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Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabès (Tunisia)
Environment International 28 (2003) 609 – 617 www.elsevier.com/locate/envint
Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabe`s (Tunisia) A. Hamza-Chaffai a,*, J. Pellerin b, J.C. Amiard c a
IPEIS, Universite de Sfax, BP 805, 3018 Sfax, Tunisia ISMer-UQAR, 310 alle´e des Ursulines, Rimouski, QC, Canada G5L 3A1 c GDR1117-CNRS-ISOMer, 2 rue de la Houssinie`re, 40322 Nantes cedex 3, France b
Abstract Metallothioneins (MTs), malonedialdehyde (MDA), and glycogen concentrations were determined in order to assess the health status of Ruditapes decussatus exposed to in situ contamination, showing the effects of time, site, and metal contamination on these three biomarkers. Metallothionein was positively correlated with Cd and Zn. If introduced in a multiple model, the factors site and time were shown to decrease significantly MT concentrations. MDA was inversely correlated with size of the animals and was affected by the factor time. Glycogen was inversely correlated with zinc and was affected by both site and time. This study constitutes a field-based validation of a multiparametric approach using specific and nonspecific biomarkers. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: Biomarkers; Glycogen; Metallothionein; Malonedialdehyde; Metals; Ruditapes decussatus
1. Introduction The Gulf of ‘‘Gabe`s’’ is situated in the southeastern coast of Tunisia. Shallow waters, weak currents, high salinity, and temperature characterise this area. Compared to other Mediterranean areas, tides are exceptionally important. From a biological point of view, coastal Posidonia and Caulerpa constitute a spawning and refuge area for fish larvae (Hamza-Chaffai, 1993). This ecosystem is known to be very rich in aquatic resources and contributes to about 65% of the national production in Tunisia (C.G.P., 1996). Unfortunately, an important industrial activity, mainly crude phosphate treatment, chemical industries, tannery, and plastic plants, is now being developed along the coasts and could threaten this marine ecosystem (Hamza-Chaffai et al., 1995, 1996, 1997; Zairi and Rouis, 1999). In fact, since 1990, destruction of Posidonia grassland and a decrease in fish production have been observed. Besides the ecosystem damages engendered by this pollution, the human health could also be affected if toxic pollutants concentration become high in edible marine organisms.
* Corresponding author. Tel.: +216-4-241-403; fax: +216-4-246-347. E-mail address: [email protected] (A. Hamza-Chaffai).
An urgent national bio-monitoring program is therefore needed to assess the degree of pollution and evaluate the quality of the marine environment. Under similar circumstances, other countries have extensively used marine molluscs such as mussels and oysters in monitoring programs, like the Mussel watch program (NAS, 1980; Phillips, 1986). In the case of Tunisia, these species are available only in few northern sites. Alternatively, Ruditapes decussatus is widely distributed along the Tunisian coast. This species is a sedentary filter feeding marine bivalve, thus satisfying criteria in biomonitoring programs for a good bioindicator of pollution (NAS, 1980; Phillips, 1986). This species as well represents an important economic endpoint, R. decussatus being mostly exported to Europe. Biomonitoring programs based on measures of contaminants in marine organisms are also interesting from a human health point of view. However, pollutant levels do not give information about their toxicological significance, particularly because xenobiotics can be stored in various forms such as insoluble precipitates and concretions (Brown, 1982; Mason and Jenkins, 1995; Murrat et al., 1998). Consequently, recent biomonitoring programs are now involving biomarkers. These are measurable parameters at different levels of biological organisation. Biomarkers reflect changes in the metabolic regulatory processes resul-
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ting from the effect of anthropogenic stressors (Lagadic et al., 1997). This approach should be multiparametric, using different and complementary biomarkers (Narbonne et al., 1989) to reflect the effects of different contaminants. However, we also need to consider variations linked to biotic and abiotic factors. In fact, the physiological state of an organism within an ecosystem is the result of an equilibrium between the influences of anthropogenic, abiotic, and biotic factors (Pellerin-Massicotte, 1994). In the present work, we have focused on three biomarkers: metallothioneins (MTs), malonedialdehyde (MDA), and glycogen, well known to be sensitive to pollutant-exposition (Bebianno et al., 1994; Pellerin-Massicotte, 1994). Metallothioneins (MTs) are low molecular weight, heat stable proteins of high cysteine content. While low basal levels of MTs are generally present in marine organisms from unpolluted areas, the synthesis of MTs is induced as a direct response to Cd, Cu, Zn, and Hg exposure (Viarengo and Nott, 1993; Roesijadi, 2000). These proteins have been identified in various tissues of vertebrates and invertebrates (Amiard and Cosson, 1997; Cosson and Amiard, 1998). Given their molecular properties and their role in metal uptake, transport, storage, and excretion, MTs offer considerable potential as a contaminant-specific biochemical indicator of metal exposure (Roesijadi, 1992, 1994; Stegeman et al., 1992). In marine invertebrates, MTs have been implicated in the storage, transport, and exchange of essential metals (Cu, Zn), and in the detoxification of nonessential metals (Ag, Cd, Hg) (Viarengo and Nott, 1993). Earlier work in our laboratory has shown the presence of metallothioneins in the digestive gland of R. decussatus in field conditions (Hamza-Chaffai et al., 1999, 2000) and after an experimental contamination (Hamza-Chaffai et al., 1998). The second biomarker is malonedialdehyde (MDA), which is a product of lipid peroxidation due to overproduction of oxyradicals in cells, following contaminant exposure or stress due to natural conditions (Pellerin-Massicotte, 1997; Cheeseman, 1982). Lipid peroxidation is considered as an important feature in cellular injury. It results from free radical reactions in biological membranes, which are rich in polyunsaturated fatty acids. MDA has been used extensively to assess detrimental effects of various pollutants (PellerinMassicotte, 1997; Viarengo et al., 1991). The third biomarker is glycogen, which is the fuel for different metabolic and physiological processes. Glycogen has been shown to respond quite well to complex and diffuse contamination situations (Pellerin et al., 1993; Pellerin-Massicotte, 1994; Gauthier-Clerc et al., 2002). Decreased levels of glycogen related to an altered growth (Pellerin and Audet, 1998) were observed in mussels (Mytilus edulis) and clams (Mya arenaria) after an exposure to pulp and paper mill effluents (Pellerin et al., 1993), as well as in a mesocosms where bivalves were exposed to oil and a silicon-based polymer. In the present work, these three biomarkers were used in order to assess the health status of R. decussatus in a
contaminated marine ecosystem. We have carried out a field experiment designed to evaluate the potential of MTs, MDA, and glycogen as biomarkers of in situ contamination. Our objective was to study the effect of site, time of transplantation, and metals (Cd, Cu, Zn) on the studied biomarkers.
