, antioxidant levels and/ or activities can greatly vary for their biosynthetic

pathway, intracellular localization, chemical nature and mode of action. In this respect, Winston et al. (1998) and Regoli and Winston (1999) developed the total oxyradical scavenging capacity assay (TOSC) which was demonstrated to have a high predictive value on the health condition of the organisms in that it allows the ability of the antioxidant defences to deal with different ROS (ROO ,  OH and HOONO radicals) in oxidative stress syndrome and their links with effects at higher levels of biological organisation such as lysosomal membrane stability (Regoli, 2000) and DNA strand breaks (Regoli et al., 2002) to be discriminated. In this paper, two studies are reported where mussels (Mytilus galloprovincialis) originating from the same population grown in a mussel farm were transplanted: (i) into the urban area of Venice and a reference site, then TOSC was measured at week 0, 1, 2 and 4 to understand the capability of the mussels to cope with oxidative pressure exerted by contaminants present in raw domestic water; (ii) throughout the lagoon at seven different sites of concern and TOSC measured after five weeks to evaluate the health condition of the bivalves.

2. Material and method 2.1. Field work 2.1.1. Urban area study In October 2002, mussels (Mytilus galloprovincialis, shell size ¼ 4.5 ± 0.5 cm) were purchased from a mussel farm located near the lagoon entrance of Malamocco (Alberoni) and transplanted to a highly anthropogenic impacted site (raw domestic sewage) in the urban area of Venice (S. Toma) and to a reference site near the lagoon entrance of Lido (Treporti). Mussels were transported in cold boxes from the farm to the boat and, after a first sorting, they were divided into groups (of about 120 individuals each), placed in cages constituted by polyethylene netting and immersed subtidally at 2.5 m depth at both sites. Animals were collected after 0, 1, 2 and 4 weeks of exposure. Upon return to laboratory, digestive glands (n ¼ 5) were immediately frozen in liquid nitrogen and stored at )80 C before TOSC analysis. In all cases, mussels were taken from the subtidal zone and were consequently placed subtidally. 2.1.2. Lagoon study In a second experiment, mussels from Alberoni were transplanted in May–June 2003, as described above, to seven different sites throughout the lagoon: Ca’Roman (reference), Palude dela Rosa, Campalto, Tresse, Sacca Sessola, Valle Millecampi and Chioggia (Fig. 1). After five weeks, mussels were retrieved and the digestive glands (n ¼ 5) excised and frozen in liquid nitrogen prior to TOSC analysis.

L. Camus et al. / Marine Pollution Bulletin 49 (2004) 801–808

Fig. 1. Location of the sampling stations in the Venice lagoon. (1) Palude della Rosa, (2) Campalto, (3) Tresse, (4) Sacca Sessola, (5) Ca’Roman, (6) Valle Millecampi and (7) Chioggia.

2.1.3. TOSC assay The method was based on Winston et al. (1998) and Regoli and Winston (1999), except that buffers were adjusted for marine invertebrates. Digestive glands of mussels were homogenised with a Potter–Elvehjem glass/Teflon homogeniser in four volumes of 100 mM KH2 PO4 buffer, 2.5% NaCl, pH 7.5. The homogenate was centrifuged at 100; 000  g for 1 h, and cytosolic fractions were aliquoted and stored at )80 C. Peroxyl radicals are generated by the thermal homolysis of 2-20 -azo-bis-(2-methyl-propionamidine)-dihydrochloride (ABAP) at 35 C. The iron-ascorbate Fenton reaction was used for hydroxyl radicals, while peroxynitrite was generated from 3-morpholinosydnomine (SIN-1), a molecule that releases concomitantly nitric oxide and superoxide anion, which rapidly combine to form HOONO. Final assay conditions were: (a) 0.2 mM aketo-c-methiolbutyric acid (KMBA), 20 mM ABAP in 100 mM potassium phosphate buffer, pH 7.4 for peroxyl radicals; (b) 1.8 lM Fe3þ , 3.6 1/4M EDTA, 0.2 mM KMBA, 180 lM ascorbic acid in 100 mM potassium phosphate buffer, pH 7.4 for hydroxyl radicals; and (c) 0.2 mM KMBA and 80 lM SIN-1 in 100 mM potassium phosphate buffer, pH 7.4 with 0.1 mM diethylenetriaminepentaacetic acid (DTPA) for peroxynitrite. Peroxyl, hydroxyl and peroxynitrite radicals can oxidize the substrate KMBA to ethylene gas which is measured by gas chromatography. With these assay conditions, the various oxyradicals induce a comparable yield of ethylene in the control reaction, thus the relative efficiency of cellular antioxidants is compared by their ability to counteract a quantitatively similar prooxidant challenge (in terms of KMBA oxidation). Reactions were carried

