The effects of zinc oxide nanoparticles on the metallome in freshwater mussels

The use of zinc oxide nanoparticles (nanoZnO) as sunscreens has raised concerns about their safety and release in the aquatic environment through swimming activities and within municipally treated wastewaters. This study's purpose was to examine the effects of nanoZnO on the elemental composition (metallome) in exposed freshwater mussels, Elliptio complanata. Mussels were exposed for 21 days to an environmentally realistic (low) concentration (2 μg/L) of nanoZnO and zinc chloride. The mussels were also exposed to a physically and chemically treated municipal effluent (ME), both alone and in the presence of both forms of Zn. The metallome profile was characterized by the following 15 elements in gills, digestive gland and gonad tissues: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Se, V and Zn. The levels of metallothioneins (MT) and lipid peroxidation (LPO) in the digestive gland were also measured as biomarkers of toxic effects. The data revealed that exposure to nanoZnO increased the total levels of Zn, MT and LPO in the digestive gland. Discriminate function analysis revealed that the digestive gland responded the most to exposure to either nanoZnO or Zn^2 +. For nanoZnO, the observed changes in Al, As and Mo in the digestive gland offered the best discrimination from dissolved Zn^2 +. Co-exposure of nanoZnO with the ME changed the metallome profile closer to dissolved Zn^2 +, suggesting a common interaction site within the ME. This was observed in changes in Ni, Cu, Se and Zn in the digestive gland of exposed mussels. Canonical analysis of essential and non-essential elements revealed that exposure to nanoZnO increased the relationships between LPO and the sum of essential elements in the digestive gland. Conversely, exposure to dissolved Zn^2 + and the ME decreased the relationship between the sum of non-essential elements and LPO and MT. In conclusion, the use of a “metallomic” approach was used to discriminate changes following exposure to nanoZnO and dissolved Zn in freshwater mussels and provided insights into the interaction of forms of Zn in ME towards mussels.     

Multiple forms of metallothionein from the digestive gland of naturally occurring and cadmium-exposed mussels, Mytilus galloprovincialis

Polymorphism of metallothioneins in the digestive gland of naturally occurring (control) and experimentally Cd-exposed mussels Mytilus galloprovincialis (200 μg Cd l^–1; 14 days) was studied by applying the conventional methods of Sephadex column liquid chromatography (G-75 and DEAE A-25), and by an electrochemical method (DPASV) for determination of Cd, Zn and Cu concentrations in chromatographic fractions. In both control and Cd-exposed mussels, two distinct molecular mass components of the metallothioneins, monomeric (MT-10) and dimeric (MT-20), were resolved by Sephadex G-75 gel filtration chromatography. In control mussels, the MT-10 component was predominantly expressed as containing markedly higher constitutive levels of Zn (100×) and Cu (10×) than of Cd. Each of these two molecular mass components was further resolved into seven metal-rich peaks by anion-exchange chromatography. In Cd-exposed mussels the larger proportion of Cd was bound to the MT-20 than to the MT-10 component, suggesting that the dimeric component may be considered as a primarily inducible metallothionein. The elution positions of metal-binding maxima of Cd-exposed and control mussels on the respective DEAE chromatographic profiles were comparable. A great similarity in elution positions of Cd maxima between the composite and single-specimen samples was also observed. Our study confirms a high multiplicity of MT forms in mussels from the Mytilus genus not only under the laboratory high-level metal exposure conditions, but also at a natural seawater metal exposure level. The ecotoxicological significance of dimeric and monomeric MT forms, as well as their possible application in the biomonitoring of seawater for trace metals, has been considered. Electronic Publication     

Heavy Metal Accumulation and Distribution in Organs of the Mussel Crenomytilus grayanus from Upwelling Areas of the Sea of Okhotsk and the Sea of Japan

The subcellular and cytosolic distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in the digestive gland and kidney of the mussel Crenomytilus grayanus from upwelling areas of the Sea of Okhotsk and the Sea of Japan was studied. Cd, Zn, Pb, and Ni have accumulated in the kidneys of mussels from these areas. When the concentrations of both essential and toxic metals in the mussel organs had increased significantly, their redistribution into the cytosol took place. Gel chromatography of cytosolic proteins in the kidneys of mussels sampled in the area with a strong stationary upwelling revealed metallothioneins. This is uncommon for bivalve mollusks from unpolluted areas. High molecular weight proteins act as preliminary ligands for Cd.     