2. Materials and methods 2.1. Study area and experimental design The transplantation experiment was carried out in two sites situated in the southeastern coast of Tunisia (Fig. 1). The experiment was designed to follow changes associated with increased metal exposure as a function of time. The two sites differed in their level of contamination. While Gargour site, situated at 17 km in the south of Sfax, is affected by industrial activity and effluents carried out by streams, Sidi Mansour site, situated at 11 km in the north of Sfax, is less affected by pollution, the two sites having similar ecological characteristics. Previous studies have demonstrated high levels of Cd, Cu, and Zn in Gargour site (Hamza-Chaffai et al., 1999, 2000). R. decussatus were available only in Gargour site. Bivalves collected from this site were caged and transferred the same day in Sidi Mansour site. One cage was kept in Gargour site. The experiment was carried out during 62 days. The cages (100 50 5 cm) were made of a polypropylene mesh (1 cm2) and attached with a nylon rope on stakes (Pellerin-Massicotte et al., 1993). Transplantation was carried out in November 1995 (t = 0). Caged clams (30 – 35 mm length) were sampled at t = 0 (the beginning of transplantation) and t = 62 days (the end of the transplantation). After sampling, clams were dissected and kept in a freezer at 70 jC until preparations and analyses. One set of animals (n = 20 per site and per sampling) was used for metal and metallothionein analyses in the digestive gland. Another set of animals (n = 12 per site and per sampling) was used for MDA (in the digestive gland) and glycogen (in the mantle) determinations. All the analyses were performed in individual animals and not pools of animals. 2.2. Fraction preparation For metal and metallothionein determinations, the digestive gland was used. In fact, while gills are capable of more rapid integration and characterisation of short-term pollution, the digestive gland is important for metal metabolism and is considered as a long-term storage tissue, reflecting persisting contamination (Duquesne and Coll, 1995). The digestive glands were homogenised with a tissue grinder (Eurostar digital, Euro-ST D) in ice-cold 50 mM Tris solution and 4.4 mM h-mercaptoethanol buffer adjusted
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.
Fig. 1. The studied sites; the transplantation experiment was done from Gargour site to Sidi Mansour site. ( ) Sampling areas, ( ! ) main effluents.
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at pH 8.6. The ratio of the Tris solution to the fresh tissue weight was 4 ml/g. The homogenates were centrifuged (50 000 g, 60 min, at 4 jC). The soluble fractions were separated from the pellet, heat-denatured (80 jC/15 min) to precipitate the heat-sensitive compounds, and subsequently centrifuged (30 000 g, 30 min, at 4 jC). The resulting supernatants were used for MTs quantification. During sample preparation, the temperature was maintained at 4 jC to minimise any risk of protein degradation.
buffer, pH 5.0 (5% w/v), and stored at 70 jC until analysis. Glycogen in crude extracts was hydrolysed with amyloglucosidase, and glucose was further analysed by enzymatic determination (Carr and Neff, 1984). Glycogen was estimated as the difference between glucose released in the incubated extracts and the glucose measured in the crude homogenates.
2.3. Metal concentrations in the digestive gland
Confidence intervals (95% level), t-test (95% level), and regression analysis were performed using Shazam program (version 6.1). Using SAS program (version 6.12), we performed three-dimensional graphs.
Each individual was treated according to the method described by Hamza-Chaffai et al. (1995). Cd, Cu, and Zn were analysed in the digestive glands by atomic absorption spectrophotometry (HITACHI Z 8200) using the Zeeman effect (Amiard et al., 1987). The analytical method was validated by intercalibration exercises involving the French laboratories (Coquery and Horvat, 1996). 2.4. Metallothionein determination Metallothioneins were analysed by the differential pulse polarographic assay based on –SH group quantification. Thompson and Cosson (1984) have described this method and Olafson and Olsson (1991) have tested its specificity. Polarographic measurements were made with a PAR Model 174A analyser, a PAR/EG&G Model 303 static mercury drop electrode (SMDE), and a flat-bed X – Y recorder (RE 0089). During MT analyses, the temperature was maintained at 4 jC. Metallothionein concentrations in R. decussatus were calculated using rabbit liver metallothionein MT-I as reference for the calibration curve. Results were expressed as milligram of MTs per gram of wet homogenised tissue.
2.7. Statistical analysis
2.8. Box –Cox transformation The relationship between MT levels and the studied factors (Cd, Cu, Zn, site, and time) was assessed by a nonlinear model. The Box – Cox transformation was used in order to determine the nonlinear functional relation between MTs and the other parameters (Hamza-Chaffai et al., 1999). The general functional form of the Box –Cox transformation can be written as Y ðkÞ ¼ a þ
i¼n X
bi Xi ðkÞ
ðIIÞ
i¼1 k
with X ðkÞ ¼ X k1 , a = 0, when we take the standardised coefficients. In our case, model (II) can be written as MTsðkÞ ¼ a þ b1 CdðkÞ þ b2 CuðkÞ þ b3 ZnðkÞ þ b4 SiteðkÞ þ b5 TimeðkÞ
2.5. Malonedialdehyde (MDA) determinations Malonedialdehyde determination was carried out in the digestive glands, using the method developed by Sunderman et al. (1985) and now known as the measure of thiobarbituric acid-reactive substances (TBARS). The two methods are based on the same principle and use the same reagents (Pellerin-Massicotte, 1997; Sunderman et al., 1985; Janero, 1998). 2.6. Glycogen determination Glycogen provides energy for different metabolic processes. Important glycogen reserves that will serve during gametogenesis are found in the mantle of bivalves (Bayne et al., 1985). Moreover, glycogen is used rapidly when organisms are under stress, and levels of this energy reserve are now commonly used as biomarkers of physiological status (Lagadic et al., 1997). The mantle was then chosen for the measure of glycogen, and for each individual, homogenised in 50 mM citrate
MTs ¼ ½b1 ðCdk 1Þ þ b2 ðCuk 1Þ þ b3 ðZnk 1Þ þ b4 ðSexk 1Þ þ b5 ðSizek 1Þ 1=k In a first stage, we obtained the least squares estimates of a, bi, and r for a fixed value of k. In a second stage, we searched for the value of k, say k0, which maximises the log likelihood function, say Lmax(k), and thus produces the optimal transformation (Box and Cox, 1964). Data were standardised to avoid the problem of unit. Lmax(k) values were determined using Shazam program (version 6.1). 2.9. The use of dummy variable The factors site and time are qualitative and nonparametric variables, thus they were introduced in the mathematical model as dummy variables. In order to distinguish between the two sites, the values 0 and 1 were arbitrary used to design Gargour and Sidi Mansour
A. Hamza-Chaffai et al. / Environment International 28 (2003) 609–617 Table 1 Variation of the whole soft tissues according to time and site
Table 2 Malonedialdehyde (MDA), glycogen, and metallothionein concentrations
Whole soft tissue masses (g) Mean Gargour t = 0 Gargour t = 62 days Sidi Mansour t = 62 days
1.64 1.70 1.79
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Standard deviation 0.26 0.26 0.28
site. It was also the case for the factor time for which we used 0 and 1 to distinguish between the beginning of the experiment (t = 0) and after 62 days of transplantation (t = 62 days).
3. Results 3.1. Variations according to time and site No significant differences in the whole soft tissue weight were observed between groups during the transplantation period (Table 1). However, metal levels (expressed in ng) increased after 62 days of transplantation in Gargour site. In clams transplanted from Gargour to Sidi Mansour site, metal levels did not show significant variation from t = 0 to t = 62 days. When comparing the two sites after 62 days of transplantation, we noticed that Gargour showed higher cadmium and copper amounts than Sidi Mansour (G>SM; t = 62 days) (Fig. 2). It can be seen in Table 2 that MDA concentrations increased significantly in both sites despite the different exposure to metals. For glycogen concentrations, a marked and significant increase was observed in both sites and according to time. Comparing the beginning of the experiment (t = 0) and after 62 days, glycogen increased by a factor 3.5 in Gargour site and a factor 2.8 in Sidi Mansour. At the same time (t = 62 days), the two sites differed by a factor 1.27 (Gargour>Sidi Mansour). Moreover, metallothionein concentrations showed a significant increase in Gargour.