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out in 10 ml rubber septa sealed vials in a final volume of 1 ml. Ethylene production was measured by gas-chromatographic analysis of 200 ll taken from the head space of the reaction vials. Ethylene formation was monitored for 96 min with a Hewlett Packard (HP 5890 series II) gas chromatograph equipped with a supelco SPB-1 capillary column (30 m · 0.32 mm · 0.25 lm) and a flame ionization detector (FID). The oven, injection and FID temperatures were 35, 160 and 220 C, respectively; helium was the carrier gas (1 ml/min flow rate) and a split ratio 20:1 was used. The data acquisition system was run by the software Millenium32 (Waters). Each analysis required the measurement of control (no antioxidant in the reaction vial) and sample reactions (biological fluid in the vial). In the presence of antioxidant, ethylene production from KMBA was reduced quantitatively and higher antioxidant concentrations resulted in longer periods in which ethylene formation was inhibited relative to controls. By plotting the absolute value of the difference between the ethylene peak areas obtained at each time point for the sample and control reaction it is possible to visualise whether the oxyradical scavenging capacity of the solution is changed. The area under the kinetic curve was calculated mathematically from the integral of the equation that best defines the experimental points for both the control and sample reactions. TOSC is then quantified according to Eq. (1): TOSC ¼ 100
ðIntSA=IntCA  100Þ

ð1Þ

where IntSA and IntCA are the integrated areas from the curve defining the sample and control reactions, respectively. Thus, a sample that displays no oxyradical scavenging capacity would give an area equal to the control (IntSA/IntCA ¼ 1) and a resulting TOSC ¼ 0. On the other hand, as IntSA/IntCA goes to 0 the hypothetical TOSC value approaches 100. Because the area obtained with the sample is related to that of the control, the obtained TOSC values are not affected by small variations in instrument sensitivity, reagents or other assay conditions. The specific TOSC value was calculated by dividing the experimental TOSC by the concentration of protein [measured according to Bradford (1976)] used for the assay. Data are expressed as TOSC unit per mg protein. 2.1.4. Statistical treatment Statistical analyses were made using JMP v3.2.6., SAS Institute, Inc., Cary, NC, USA. Normal distribution and homogeneity of variances was established before statistical treatment. The Student’s t-test was performed for each set of data to test differences between exposed and control groups (Fig. 2). Dunnett’s test was used for testing change of TOSC following transplantation (within control or within exposed

L. Camus et al. / Marine Pollution Bulletin 49 (2004) 801–808

TOSC unit / mg protein

600

350

*

ROO .

Exposed

300 TOSC unit / mg protein

804

500

Control 400 300 200

250

ROO. * *

200

*

150 100

100 50

0 0

1

2

3

0

4

Palude della Campalto Rosa

Time (week)

*

. OH

300

1000 Exposed

800 600

**

400

Sacca Sessola

Valle Ca'Roman Millecampi (Control)

Chioggia

Sites

. OH

Control TOSC unit / mg protein

TOSC unit / mg protein

1200

Tresse

250 200

*

*

150 100

200

50

0

0

0

1

2

3

4

Palude della Campalto Rosa

Tresse

Sacca Sessola

Valle Ca'Roman Millecampi (Control)

Chioggia

Time (week) 250

HOONO

500

Control

400

Exposed

*

300 200

TOSC unit / mg protein

TOSC unit / mg protein

600

Sites

HOONO *

200 150

100

100

50

0 0

1

2

3

0

4

Palude della Campalto Rosa

Time (week)

Tresse

Sacca Sessola

Valle Ca'Roman Millecampi (Control)

Chioggia

Sites

Fig. 2. Total oxyradical scavenging capacity towards peroxyl (ROO ), hydroxyl ( OH) and peroxynitrite (HOONO) of digestive gland cytosolic fraction of mussels (Mytilus galloprovincialis) after 0, 1, 2 and 4 weeks of transplantation into the urban area of Venice and the control site Treporti. Values are expressed as TOSC unit/mg protein (mean ± SD, n ¼ 5). Asterisks indicate significant difference (t-test) between exposed and control ( P < 0:05,  P < 0:01).