The distribution of Pb,Zn, Cd and Cu within the pulmonate mollusc Helix aspersa müller

Summary The distribution of Pb, Zn, Cd and Cu throughout the body tissues of Helix aspersa was determined. The digestive gland of the mollusc was shown to be an important store of Pb, Zn and Cd while Cu was found to be more evenly distributed throughout the tissues of the animal. These results are discussed with relation to previous work concerning heavy metal content in molluscs. The selectivity of the digestive gland for Cd was higher than it was for either Pb or Zn and it is suggested that analysis of the digestive gland of molluscs may be useful in studying levels of Cd at different sites.At a contaminated site the levels of Cd in Helix aspersa were shown to be 13 times greater than at a relatively uncontaminated site.     

Comparative study of the bioaccumulation and elimination of trace metals (Cd,Pb, Zn,Mn and Fe) in the digestive gland,gills and muscle of bivalve Pinna nobilis during a field transplant experiment

The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.     

Heavy metal effects on lipid peroxidation in the tissues of mytilus gallopro vincialis lam.

1. The effects of heavy metals on lipid peroxidation in the gills and digestive gland of mussels exposed for six days to Cu²⁺, Cd²⁺ or Zn²⁺ (40 μg/l/animal) were investigated.2. In the tissues of Cu-exposed mussels a significant increase of the level of malondialdehyde (MDA), which is indicative of the peroxidative process, and a decrease of the concentration of glutathione were observed.3. Moreover, in the digestive gland of mussels, copper exposure results in an increase of other carbonyl compounds and in the lysosomal accumulation of lipofuscin granules.4. The exposure of mussels to Zn or to Cd did not elicit any of the above effects.5. The results are discussed in relation to the possible role that Cu-induced lysosomal lipofuscin accumulation may play in heavy metal detoxification.     

The potential role of spherocrystals in the detoxification of essential trace metals following exposure to Cu and Zn in the fighting conch <Emphasis Type="Italic">Strombus</Emphasis> (<Emphasis Type="Italic">Lobatus</Emphasis>) <Emphasis Type="Italic">pugilis</Emphasis>

Crypt cells—one of the three cell types composing Strombidae digestive tubules—are characterized by the presence of numerous metal-containing phosphate granules termed spherocrystals. We explored the bioaccumulation and detoxification of metals in Strombidae by exposing wild fighting conch Strombus pugilis for 9 days to waterborne CuSO₄ and ZnSO₄. The total amount of Cu and Zn was determined in the digestive gland and in the rest of the body by Inductively Coupled Plasma (ICP) analyses. The digestive gland spherocrystal metal content was investigated based on the semi-quantitative energy dispersive X-ray (EDX) elemental analysis. ICP analyses of unexposed individuals revealed that 87.0?±?5.9% of the Zn is contained in the digestive gland, where its concentration is 36 times higher than in the rest of the body. Regarding Cu, 25.8?±?16.4% of the metal was located in the digestive gland of the control individuals, increasing to 61.5?±?16.4% in exposed individuals. Both Cu and Zn concentrations in the digestive gland increased after exposures, pointing to a potential role of this organ in the detoxification of these metals. EDX analysis of spherocrystals revealed the presence of Ca, Cl, Fe, K, Mg, P, and Zn in unexposed individuals. No difference was found in the relative proportion of Zn in spherocrystals of exposed versus control individuals. Contrastingly, copper was never detected in the spherocrystals from controls and Zn-exposed individuals, but the relative proportion of Cu in spherocrystals of Cu-exposed individuals varied from 0.3 to 5.7%. Our results show the direct role of spherocrystals in Cu detoxification.     

Assessment of the adaptation capabilities of the bivalves Modiolus modiolus (Linnaeus, 1758) and Crenomytilus grayanus (Dunker, 1853) under increased levels of heavy metals in the environment

The active biomonitoring method was used to examine the changes in heavy-metal (Fe, Zn, Cu, Cd, Mn, Pb, and Ni) contents in the organs (muscle, gonads, mantle, gills, digestive gland, and kidney) of the mussels Crenomytilus grayanus and Modiolus modiolus. The dynamics of trace element concentrations in mussel organs during the experiment were compared with their subcellular distribution. The defense strategy of M. modiolus consisted of the threshold accumulation of toxic metals in all organs followed by their excretion, whereas the strategy of C. grayanus involved short-term isolation from adverse environmental influence. Under chronic pollution, in C. grayanus the main loads occurred in the digestive gland and kidney. Under acute changes in environmental conditions, the processes of regulation (detoxification/excretion) of Fe, Mn, and Pb in this species were impaired substantially.     