MDA Glycogen MTs (mM/g w/w tissue) (mg/g w/w tissue) (mg/g w/w tissue) Gargour t = 0 (G0) Gargour t = 62 days (G1) Sidi Mansour t=0 (SM0 = G0) Sidi Mansour t = 62 days (SM1)
240.70 (42.41)
1.74 (0.68)
2.118 (0.588)
270.99 (46.86)
6.19 (3.31)
3.293 (1.186)
240.70 (42.41)
1.74 (0.68)
2.118 (0.588)
296.47 (67.00)
4.87 (2.99)
1.434 (0.301)
Data in parentheses are standard deviation (SD).
However, a significant decrease in MTs was observed in Sidi Mansour between t = 0 and t = day 62. 3.2. Multiparametric variation of MDA and glycogen 3.2.1. Malonedialdehyde MDA variations according to site and time are represented by Fig. 3. The linear multiple model is given by the following equation. MDA ¼ 0:43 Sizeðt¼4:21; P<0:01Þ 0 :27Siteðt¼1:95; P<0:01Þ þ 0 :38Timeðt¼2:28; P<0:01Þ
R2 ¼ 0:48
ðAÞ
MDA was inversely correlated to size. Another interesting observation is the strong correlation between MDA levels and the factor time (t = 2.28). The factor site also affected MDA. This is well explained by other findings about the pulse contamination occurring during the studied period and which was confirmed by metal analysis (Fig. 2). 3.2.2. Glycogen According to our objective that is to detect physiological changes after in situ exposure to contamination, we established a correlation between glycogen and the studied
Fig. 2. Variation of soluble metals (ng) according to time and site. Cadmium concentrations were multiplied by 10. G0: Gargour at t = 0. G1: Gargour at t = 62 days. SM0: Sidi Mansour at t = 0. SM1: Sidi Mansour at t = 62 days. **—Significant difference at 95% level; ns—no significant difference.
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Fig. 4. Three-dimensional representation of metallothionein = f (Cd, Zn).
Fig. 3. MDA, MTLP, and glycogen concentrations (means and SD) in R. descussatus from the two sites and at t = 0 and t = 62 days.
parameters. The following model indicates that only zinc, site, and time affected significantly glycogen concentrations. Glycogen ¼ 0:4Znðt¼2:39; P<0:01Þ 0 :55Siteðt¼3:11;P<0:01Þ þ 1 :02 Timeðt¼5:66; P<0:01Þ
R2 ¼ 0:54
ðBÞ
The negative sign of zinc coefficient indicates that zinc was inversely correlated to glycogen (t = 2.935, P < 0.01), thus showing a decrease in the storage of energy due probably to zinc (Pellerin-Massicotte et al., 1993). 3.3. Relationship between metallothionein and metals, MTs=f(Cd, Cu, and Zn) We established a nonlinear relationship between MTs and metals. It can be seen that MT levels are mainly affected by cadmium followed by zinc. For copper, the correlation was less significant. The relationship was established as shown in model (I),
The representation of MTs = f(Cd, Zn) (Fig. 4) showed that for high cadmium and high zinc levels, MTs increase (Fig. 4, area A). The lowest MT levels are observed in an area corresponding to low zinc and cadmium concentrations (area B). Minimal levels of MTs were obtained for lowest cadmium concentrations. The important effect of Cd is visualised in the three-dimensional representation by area A* corresponding to highest cadmium concentrations. Nevertheless, area B* reflected highest zinc concentrations. When comparing these two areas, we notice the important effect of cadmium on MT levels compared to those of zinc. According to the same model (I), an increase in cadmium concentrations leads to an increase in MT levels (the coefficient of cadmium is positive and highly significant, t = 4.36). However, copper acted negatively. Fig. 5 shows that highest MT levels are observed in areas corresponding to high Cd and low Cu concentrations (area C). However, the lowest MT levels are observed in area D that corresponds to highest Cd and Cu concentrations. This is an expected result because in the model, Cd and Cu coefficients have not the same sign and therefore act inversely.
MTsk ¼ 0:46Cdkðt¼4:36; P<0:01Þ 1 :18 Cukðt¼1:72; P<0:05Þ þ 0 :23 Znkðt¼2:10; P<0:01Þ ðR2 ¼ 0:42 k ¼ 0:73Þ ðIÞ
3.4. A three-dimensional representation for metallothionein and metals According to the nonlinear model giving MTs as a function of metal (model (I)), a three-dimensional representation was performed in order to visualise the combined effect of metals on MTs. Representations are shown in Figs. 4 and 5.
Fig. 5. Three-dimensional representation of metallothionein = f (Cd, Cu).
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3.5. Metallothionein variations with metals, size, time, and site, MTs=f(Cd, Cu, Zn, site, and time) We used a nonlinear model, based on Box –Cox transformation, to establish possible relationship between MTs, Cd, Cu, Zn, site, and time. Moreover, the variables site and time were introduced as dummy variables in the matrix. They take the value 1 or 0 to distinguish sites and times. MTsk ¼ 0:22Cdkðt¼2:34Þ 0 :13Cukðt¼1:48Þ þ 0 :13Znkðt¼1:46Þ 0 :64 Sitekðt¼6:94Þ þ 0 :46 Timekðt¼5:45Þ ðR2 ¼ 0:59 k ¼ 0:72Þ
ðIIÞ
As shown in model (II), the high values of t (Student) indicate that metallothionein levels are highly affected by the factor time (t = 5.54, significant at 99% level) and site (t = 6.94, significant at 99% level). When comparing the studied metals, cadmium could be considered as the metal that affects more metallothionein levels.
4. Discussion Compared to in situ sampling and to laboratory experiments, the present work constitutes a field experiment with the advantages of keeping organisms in their natural environment and to use animals of the same size, originating from the same population. The three studied biomarkers seemed to respond to metal contamination and vary according to site and time as it was shown in models A, B, and II. In fact, the factors site and time are related to metal contamination in Gargour site in which cadmium and copper increased after 62 days of transplantation (Fig. 2) leading to an increase of MT levels (Fig. 3). Our explanation is that marine coastal streams are carrying industrial effluents with high metal content as it was demonstrated by Illou (1999) and Hamza-Chaffai et al. (2000). Malonedialdehyde was inversely correlated to size in R. decussatus even if no significant differences in the whole soft tissue weights were observed between groups during the transplantation period. MDA levels were, however, strongly correlated to the factor time, an interesting finding which could be explained by two things, the pulse contamination registered at t = 62 days, and the effect of the reproductive state of the animals. The second explanation was brought by Viarengo et al. (1991) in the digestive gland of mussels where MDA levels fluctuated significantly with the factor month. During the studied period, an inter-site and an intra-site increase in glycogen concentrations was observed. There are two possible explanations: (i) The trophic status of Gargour site could be more favourable for glycogen storage than in Sidi Mansour.