groups through time) into the urban area of Venice (Table 1) and throughout the lagoon (Fig. 3). Data are

Fig. 3. Total oxyradical scavenging capacity towards peroxyl (ROO ), hydroxyl ( OH) and peroxynitrite (HOONO) of digestive gland cytosolic of mussels (Mytilus galloprovincialis) after five weeks of transplantation at various sites throughout the lagoon of Venice. Values are expressed as TOSC unit/mg protein (mean ± SD, n ¼ 5). Asterisks indicate significant difference (Dunnett’s test) between exposed and reference site (Ca’Roman) ( P < 0:05).

plotted as mean and standard deviation of the mean. The significance level was P < 0:05.

Table 1 Statistical differences (Dunnett’s test) between transplanted groups (control and exposed at week 1, 2, and 4) and the group before transplantation ‘‘week 0’’ Control



ROO (week 0) OH (week 0) HOONO (week 0) 

Exposed

Week 1

Week 2

Week 4

Week 1

Week 2

Week 4

ns ns ns

* ns ns

ns ** ns

* ns *

ns * ns

ns ns ns

Asterisks indicate significant difference between exposed and control ( P < 0:05,



P < 0:01 and ns ¼ non significant).

L. Camus et al. / Marine Pollution Bulletin 49 (2004) 801–808

3. Results 3.1. Urban area study The TOSC values toward the various oxyradicals decreased in the first week following transplantation concomitantly in both control and urban sites (Fig. 2); this decline at week 1 was significantly different from week 0 (P < 0:05) for peroxyl and peroxynitrite radicals of the exposed group only (Table 1) but not from the control group (P > 0:05). While the scavenging capacity of the mussels recovered for peroxyl and peroxynitrite radicals at week 2 in both groups (Fig. 2), TOSC toward hydroxyl radicals further decreased to become significantly different from week 0 (P < 0:01) (Table 1) and from control (P < 0:01). Recovery of the TOSC value toward hydroxyl radicals returned to initial values at week 4 in the exposed group while a significant increase (P < 0:01) was measured in the control group (Table 1). The significant difference (P < 0:05) in TOSC toward peroxyl between exposed versus control groups at week 2 was attributed to a TOSC induction of the control group (Fig. 2). Finally, difference between control and exposed group for TOSC toward peroxynitrite was characterised by a significant increase of the exposed group (P < 0:05) at week 4 (Fig. 2). 3.2. Lagoon study After five weeks of exposure, significant TOSC reduction was measured (P < 0:05) compared to the reference site (Ca’Roman) toward peroxyl for Palude della Rosa, Chioggia and Valle Millecampi, toward hydroxyl for Valle Millecampi and Campalto, toward peroxynitrite for Valle Millecampi (Fig. 3).

4. Discussion 4.1. Urban area study The main objective of biomonitoring with animals originating from the same population (i.e. farmed organisms) grown in a clean site and transplanted into the area of environmental concern is to reduce the physiological variability between population of indigenous organisms in order to better draw conclusions on the real biological effects of contaminants. While numerous studies have reported the benefit of the transplantation procedure for biomonitoring (Regoli and Principato, 1995; Nasci et al., 1999, 2000, 2002; Regoli, 2000; Gowland et al., 2002; Shaw et al., 2002; Da Ros et al., 2002; Romeo et al., 2003), the present study revealed that moving an animal from its environment to another can be a source of stress which can