Changes in the Microelement Composition in Organs and Tissues of the Bivalve <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> Acclimatized in a Biotope with Long-Term Heavy Metal Contamination

This paper deals with changes in the Fe, Zn, Cu, Cd, Mn, Pb, and Ni concentration in the muscle, gonad, mantle, gills, digestive gland, and kidneys of Gray mussels transplanted from two sites differing in the extent of metal contamination to a biotope with the impact from anthropogenic conditions in the course of their acclimatization. The rate of metal accumulation depended on the initial element concentration in the mussel organs. An abrupt change in the metal concentration in the environment was followed by a disturbance in the microelement balance in some organs. The microelement metabolism was shown to depend on mussel preadaptation to a changeable environment.Original Russian Text Copyright © 2005 by Biologiya Morya, Kavun, Shulkin.     

Comparative Analysis of Subcellular Distribution of Heavy Metals in Organs of the Bivalve Mollusks <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> and <Emphasis Type="Italic">Modiolus modiolus</Emphasis> in a Continuously Polluted Environment

The distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in subcellular fractions, and of Cd, Zn, and Cu in cytoplasm proteins of the kidney and digestive gland of the mussels Crenomytilus grayanus and Modiolus modiolus, sampled from contaminated and conditionally clean areas, was studied. It was found that, in a contaminated environment, the organs of mussels were more highly enriched with metals. It was shown that essential trace elements were accumulated mostly in the cytosol of organs of both molluscan species from contaminated areas, whereas in the background areas the trace elements were associated mostly with membrane structures in Gray's mussel, C. grayanus, and with the cytosol in M. modiolus, the northern horse mussel. The lead was bound mostly to membrane structures in organs of both mussel species at all stations. The method of gel chromatography enabled us to isolate metallothionein-like proteins from the kidney of the northern horse mussel sampled in contaminated areas, whereas their concentration in the kidney of Gray's mussels was lower than the limiting error of the method. It is supposed that in the kidney of Gray's mussel the synthesis of metallothionein-like proteins was quenched by the integrated effect of the accumulated metals.     

Influence of some metal concentrations on the activity of antioxidant enzymes and concentrations of vitamin E and SH-groups in the digestive gland and gills of the freshwater bivalve Unio tumidus from the Serbian part of Sava River

We examined whether the freshwater bivalve Unio tumidus from the Sava River can serve as a bioindicator organism for long-term biomonitoring of river ecosystems for the presence of metal pollutants. To this end, we assessed in the digestive glands and gills of mussels, changes in activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST)), and changes in the concentrations of the non-enzymatic components of the antioxidant system (vitamin E and sulfhydryl groups (-SH), after exposure to metals in the environment. Mussels were collected at four sites where the concentrations of dissolved metals (Cu, Cd, Zn, Fe, Mn, Hg, Ni, As, Pb) were quantified. Cu, Ni and As exerted concentration-dependent inhibitory effects on CAT and GST activities. Increasing concentrations of Cd promoted increases in GSH-Px activity and -SH concentration. In response to increased Zn concentration GR activity increased whereas Fe promoted decreased enzymatic activity. Negative correlations between the concentrations of Cu and Cd and vitamin E, and a positive correlation between Mn and vitamin E concentrations were detected. The described correlations between components of the antioxidant system and metal levels in the environment reveal a high physiological sensitivity of freshwater mussels to pollution, supporting their use in biomonitoring of metal contamination in river ecosystems.     

Effects of Total Soft Tissue and Shell Thickness on the Accumulation of Heavy Metals (Cd,Cu, Pb,and Zn) in the Green-Lipped Mussel Perna viridis

The effects of the total soft tissue dry weight and shell thickness and on the accumulation of Cd, Cu, Pb, and Zn were determined in the green-lipped mussel Perna viridis. In agreement with Boyden's formula (1977), our results showed that the plotting of metal concentrations against the total soft tissue dry weight and shell thickness of the mussel on a double logarithmic basis gave negative coefficients especially for Cd, Pb, and Zn. Therefore, the smaller mussels (lower total soft tissue dry weight) had higher concentrations of Cd, Pb, and Zn than the larger ones. Since shell thickness could be considered to estimate of the age of the mussel, it was also found that the younger mussels accumulated more Cd, Pb, and Zn than the older ones. This indicated that P. viridis has a different metabolic strategy for each of the metals studied which may be related to age. However, the accumulation of Cu was hardly affected by the sizes and ages of the mussel. This indicated that the accumulation pathways of Cu and the processes affecting the bioavailability of Cu to the mussel are different from those for Cd, Pb, and Zn.     