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(ii) Since Gargour site receives industrial pollutants, the energy allocation could be blocked for other physiological processes like reproduction (Gauthier-Clerc et al., 2002). In fact, some contaminants act as endocrine disrupters on metabolic pathways related to gametogenesis (Safe et al., 1991) that needs glycogen transformation to lipids (Gabbott, 1975). Glycogen decreased with zinc concentrations. It is not the first time that such result was found; the same correlation was observed in a temperate ecosystem in M. edulis and M. arenaria (Pellerin-Massicotte et al., 1993; Saint-Hilaire and Pellerin, 1995). This variation is very interesting from two points of views: (i) It reinforces the use of biochemical parameters related to energy storage as biomarkers of health condition. (ii) This decrease of glycogen related to Zn contamination could also serve as a predictive indicator for the ecosystem dysfunction provoked by anthropogenic influences. It should be noted that in the studied sites, industries, mainly those for phosphate treatment, release high levels of zinc (6 t per year) and cadmium (1 t per year) in the coastal environment (Hamza-Chaffai, 1993; Sarbaji, 1991). Glycogen was also highly correlated to site, indicating at least an inter-site variation of energy storage. Since contamination status was different in the two studied sites, it then appears clearly again that there is a strong link between zinc and glycogen. Moreover, a strong correlation was observed between glycogen and time. This could be explained at least partly by the reproductive status of R. decussatus, showing an increased coefficient index between November and January (Viarengo et al., 1991) and/or by the observed increased contamination. Metallothionein concentrations are correlated principally to cadmium and zinc. This finding was also obtained previously in a natural population of R. decussatus from Gargour site (Hamza-Chaffai et al., 1999). The strong relationship between MTs and cadmium was also evidenced after in vivo contamination of R. decussatus (Bebianno et al., 1993, 1994). The importance of Cd at the transcriptional level of MTs (mRNA) was evidenced in molluscs (Unger and Roesijadi, 1996). It is actually well known that MT levels are not only affected by metals but also by other factors such as size, site, or time. Therefore, we established a nonlinear model relating all these parameters. When considered together (Cd, Cu, Zn, site, time), MT levels were highly correlated to cadmium, site, and time as shown in model II. These observations are in agreement with previous results about MTs and metals in the digestive gland of R. decussatus (HamzaChaffai et al., 1999). The strong link between MTs site and time could be explained by the variation of metal contamination with time and site.
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The methods used in statistical treatments present some advantages. In fact, the Box –Cox transformation allowed the estimation of the nonlinear model reflecting the relationship between metallothionein and metals. Moreover, the use of dummy variables permitted the evaluation of the effect of factors site and time, which are qualitative variables. It is evident that factors other than contamination affect biomarker levels and we should evaluate their importance in order to validate biomarkers in field conditions. The advantage of the three-dimensional representation was to visualise the combined effect of metals on MT levels and the importance of each one.
5. Conclusion The in situ measure of metallothioneins, MDA, and glycogen levels in R. decussatus has many advantages particularly to promote the possible use of this species in a national biomonitoring program based on biomarkers. The reproductive cycle of R. decussatus should be therefore thoroughly investigated in sites differing by their levels of contaminants. This will enlighten the causes of glycogen increase and will clarify the deleterious effects of pollution on reproduction.
Acknowledgements Two research programs, FICU2000/PAS/42 and IFS grant A/3061-1, supported this work. The authors thank Mohamed Chaffai for statistical help and Mohamed Makloufi for his technical assistance. References Amiard JC, Cosson RP. Les me´tallothione´ines. In: Lagadic L, Caquet T, Amiard JC, Ramade F, editors. Biomarqueurs en e´cotoxicologie Aspects fondamentaux. Paris: Masson; 1997. p. 53 – 66. Amiard JC, Pineau A, Boiteau H, Me´tayer C, Amiard-Triquet C. Application de la spectrome´trie d’absorption atomique Zeeman aux dosages de huit e´le´ments traces (Ag, Cd, Cr, Cu, Mn, Ni, Pb et Se) dans des matrices biologiques solides. Water Res 1987;21:693 – 7. Bayne BL, Brown DA, Burns K, Dixon DR. The effect of stress and pollution on marine animals. New York: Praeger Publishers; 1985. p. 384. Bebianno MJ, Nott JA, Langston WJ. Cadmium metabolism in the clam Ruditapes decussata, the role of metallothioneins. Aquat Toxicol 1993;27:315 – 34. Bebianno MJ, Serafin MAP, Rita MF. Involvement of metallothionein in cadmium accumulation and elimination in the clam Ruditapes decussata. Bull Environ Contam Toxicol 1994;53:726 – 32. Box GEP, Cox DR. An analysis of transformation. J R Stat Soc 1964; 211 – 52. Brown BE. The form and function of metal-containing ‘‘granules’’ in invertebrate tissues. Biol Rev 1982;57:621 – 67. Carr RS, Neff JM. Quantitative semi-automated enzymatic assay for tissue glycogen. Comp Biochem Physiol 1984;77B(3):447 – 9. C.G.P. Annuaire des statistiques des peˆches en Tunisie. Tunisie: Ministe`re de l’agriculture; 1996.
Cheeseman KM. Effect of scavengers and inhibitors on lipid peroxidation in rat liver microsomes. In: Mac Brien DC, Salter TF, editors. Free radicals, lipid peroxidation and cancer. New York: Academic Press; 1982. p. 196 – 211. Coquery M, Horvat M. The analytical performance study for MEDPOl area: determination of trace elements in marine sediment SD-MEDPOL-1/TM and in fish homogenate MA-MEDPOL-1/TM. Report IAEA, Monaco; 1996. p. 85. Cosson RP, Amiard JC. Utilisation des me´tallothione´ines comme biomarqueurs d’exposition aux me´taux. In: Lagadic L, Caquet T, Amiard JC, Ramade F, editors. Utilisation des biomarqueurs pour la surveillance de la qualite´ de l’environnement. Londres: Lavoisier; 1998. p. 77 – 109. Duquesne SJ, Coll JC. Metal accumulation in the clam Tridacna corcea under natural and experimental conditions. Aquat Toxicol 1995;32: 239 – 53. Gabbott PA. Storage cycles in marine bivalve molluscs, a hypothesis concerning the relationship between glycogen metabolism and gametogenesis. In: Barnes H, editor. Proc 9th Europ Mar Biol Symp, Aberdeen Univ Press; 1975. p. 191 – 211. Gauthier-Clerc S, Pellerin J, Blaise C, Gagne´ F. Delayed gametogenesis of Mya arenaria in the sagueay fjord (Canada): a consequence of endocrine disrupters. Comp Biochem Physiol C 2002;131(4):457 – 67. Hamza-Chaffai A. Bioaccumulation me´tallique et me´tallothione´ines chez des poissons de la coˆte de Sfax (Golfe de Gabe`s), Doctorat de spe´cialite´, Universite´ de Sfax; 1993. 168 pp. Hamza-Chaffai A, Cosson RP, Amiard-Triquet C, El Abed A. Physicochemical forms of storage of metals (Cd, Cu and Zn) and metallothionein like proteins in fish from the Tunisian coast, ecotoxicological consequences. Comp Biochem Physiol 1995;111C(2):329 – 41. Hamza-Chaffai A, Rome´o M, El Abed A. Heavy metals in different fishes from the middle eastern coast of Tunisia. Bull Environ Contam Toxicol 1996;56:766 – 73. Hamza-Chaffai A, Amiard-Triquet C, El Abed A. Metallothionein-like protein, is it an efficient biomarker of metal contamination? A case study based on fish from the Tunisian coast. Arch Environ Contam Toxicol 1997;33:53 – 62. Hamza-Chaffai A, Rome´o M, Gnassia-Barelli M, El Abed A. Effect of copper and lindane on some biomarkers measured in the clam Ruditapes decussatus. Bull Environ Contam Toxicol 1998;61:397 – 404. Hamza-Chaffai A, Amiard JC, Cosson RP. Relationship between metallothionein and metals in a natural population of clam Ruditapes decussatus from Sfax coast, a non-linear model using Box – Cox transformation. Comp Biochem Physiol 1999;123:153 – 63. Hamza-Chaffai A, Amiard JC, Pellerin J, Joux L, Berthet B. The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure. Comp Biochem Physiol 2000;127: 185 – 97. Illou S. Impact des rejets telluriques d’origines domestiques et industrielle sur les environnement coˆtiers: cas du littoral nord de la ville de Sfax (Tunisie). Doctorat de spe´cialite´, Universite´ de Tunis II; 1999. 259 pp. Janero D. Malonedialdehyde and thiobarbutiric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissues injury. Free Radic Biol Med 1998;9:515 – 40. Lagadic L, Caquet T, Amiard JC. Biomarqueurs en e´cotoxicologie, principes et de´finitions. In: Lagadic L, Caquet T, Amiard JC, Ramade F, editors. Biomarqueurs en e´cotoxicologie. Aspects fondamentaux. Paris: Masson; 1997. p. 1 – 9. Mason AZ, Jenkins KD. Metal detoxification in aquatic organisms. In: Tessier A, Turner DR, editors. Metal speciation and bioavailability in aquatic systems. Chichester: Wiley; 1995. p. 479 – 607. Murrat T, Brown T, Depeledge H. Determinant of trace metal concentrations in marine organisms. In: Langston B, Bebianno MJ, editors. Metal metabolism in aquatic environments. London: Chapman & Hall; 1998. p. 185 – 217. Narbonne JF, Daubeze M, Clerandeau C, Garrigues P. Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts. Biomarkers 1989;4:415 – 25.