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mislead the interpretation of the investigator; herein, a reduction of the capability of the mussels from both control and urban sites, to neutralize the three oxyradicals of concern was observed one week after transplantation. Although the decrease of the TOSC values of the control group is not statistically significant, the concomitant decline of both control and exposed groups resulted in a non significant difference of TOSC values after one week of exposure leading to the conclusion that mussels transplanted into the urban area did not experience any prooxidant pressure exerted by the presence of contaminants. Indeed, it can be suspected that significant difference would exist if the control group had remained steady. Generally, a decline in TOSC indicates a depletion of low molecular weight scavengers consumed to neutralize oxyradicals produced by the cells or an inhibition in enzyme activity (Regoli, 2000, Regoli et al., 2002). Two probable potential sources of stress associated to transplantation that could affect the TOSC level of the mussels were foreseen: (i) handling, emersion and transport have induced valves closure leading to anaerobiosis (Sadok et al., 1999) and subsequent depression of most antioxidant defences (Pannunzio and Storey, 1998); in addition recent work by Lacoste et al. (2002) with oyster (Crassostrea gigas) revealed that physical stress such as handling provokes an immune reaction, notably on the oxidative burst generated by haemocytes which can consequently affect antioxidant defences (ii) the re-immersion of the bivalves on the transplantation site following a short period of anoxia taking place during transport could have led to an oxidative burst and a rapid depletion in low molecular weight scavengers to neutralize the ROS. According to Sadok et al. (1999) and Uglow and Williams (2001), it is important to note that the life history of the mussels can play an essential role in mitigating the degree of impact of these oxidative stresses. Indeed, mussels inhabiting the subtidal zone, as the mussels of the present report, do not experience this successive period of anoxia and oxidative burst engendered by the tide cycle, hence, they are not physiologically adapted to cope with variation in oxygen availability while marine invertebrates living on the upper shore are according to Pannunzio and Storey (1998). Therefore, the mussels employed in this study may be highly susceptible to oxidative stress triggered by the transplantation procedure. Although the TOSC decline of the control group week 1 is not significant compared to week 0, a significant decrease is shown in the urban group after one week for peroxyl and peroxynitrite and two weeks for hydroxyl radicals; this suggests that the oxidative pressure endured by the organisms was more elevated than for the control group, and could be attributed to the presence of pollutants as observed by Regoli (2000). The equivocal high toxicity of the hydroxyl radicals (Regoli,

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2000; Regoli et al., 2002; Machella et al., 2004) and the poor efficiency of the cell to scavenge this reactive oxygen species (Regoli and Winston, 1999) led Regoli et al. (2002) to hypothesise that in oxidative metabolism, bivalves have developed a strategy to prevent the formation of the highly potent hydroxyl radicals in order to avoid any biological damages, and a fundamental role has been postulated for catalase in inhibiting the Fenton reaction by removing H2 O2 . The week delay to measure a significant TOSC diminution toward hydroxyl compared to peroxyl and peroxynitrite radicals supported the hypothesis that the cells possess a system that minimizes the production of hydroxyl radicals. However, failure of preventing the hydroxyl radicals formation can occur when the oxidative pressure is too high and overwhelmed the cell antioxidant machinery. For instance, the activation of the lysosomal response as shown in Mytilus galloprovincialis sampled from the urban area of Venice (Lowe et al., 1995, Lowe and Fossato, 2000) can participate to the enhancement of the generation of hydroxyl radicals owing to the simultaneous presence of essential metals that catalyze the Haber-Weiss and Fenton reactions and the generation of the superoxide anion in and out the lysosomal membrane (Winston et al., 1996, Regoli, 2000). Additionally, overproduction of H2 O2 and/or inhibition of the catalase enzyme may favour the Fenton reaction to take place and, therefore, enhance the production of hydroxyl radicals. Regoli (2000) demonstrated that soluble antioxidant generally accounted for 70% of the total scavenging capacity toward peroxyl radicals but their contribution was only 50% toward hydroxyl radicals in Mytilus galloprovincialis. In addition, classic low molecular weight antioxidants (glutathione, ascorbic acid, uric acid, vitamin E) are relatively poorer scavengers of this ROS, with an efficiency often one order of magnitude lower than that exhibited toward peroxyl radicals (Regoli and Winston, 1999). On the other hand, numerous glutathione dependant enzymes (Glutathione S-Transferase, Glutathione Peroxidase and Reductase) participate to the removal of peroxyl radicals precursors. The fact that the TOSC toward hydroxyl radicals further decreased at week 2 while TOSC toward peroxyl regained the initial value, supported the hypothesis that the formation of the potent hydroxyl radicals is the consequence of the combination of a strong oxidative pressure exerted by pollutants and also of a relatively poorer responsive scavenging machinery of the cells against this ROS. The increase in TOSC toward peroxyl (week 2) and toward hydroxyl (week 4) in the control group can correspond to variation of single antioxidants that deal with natural production of oxyradicals associated to normal metabolic functions (feeding, respiration) while the lack of TOSC induction in the exposed group indicates that the mussels experience a high oxidative pres-