Impact of Engineered Zinc Oxide Nanoparticles on the Individual Performance of Mytilus galloprovincialis

The increased use of engineered nanoparticles (ENPs) in consumer products raises the concern of environmental release and subsequent impacts in natural communities. We tested for physiological and demographic impacts of ZnO, a prevalent metal oxide ENP, on the mussel Mytilus galloprovincialis. We exposed mussels of two size classes, <4.5 and ≥4.5 cm shell length, to 0.1–2 mg l⁻¹ ZnO ENPs in seawater for 12 wk, and measured the effect on mussel respiration, accumulation of Zn, growth, and survival. After 12 wk of exposure to ZnO ENPs, respiration rates of mussels increased with ZnO concentration. Mussels had up to three fold more Zn in tissues than control groups after 12 wk of exposure, but patterns of Zn accumulation varied with mussel size and Zn concentrations. Small mussels accumulated Zn 10 times faster than large mussels at 0.5 mg l⁻¹, while large mussels accumulated Zn four times faster than small mussels at 2 mg l⁻¹. Mussels exposed to 2 mg l⁻¹ ZnO grew 40% less than mussels in our control group for both size classes. Survival significantly decreased only in groups exposed to the highest ZnO concentration (2 mg l⁻¹) and was lower for small mussels than large. Our results indicate that ZnO ENPs are toxic to mussels but at levels unlikely to be reached in natural marine waters.     

Cadmium accumulation in tissues of the mussel Mytilus galloprovincialis

Studies have been made on cadmium accumulation in tissues of mussels kept within 20-60 days in water artificially enriched by Cd up to 20-100 micrograms/l. Irrespectively of cadmium concentration in the medium, its accumulation in tissues decreases in the following order: mid-gut gland, gills, gonads, mantle, adductor. Maximum concentration of Cd was found in the digestive tubuli of the mid-gut gland by X-ray microanalysis. The increase in S and, to a lower extent, P concentrations in these tubuli was also observed. It is suggested that the latter is due to immobilization of Cd by metal-binding proteins as well as to lyzosomal vesicles involved into detoxication of Cd. The increase in the external cadmium up to 100 micrograms/l did not affect the level of K, Ca and Mg in tissues of the mussel.     

Mussels as bioindicators of trace metal pollution in the Danube Delta of Romania

Specimens of mussels species Anodonta anatina, Unio pictorum, U. tumidus and surfical sediment samples were collected in the summers of 1994 and 1995 from twelve lakes in the Danube Delta, Romania. Whole mussel tissues were analyzed for metals (Ag, As, Cd, Co, Cu, Cr, Ni, Pb, Se and Zn), and sediment samples were subjected to weak acid extraction and to a sequential extraction procedure, and analyzed for Cr, Cu, Ni and Zn. Total mean Cu and Ag concentrations were statistically greater in Unio species than in Anodonta species, but other metal levels did not significantly differ between mussel species. There was a ten fold variation for Cd and Pb and five fold variation for Zn, Ag and Co concentrations in mussels among lakes. Mean concentrations of As, Cu, Se and Zn in mussel tissues varied two fold among lakes. Zn, Co and Cd concentrations in mussels were significantly negatively correlated with distance gradients from the origin of the Delta and with distance from main channels of the Danube. Other metal concentrations in mussels did not correlate with either distance gradient. Trace metal levels in mussel tissues were correlated with those in weak acid and in sequential extractions of sediment. Zn concentrations in the carbonate fraction was the best predictor of Zn concentrations in mussels. Cu and Ni levels in the organic matter and sulfide fraction were the best estimators of Cu and Ni concentrations in mussels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lysosomal enlargement in digestive cells of mussels exposed to cadmium, benzo[a]pyrene and their combination