A. Hamza-Chaffai et al. / Environment International 28 (2003) 609–617 NAS. The international Mussel Watch. Washington (DC): US Nat Acad Sci; 1980. Olafson RW, Olsson PE. Electrochemical detection of metallothionein. Methods Enzymol 1991;205:205 – 13. Pellerin J, Audet C. Les indicateurs physiologiques, leur utilite´ en e´cotoxicologie. Proceedings, physiologie et biochimie compare´e, 37e Congre`s Annuel de la Soc. Can. Zoologie, Kelowna, CB [Mai, 1998]. Pellerin J, Vincent B, Pelletier E. Evaluation e´cotoxicologique de la qualite´ de la baie des Anglais (Que´bec). Water Pollut Res J Can 1993; 28:665 – 89. Pellerin-Massicotte J. Oxidative processes as indicators of chemical stress in marine bivalves. J Aquat Ecosyst Health 1994;3:101 – 11. Pellerin-Massicotte J. Influence of elevated temperature and air exposure on MDA levels and catalase activities in digestive glands of the blue mussel (Mytilus edulis). J Rech Oce´anogr 1997;22(3):91 – 8. Pellerin-Massicotte J, Vincent B, Pelletier E. Evaluation e´cotoxicologique de la baie des anglais a` Baie-Comeau (Que´bec). Water poll Res J Canada 1993;28(4):665 – 686. Phillips DJH. The use of bio-indicators in monitoring conservative contaminants. Program design imperatives. Mar Pollut Bull 1986;17:10 – 7. Roesijadi G. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat Toxicol 1992;22:81 – 114. Roesijadi G. Metallothionein induction as a measure of response to metal exposure in aquatic animals. Environ Health Prospect 1994;102(2): 3293 – 405. Roesijadi G. Metal transfer as a mechanism for metallothionein-mediated metal detoxification. Cell Mol Biol 2000;46:81 – 114. Safe S, Astroff B, Harris T, Zacharewski R, Dockerson M, Romkes M, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds as
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antioestrogens, characterization and mechanism of action. Pharmacol Toxicol 1991;69:400 – 9. Saint-Hilaire N, Pellerin J. Variations of scope for growth in Mytilus edulis L and Mya arenaria after a chronic exposure to TBT. 22e annual of the Aquatic Toxicity Workshop, St Andrews, NB [October, 1995]. Sarbaji MM. Contribution a` l’e´tude de l’impact des rejets industriels et urbains sur l’environnement marin de la re´gion de Sfax-essais de traitements. DEA Universite´ de Tunis, Tunis; 1991 [139 p.]. Stegeman JJ, Brouwer M, Di Giulio RT, Fo¨rlin L, Fowler BA, Sanders BM, et al. Enzyme and protein synthesis as indicators of contaminant exposure and effect. In: Hugget RJ, Kimerle RA, Bergman HL, editors. Biomarkers-biochemical, physiological and histological markers of anthropogenic stress. Chelsea, MI: Lewis Publishers; 1992. p. 235 – 335. Sunderman FW, Marzoul A, Hopfer SM, Zaharia O, Reid MC. Increased lipid peroxidation in tissues of nickel chloride-treated rats. Ann Clin Lab Sci 1985;15:229 – 36. Thompson JAJ, Cosson RP. An improved electrochemical method for the quantification of metallothionein in marine organisms. Mar Environ Res 1984;11:137 – 52. Unger ME, Roesijadi G. Increase in metallothionein m RNA accumulation during Cd challenge in oyster pre-exposed to Cd. Aquat Toxicol 1996; 34:185 – 93. Viarengo A, Nott JA. Mechanisms of heavy metal cation homeostasis in marine invertebrates. Comp Biochem Physiol 1993;104C(3):355 – 72. Viarengo A, Canesi L, Pertica M, Livingstone DR. Seasonal variation in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels. Comp Biochem Physiol 1991;100C(1/2):187 – 90. Zairi M, Rouis MJ. Impact environnementaux du stockage du phosphogypse a` Sfax (Tunisie). Bull Labo Ponts Chausse´es 1999;219:29 – 40.
Health assessment of a marine bivalve Ruditapes decussatus from the Gulf of Gabe`s (Tunisia) A. Hamza-Chaffai a,*, J. Pellerin b, J.C. Amiard c a
IPEIS, Universite de Sfax, BP 805, 3018 Sfax, Tunisia ISMer-UQAR, 310 alle´e des Ursulines, Rimouski, QC, Canada G5L 3A1 c GDR1117-CNRS-ISOMer, 2 rue de la Houssinie`re, 40322 Nantes cedex 3, France b
Abstract Metallothioneins (MTs), malonedialdehyde (MDA), and glycogen concentrations were determined in order to assess the health status of Ruditapes decussatus exposed to in situ contamination, showing the effects of time, site, and metal contamination on these three biomarkers. Metallothionein was positively correlated with Cd and Zn. If introduced in a multiple model, the factors site and time were shown to decrease significantly MT concentrations. MDA was inversely correlated with size of the animals and was affected by the factor time. Glycogen was inversely correlated with zinc and was affected by both site and time. This study constitutes a field-based validation of a multiparametric approach using specific and nonspecific biomarkers. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: Biomarkers; Glycogen; Metallothionein; Malonedialdehyde; Metals; Ruditapes decussatus
1. Introduction The Gulf of ‘‘Gabe`s’’ is situated in the southeastern coast of Tunisia. Shallow waters, weak currents, high salinity, and temperature characterise this area. Compared to other Mediterranean areas, tides are exceptionally important. From a biological point of view, coastal Posidonia and Caulerpa constitute a spawning and refuge area for fish larvae (Hamza-Chaffai, 1993). This ecosystem is known to be very rich in aquatic resources and contributes to about 65% of the national production in Tunisia (C.G.P., 1996). Unfortunately, an important industrial activity, mainly crude phosphate treatment, chemical industries, tannery, and plastic plants, is now being developed along the coasts and could threaten this marine ecosystem (Hamza-Chaffai et al., 1995, 1996, 1997; Zairi and Rouis, 1999). In fact, since 1990, destruction of Posidonia grassland and a decrease in fish production have been observed. Besides the ecosystem damages engendered by this pollution, the human health could also be affected if toxic pollutants concentration become high in edible marine organisms.
* Corresponding author. Tel.: +216-4-241-403; fax: +216-4-246-347. E-mail address: [email protected] (A. Hamza-Chaffai).