sure overwhelming the antioxidant defence system, preventing the replenishment in low molecular weight scavengers (i.e. Glutathione) and, also, leading to the inhibition of the activity of enzymes as shown by others for catalase and superoxide dismutase (Regoli and Principato, 1995; Regoli, 2000; Nasci et al., 2002). The reduction of oxyradical scavenging capacity toward the three oxyradicals of the exposed group returned to initial value at week 4 indicating a transient response of antioxidants to pollutants, corroborating similar observations reported by other authors (Wenning et al., 1988; DiGiulio et al., 1993; Livingstone et al., 1993; Doyotte et al., 1997; Cavaletto et al., 2002). 4.2. Lagoon study TOSC measured in mussels transplanted at various sites throughout the lagoon revealed that after five weeks of exposure a reduction was observed toward peroxyl for Palude della Rosa, Chioggia and Valle Millecampi, toward hydroxyl for Valle Millecampi and Campalto, toward peroxynitrite for Valle Millecampi. Considering that TOSC toward all three oxyradicals of concern was reduced in mussels transplanted to the site of Valle Millecampi, it can be concluded that bivalves suffered from intensive oxidative stress which can be attributed to chemicals, notably employed in agriculture such as pesticides. Due to the high toxicity of hydroxyl radicals and the capability of bivalves to reduce its formation it can be concluded that the TOSC decrease toward hydroxyl radicals in mussels located at Campalto was the result of the presence of high levels of pollutants, notably polychlorinated biphenyls, which can originate from an industrial landfill located in this area (Volpi et al., 1999). The present findings at Chioggia support the study of Nasci et al. (2002) that reported that this urban site is characterized by the presence of domestic sewage and contaminants associated to intense harbour activities known to generate oxidative stress in transplanted mussels. Also, Palude della Rosa is known as a relatively low contaminated site but some biochemical markers were shown to respond in the clam Tapes philippinarum transplanted into this site (Nasci et al., 2000), thereby corroborating the present decrease in TOSC toward peroxyl in mussels. The ecological relevance of biomarkers is incontestably greater when they indicate adverse effects at the organism level (Depledge, 1994). The relatively strong correlation between TOSC and the lysosomal response (Regoli, 2000) and, also, DNA strand break measured as comet assay (Regoli et al., 2002) highlights that analysis of TOSC toward different forms of oxyradicals appears an useful biomarker with predictive validity at the organism level. Reduced capability in neutralizing various ROS, as shown in the lagoon study, is found in exposed animals characterized by an increased suscep-

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tibility to oxidative stress disease (Regoli, 2000, Regoli et al., 2002). Mussels transplanted into the sites Palude della Rosa, Chioggia, Campalto and Valle Millecampi may suffer from several oxyradical mediated forms of toxicity. These latter range from DNA damages, lysosomal alteration with consequences on the immune functions and, physiological impairments. However, owing to the TOSC transient response elicited by the mussels four weeks after transplantation in the first experiment, it is likely that only sites with strong environmental pollution were characterized by a TOSC reduction. Furthermore, results may reflect antagonistic and/or synergistic effects due to complex contaminant mixtures (Lowe et al., 1995, Nasci et al., 2000, Orbea et al., 2002) present in the impacted areas, or a time- and dose-dependant response as in part suggested by the first experiment, thereby precluding a clear conclusion to be drawn. Therefore, additional biomarkers of exposure and effects should be run in parallel to support the TOSC data in order to elucidate the health status of the mussels transplanted for five weeks at various sites throughout the lagoon. In conclusion, mussels transplanted into the urban area were characterized by a decrease in TOSC indicative of oxidative stress generated by pollutants discharged with the raw sewage water of the city of Venice. Biomonitoring with the transplantation method requires a good practice to minimize stress that can affect the balance between prooxidant and antioxidant forces of the mussels. A control group run in parallel with the exposed group is necessary for evaluating the effects of the transplantation procedure on the oxidative stress. According to the present study, two weeks of exposure were required to measure a significant difference between control and exposed groups. After four weeks, TOSC values returned to initial values indicating a transient response and an adaptation of the mussels. Finally, in the lagoon monitoring study, TOSC values were decreased at the sites Palude della Rosa, Chioggia, Campalto and Valle Millecampi after five weeks of transplantation. Although this may indicate that the mussels experienced higher levels of oxidant challenge which was reflected in the partial depletion of their antioxidant stores, further investigations with other parameters of biological effects are required to draw a conclusion on the health condition of the mussel.

Acknowledgements This study was part of the project WATERS-CORILA, funded by the Venice Water Authority through Consorzio Venezia Nuova. Additional funding was provided by the Norwegian Research Council under the program ‘‘Marine Resource, Environment and Man-

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agement’’, project no. 146478/120 and by the Norwegian marine research laboratory RF-Akvamiljø.

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