An urgent national bio-monitoring program is therefore needed to assess the degree of pollution and evaluate the quality of the marine environment. Under similar circumstances, other countries have extensively used marine molluscs such as mussels and oysters in monitoring programs, like the Mussel watch program (NAS, 1980; Phillips, 1986). In the case of Tunisia, these species are available only in few northern sites. Alternatively, Ruditapes decussatus is widely distributed along the Tunisian coast. This species is a sedentary filter feeding marine bivalve, thus satisfying criteria in biomonitoring programs for a good bioindicator of pollution (NAS, 1980; Phillips, 1986). This species as well represents an important economic endpoint, R. decussatus being mostly exported to Europe. Biomonitoring programs based on measures of contaminants in marine organisms are also interesting from a human health point of view. However, pollutant levels do not give information about their toxicological significance, particularly because xenobiotics can be stored in various forms such as insoluble precipitates and concretions (Brown, 1982; Mason and Jenkins, 1995; Murrat et al., 1998). Consequently, recent biomonitoring programs are now involving biomarkers. These are measurable parameters at different levels of biological organisation. Biomarkers reflect changes in the metabolic regulatory processes resul-
0160-4120/02/$ - see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 0 - 4 1 2 0 ( 0 2 ) 0 0 1 0 2 - 2
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ting from the effect of anthropogenic stressors (Lagadic et al., 1997). This approach should be multiparametric, using different and complementary biomarkers (Narbonne et al., 1989) to reflect the effects of different contaminants. However, we also need to consider variations linked to biotic and abiotic factors. In fact, the physiological state of an organism within an ecosystem is the result of an equilibrium between the influences of anthropogenic, abiotic, and biotic factors (Pellerin-Massicotte, 1994). In the present work, we have focused on three biomarkers: metallothioneins (MTs), malonedialdehyde (MDA), and glycogen, well known to be sensitive to pollutant-exposition (Bebianno et al., 1994; Pellerin-Massicotte, 1994). Metallothioneins (MTs) are low molecular weight, heat stable proteins of high cysteine content. While low basal levels of MTs are generally present in marine organisms from unpolluted areas, the synthesis of MTs is induced as a direct response to Cd, Cu, Zn, and Hg exposure (Viarengo and Nott, 1993; Roesijadi, 2000). These proteins have been identified in various tissues of vertebrates and invertebrates (Amiard and Cosson, 1997; Cosson and Amiard, 1998). Given their molecular properties and their role in metal uptake, transport, storage, and excretion, MTs offer considerable potential as a contaminant-specific biochemical indicator of metal exposure (Roesijadi, 1992, 1994; Stegeman et al., 1992). In marine invertebrates, MTs have been implicated in the storage, transport, and exchange of essential metals (Cu, Zn), and in the detoxification of nonessential metals (Ag, Cd, Hg) (Viarengo and Nott, 1993). Earlier work in our laboratory has shown the presence of metallothioneins in the digestive gland of R. decussatus in field conditions (Hamza-Chaffai et al., 1999, 2000) and after an experimental contamination (Hamza-Chaffai et al., 1998). The second biomarker is malonedialdehyde (MDA), which is a product of lipid peroxidation due to overproduction of oxyradicals in cells, following contaminant exposure or stress due to natural conditions (Pellerin-Massicotte, 1997; Cheeseman, 1982). Lipid peroxidation is considered as an important feature in cellular injury. It results from free radical reactions in biological membranes, which are rich in polyunsaturated fatty acids. MDA has been used extensively to assess detrimental effects of various pollutants (PellerinMassicotte, 1997; Viarengo et al., 1991). The third biomarker is glycogen, which is the fuel for different metabolic and physiological processes. Glycogen has been shown to respond quite well to complex and diffuse contamination situations (Pellerin et al., 1993; Pellerin-Massicotte, 1994; Gauthier-Clerc et al., 2002). Decreased levels of glycogen related to an altered growth (Pellerin and Audet, 1998) were observed in mussels (Mytilus edulis) and clams (Mya arenaria) after an exposure to pulp and paper mill effluents (Pellerin et al., 1993), as well as in a mesocosms where bivalves were exposed to oil and a silicon-based polymer. In the present work, these three biomarkers were used in order to assess the health status of R. decussatus in a
contaminated marine ecosystem. We have carried out a field experiment designed to evaluate the potential of MTs, MDA, and glycogen as biomarkers of in situ contamination. Our objective was to study the effect of site, time of transplantation, and metals (Cd, Cu, Zn) on the studied biomarkers.
2. Materials and methods 2.1. Study area and experimental design The transplantation experiment was carried out in two sites situated in the southeastern coast of Tunisia (Fig. 1). The experiment was designed to follow changes associated with increased metal exposure as a function of time. The two sites differed in their level of contamination. While Gargour site, situated at 17 km in the south of Sfax, is affected by industrial activity and effluents carried out by streams, Sidi Mansour site, situated at 11 km in the north of Sfax, is less affected by pollution, the two sites having similar ecological characteristics. Previous studies have demonstrated high levels of Cd, Cu, and Zn in Gargour site (Hamza-Chaffai et al., 1999, 2000). R. decussatus were available only in Gargour site. Bivalves collected from this site were caged and transferred the same day in Sidi Mansour site. One cage was kept in Gargour site. The experiment was carried out during 62 days. The cages (100 50 5 cm) were made of a polypropylene mesh (1 cm2) and attached with a nylon rope on stakes (Pellerin-Massicotte et al., 1993). Transplantation was carried out in November 1995 (t = 0). Caged clams (30 – 35 mm length) were sampled at t = 0 (the beginning of transplantation) and t = 62 days (the end of the transplantation). After sampling, clams were dissected and kept in a freezer at 70 jC until preparations and analyses. One set of animals (n = 20 per site and per sampling) was used for metal and metallothionein analyses in the digestive gland. Another set of animals (n = 12 per site and per sampling) was used for MDA (in the digestive gland) and glycogen (in the mantle) determinations. All the analyses were performed in individual animals and not pools of animals. 2.2. Fraction preparation For metal and metallothionein determinations, the digestive gland was used. In fact, while gills are capable of more rapid integration and characterisation of short-term pollution, the digestive gland is important for metal metabolism and is considered as a long-term storage tissue, reflecting persisting contamination (Duquesne and Coll, 1995). The digestive glands were homogenised with a tissue grinder (Eurostar digital, Euro-ST D) in ice-cold 50 mM Tris solution and 4.4 mM h-mercaptoethanol buffer adjusted
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.
Fig. 1. The studied sites; the transplantation experiment was done from Gargour site to Sidi Mansour site. ( ) Sampling areas, ( ! ) main effluents.
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at pH 8.6. The ratio of the Tris solution to the fresh tissue weight was 4 ml/g. The homogenates were centrifuged (50 000 g, 60 min, at 4 jC). The soluble fractions were separated from the pellet, heat-denatured (80 jC/15 min) to precipitate the heat-sensitive compounds, and subsequently centrifuged (30 000 g, 30 min, at 4 jC). The resulting supernatants were used for MTs quantification. During sample preparation, the temperature was maintained at 4 jC to minimise any risk of protein degradation.
buffer, pH 5.0 (5% w/v), and stored at 70 jC until analysis. Glycogen in crude extracts was hydrolysed with amyloglucosidase, and glucose was further analysed by enzymatic determination (Carr and Neff, 1984). Glycogen was estimated as the difference between glucose released in the incubated extracts and the glucose measured in the crude homogenates.
2.3. Metal concentrations in the digestive gland
Confidence intervals (95% level), t-test (95% level), and regression analysis were performed using Shazam program (version 6.1). Using SAS program (version 6.12), we performed three-dimensional graphs.
Each individual was treated according to the method described by Hamza-Chaffai et al. (1995). Cd, Cu, and Zn were analysed in the digestive glands by atomic absorption spectrophotometry (HITACHI Z 8200) using the Zeeman effect (Amiard et al., 1987). The analytical method was validated by intercalibration exercises involving the French laboratories (Coquery and Horvat, 1996). 2.4. Metallothionein determination Metallothioneins were analysed by the differential pulse polarographic assay based on –SH group quantification. Thompson and Cosson (1984) have described this method and Olafson and Olsson (1991) have tested its specificity. Polarographic measurements were made with a PAR Model 174A analyser, a PAR/EG&G Model 303 static mercury drop electrode (SMDE), and a flat-bed X – Y recorder (RE 0089). During MT analyses, the temperature was maintained at 4 jC. Metallothionein concentrations in R. decussatus were calculated using rabbit liver metallothionein MT-I as reference for the calibration curve. Results were expressed as milligram of MTs per gram of wet homogenised tissue.
2.7. Statistical analysis
2.8. Box –Cox transformation The relationship between MT levels and the studied factors (Cd, Cu, Zn, site, and time) was assessed by a nonlinear model. The Box – Cox transformation was used in order to determine the nonlinear functional relation between MTs and the other parameters (Hamza-Chaffai et al., 1999). The general functional form of the Box –Cox transformation can be written as Y ðkÞ ¼ a þ
i¼n X
bi Xi ðkÞ
ðIIÞ
i¼1 k
with X ðkÞ ¼ X k1 , a = 0, when we take the standardised coefficients. In our case, model (II) can be written as MTsðkÞ ¼ a þ b1 CdðkÞ þ b2 CuðkÞ þ b3 ZnðkÞ þ b4 SiteðkÞ þ b5 TimeðkÞ
2.5. Malonedialdehyde (MDA) determinations Malonedialdehyde determination was carried out in the digestive glands, using the method developed by Sunderman et al. (1985) and now known as the measure of thiobarbituric acid-reactive substances (TBARS). The two methods are based on the same principle and use the same reagents (Pellerin-Massicotte, 1997; Sunderman et al., 1985; Janero, 1998). 2.6. Glycogen determination Glycogen provides energy for different metabolic processes. Important glycogen reserves that will serve during gametogenesis are found in the mantle of bivalves (Bayne et al., 1985). Moreover, glycogen is used rapidly when organisms are under stress, and levels of this energy reserve are now commonly used as biomarkers of physiological status (Lagadic et al., 1997). The mantle was then chosen for the measure of glycogen, and for each individual, homogenised in 50 mM citrate
MTs ¼ ½b1 ðCdk 1Þ þ b2 ðCuk 1Þ þ b3 ðZnk 1Þ þ b4 ðSexk 1Þ þ b5 ðSizek 1Þ 1=k In a first stage, we obtained the least squares estimates of a, bi, and r for a fixed value of k. In a second stage, we searched for the value of k, say k0, which maximises the log likelihood function, say Lmax(k), and thus produces the optimal transformation (Box and Cox, 1964). Data were standardised to avoid the problem of unit. Lmax(k) values were determined using Shazam program (version 6.1). 2.9. The use of dummy variable The factors site and time are qualitative and nonparametric variables, thus they were introduced in the mathematical model as dummy variables. In order to distinguish between the two sites, the values 0 and 1 were arbitrary used to design Gargour and Sidi Mansour
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Table 2 Malonedialdehyde (MDA), glycogen, and metallothionein concentrations
Whole soft tissue masses (g) Mean Gargour t = 0 Gargour t = 62 days Sidi Mansour t = 62 days
1.64 1.70 1.79
613
Standard deviation 0.26 0.26 0.28
site. It was also the case for the factor time for which we used 0 and 1 to distinguish between the beginning of the experiment (t = 0) and after 62 days of transplantation (t = 62 days).
3. Results 3.1. Variations according to time and site No significant differences in the whole soft tissue weight were observed between groups during the transplantation period (Table 1). However, metal levels (expressed in ng) increased after 62 days of transplantation in Gargour site. In clams transplanted from Gargour to Sidi Mansour site, metal levels did not show significant variation from t = 0 to t = 62 days. When comparing the two sites after 62 days of transplantation, we noticed that Gargour showed higher cadmium and copper amounts than Sidi Mansour (G>SM; t = 62 days) (Fig. 2). It can be seen in Table 2 that MDA concentrations increased significantly in both sites despite the different exposure to metals. For glycogen concentrations, a marked and significant increase was observed in both sites and according to time. Comparing the beginning of the experiment (t = 0) and after 62 days, glycogen increased by a factor 3.5 in Gargour site and a factor 2.8 in Sidi Mansour. At the same time (t = 62 days), the two sites differed by a factor 1.27 (Gargour>Sidi Mansour). Moreover, metallothionein concentrations showed a significant increase in Gargour.
MDA Glycogen MTs (mM/g w/w tissue) (mg/g w/w tissue) (mg/g w/w tissue) Gargour t = 0 (G0) Gargour t = 62 days (G1) Sidi Mansour t=0 (SM0 = G0) Sidi Mansour t = 62 days (SM1)
240.70 (42.41)
1.74 (0.68)
2.118 (0.588)
270.99 (46.86)
6.19 (3.31)
3.293 (1.186)
240.70 (42.41)
1.74 (0.68)
2.118 (0.588)
296.47 (67.00)
4.87 (2.99)
1.434 (0.301)
Data in parentheses are standard deviation (SD).
However, a significant decrease in MTs was observed in Sidi Mansour between t = 0 and t = day 62. 3.2. Multiparametric variation of MDA and glycogen 3.2.1. Malonedialdehyde MDA variations according to site and time are represented by Fig. 3. The linear multiple model is given by the following equation. MDA ¼ 0:43 Sizeðt¼4:21; P<0:01Þ 0 :27Siteðt¼1:95; P<0:01Þ þ 0 :38Timeðt¼2:28; P<0:01Þ
R2 ¼ 0:48
ðAÞ
MDA was inversely correlated to size. Another interesting observation is the strong correlation between MDA levels and the factor time (t = 2.28). The factor site also affected MDA. This is well explained by other findings about the pulse contamination occurring during the studied period and which was confirmed by metal analysis (Fig. 2). 3.2.2. Glycogen According to our objective that is to detect physiological changes after in situ exposure to contamination, we established a correlation between glycogen and the studied
Fig. 2. Variation of soluble metals (ng) according to time and site. Cadmium concentrations were multiplied by 10. G0: Gargour at t = 0. G1: Gargour at t = 62 days. SM0: Sidi Mansour at t = 0. SM1: Sidi Mansour at t = 62 days. **—Significant difference at 95% level; ns—no significant difference.
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Fig. 4. Three-dimensional representation of metallothionein = f (Cd, Zn).
Fig. 3. MDA, MTLP, and glycogen concentrations (means and SD) in R. descussatus from the two sites and at t = 0 and t = 62 days.
parameters. The following model indicates that only zinc, site, and time affected significantly glycogen concentrations. Glycogen ¼ 0:4Znðt¼2:39; P<0:01Þ 0 :55Siteðt¼3:11;P<0:01Þ þ 1 :02 Timeðt¼5:66; P<0:01Þ
R2 ¼ 0:54
ðBÞ
The negative sign of zinc coefficient indicates that zinc was inversely correlated to glycogen (t = 2.935, P < 0.01), thus showing a decrease in the storage of energy due probably to zinc (Pellerin-Massicotte et al., 1993). 3.3. Relationship between metallothionein and metals, MTs=f(Cd, Cu, and Zn) We established a nonlinear relationship between MTs and metals. It can be seen that MT levels are mainly affected by cadmium followed by zinc. For copper, the correlation was less significant. The relationship was established as shown in model (I),
The representation of MTs = f(Cd, Zn) (Fig. 4) showed that for high cadmium and high zinc levels, MTs increase (Fig. 4, area A). The lowest MT levels are observed in an area corresponding to low zinc and cadmium concentrations (area B). Minimal levels of MTs were obtained for lowest cadmium concentrations. The important effect of Cd is visualised in the three-dimensional representation by area A* corresponding to highest cadmium concentrations. Nevertheless, area B* reflected highest zinc concentrations. When comparing these two areas, we notice the important effect of cadmium on MT levels compared to those of zinc. According to the same model (I), an increase in cadmium concentrations leads to an increase in MT levels (the coefficient of cadmium is positive and highly significant, t = 4.36). However, copper acted negatively. Fig. 5 shows that highest MT levels are observed in areas corresponding to high Cd and low Cu concentrations (area C). However, the lowest MT levels are observed in area D that corresponds to highest Cd and Cu concentrations. This is an expected result because in the model, Cd and Cu coefficients have not the same sign and therefore act inversely.
MTsk ¼ 0:46Cdkðt¼4:36; P<0:01Þ 1 :18 Cukðt¼1:72; P<0:05Þ þ 0 :23 Znkðt¼2:10; P<0:01Þ ðR2 ¼ 0:42 k ¼ 0:73Þ ðIÞ
3.4. A three-dimensional representation for metallothionein and metals According to the nonlinear model giving MTs as a function of metal (model (I)), a three-dimensional representation was performed in order to visualise the combined effect of metals on MTs. Representations are shown in Figs. 4 and 5.
Fig. 5. Three-dimensional representation of metallothionein = f (Cd, Cu).
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3.5. Metallothionein variations with metals, size, time, and site, MTs=f(Cd, Cu, Zn, site, and time) We used a nonlinear model, based on Box –Cox transformation, to establish possible relationship between MTs, Cd, Cu, Zn, site, and time. Moreover, the variables site and time were introduced as dummy variables in the matrix. They take the value 1 or 0 to distinguish sites and times. MTsk ¼ 0:22Cdkðt¼2:34Þ 0 :13Cukðt¼1:48Þ þ 0 :13Znkðt¼1:46Þ 0 :64 Sitekðt¼6:94Þ þ 0 :46 Timekðt¼5:45Þ ðR2 ¼ 0:59 k ¼ 0:72Þ
ðIIÞ
As shown in model (II), the high values of t (Student) indicate that metallothionein levels are highly affected by the factor time (t = 5.54, significant at 99% level) and site (t = 6.94, significant at 99% level). When comparing the studied metals, cadmium could be considered as the metal that affects more metallothionein levels.
4. Discussion Compared to in situ sampling and to laboratory experiments, the present work constitutes a field experiment with the advantages of keeping organisms in their natural environment and to use animals of the same size, originating from the same population. The three studied biomarkers seemed to respond to metal contamination and vary according to site and time as it was shown in models A, B, and II. In fact, the factors site and time are related to metal contamination in Gargour site in which cadmium and copper increased after 62 days of transplantation (Fig. 2) leading to an increase of MT levels (Fig. 3). Our explanation is that marine coastal streams are carrying industrial effluents with high metal content as it was demonstrated by Illou (1999) and Hamza-Chaffai et al. (2000). Malonedialdehyde was inversely correlated to size in R. decussatus even if no significant differences in the whole soft tissue weights were observed between groups during the transplantation period. MDA levels were, however, strongly correlated to the factor time, an interesting finding which could be explained by two things, the pulse contamination registered at t = 62 days, and the effect of the reproductive state of the animals. The second explanation was brought by Viarengo et al. (1991) in the digestive gland of mussels where MDA levels fluctuated significantly with the factor month. During the studied period, an inter-site and an intra-site increase in glycogen concentrations was observed. There are two possible explanations: (i) The trophic status of Gargour site could be more favourable for glycogen storage than in Sidi Mansour.
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(ii) Since Gargour site receives industrial pollutants, the energy allocation could be blocked for other physiological processes like reproduction (Gauthier-Clerc et al., 2002). In fact, some contaminants act as endocrine disrupters on metabolic pathways related to gametogenesis (Safe et al., 1991) that needs glycogen transformation to lipids (Gabbott, 1975). Glycogen decreased with zinc concentrations. It is not the first time that such result was found; the same correlation was observed in a temperate ecosystem in M. edulis and M. arenaria (Pellerin-Massicotte et al., 1993; Saint-Hilaire and Pellerin, 1995). This variation is very interesting from two points of views: (i) It reinforces the use of biochemical parameters related to energy storage as biomarkers of health condition. (ii) This decrease of glycogen related to Zn contamination could also serve as a predictive indicator for the ecosystem dysfunction provoked by anthropogenic influences. It should be noted that in the studied sites, industries, mainly those for phosphate treatment, release high levels of zinc (6 t per year) and cadmium (1 t per year) in the coastal environment (Hamza-Chaffai, 1993; Sarbaji, 1991). Glycogen was also highly correlated to site, indicating at least an inter-site variation of energy storage. Since contamination status was different in the two studied sites, it then appears clearly again that there is a strong link between zinc and glycogen. Moreover, a strong correlation was observed between glycogen and time. This could be explained at least partly by the reproductive status of R. decussatus, showing an increased coefficient index between November and January (Viarengo et al., 1991) and/or by the observed increased contamination. Metallothionein concentrations are correlated principally to cadmium and zinc. This finding was also obtained previously in a natural population of R. decussatus from Gargour site (Hamza-Chaffai et al., 1999). The strong relationship between MTs and cadmium was also evidenced after in vivo contamination of R. decussatus (Bebianno et al., 1993, 1994). The importance of Cd at the transcriptional level of MTs (mRNA) was evidenced in molluscs (Unger and Roesijadi, 1996). It is actually well known that MT levels are not only affected by metals but also by other factors such as size, site, or time. Therefore, we established a nonlinear model relating all these parameters. When considered together (Cd, Cu, Zn, site, time), MT levels were highly correlated to cadmium, site, and time as shown in model II. These observations are in agreement with previous results about MTs and metals in the digestive gland of R. decussatus (HamzaChaffai et al., 1999). The strong link between MTs site and time could be explained by the variation of metal contamination with time and site.
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The methods used in statistical treatments present some advantages. In fact, the Box –Cox transformation allowed the estimation of the nonlinear model reflecting the relationship between metallothionein and metals. Moreover, the use of dummy variables permitted the evaluation of the effect of factors site and time, which are qualitative variables. It is evident that factors other than contamination affect biomarker levels and we should evaluate their importance in order to validate biomarkers in field conditions. The advantage of the three-dimensional representation was to visualise the combined effect of metals on MT levels and the importance of each one.
5. Conclusion The in situ measure of metallothioneins, MDA, and glycogen levels in R. decussatus has many advantages particularly to promote the possible use of this species in a national biomonitoring program based on biomarkers. The reproductive cycle of R. decussatus should be therefore thoroughly investigated in sites differing by their levels of contaminants. This will enlighten the causes of glycogen increase and will clarify the deleterious effects of pollution on reproduction.
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