Digestive cell lysosomes in mussels are known to respond to individual organic chemicals and metals after experimental exposure under laboratory conditions but reports dealing with the response to mixtures of pollutants are scarce. The aim of the present investigation was to compare the lysosomal responses elicited by exposure to a model organic chemical compound (benzo(a)pyrene, B[a]P), a model toxic metal (Cd) and their combination (B[a]P+Cd) under controlled laboratory conditions. Dimethylsulfoxide (DMSO) was used as vehicle to dissolve organic chemicals into seawater. Control mussels were either kept untreated in clean seawater or treated with DMSO. Digestive glands were excised on Day 21. beta-Glucuronidase activity was demonstrated in 8 mum cryotome sections. Lysosomal volume, surface and numerical densities (Vv, Sv and Nv), and surface-to-volume ratio (S/V) were quantified by image analysis. Lysosomal enlargement was evident in digestive cells of mussels exposed to either Cd, B[a]P or B[a]P+Cd. Such enlargement was more marked after exposure to B[a]P+Cd than to B[a]P, but did not reach the levels recorded after Cd exposure. It seems therefore that the presence of B[a]P reduced to some extent the effects of Cd on digestive cell lysosomes in mussels.     

Molecular fractionation of Zn, Cu, Cd and Pb in the midgut gland of Helix pomatia L

Specimens of Helix pomatia were fed lettuce enriched with Zn, Cu, Cd and Pb for 30-35 days, after which time the distribution of the four metals in the midgut gland was studied by centrifugation, ion exchange and gel chromatography. In control snails the distribution between the soluble and particulate fraction of the midgut gland is relatively uniform, with a ratio of approximately 3:1 for Cu, Cd and Pb, and of 1:1 for Zn. However, after metal-loading about two-thirds of total Zn and Pb appears in the particulate fraction whereas little change in relative distribution is observed with Cu and Cd. After ion exchange chromatography of the supernatant both Cd and Zn eluted in one distinct peak, Cu in two peaks, whereas Pb seemed to be distributed unspecifically throughout the elution volume. If ion exchange is followed by gel chromatography, Zn, Cu and Cd are found associated with from one to four molecular species, ranging in mol. wt from 1000 to greater than 20,000, whereas no complex was found for Pb. The molecular patterns are specific for each of the three metals and there are some specific shifts in abundance, usually involving one molecular fraction, if control snails are compared with metal-loaded snails.     

Antioxidant and lipid peroxidation responses in Mytilus galloprovincialis exposed to mixtures of benzo(a)pyrene and copper

This study aimed to assess the antioxidant system potential and lipid peroxidative effects, in the gill and digestive gland of Mytilus galloprovincialis exposed to individual and binary mixtures of benzo(a)pyrene (BaP) and Cu for 7 days. Data demonstrated that in mussels exposed to BaP antioxidant enzymes (catalase--CAT, total glutathione peroxidase--tGPx, glutathione S-transferase--GST and glutathione reductase--GR) and lipid peroxidation (LPO) increased in the gill. On the contrary, in the digestive gland inhibitory antioxidant effects (superoxide dismutase-SOD, GR, metallothioneins-MT) and no changes in LPO levels were detected. Cu was also a potent oxidant agent since MT and LPO levels increased in mussel gill, despite no LPO effect in the digestive gland. For both single contaminants the organ specificity and distinct physiologic/metabolism roles were evident in terms of antioxidant capacity. Gill SOD inhibition, MT and GST unchanged was a result of "simple independent action" of exposure to BaP and Cu. "Interactions" in the binary mixtures, led to absence of changes in LPO effects. In the digestive gland, BaP and Cu interactions were also responsible for the GST and LPO enhancement (antagonistic effects). The current findings demonstrate the differences in antioxidant responses where the organ dependency highlights each contaminant particular mode of action. Generally, in the gill "non-interactive" effects occurred with the lowest Cu concentration while "interactions" exist for the mixture with the highest Cu concentrations. In the digestive gland, "interactions" and "no interaction" effects occurred in all the binary mixtures. Complex contaminant mixtures interact differently based on target tissue which may lead to an imbalance in the mussels health status.     

Occupational exposure of foundry workers assessed by the urinary concentrations of 18 elements and arsenic species

BackgroundSome raw materials applied in Fe foundry industries may contain potentially toxic elements. Thus, foundry worker’s occupational exposure is a constant health concern.MethodIn this study, 194 urine samples from foundry workers were analyzed by inductively coupled plasma mass spectrometry for biomonitoring of Al, As, Ba, Cd, Co, Cr, Cs, Cu, Fe, I, Mn, Ni, Pb, Sb, Sn, Se, U and Zn. Moreover, arsenic speciation was performed in representative samples of production sector workers (group A) and administration sector workers (group B).ResultsConcentrations of As, Pb, Cd, Cu, Cs, I, Sb in urines from group A were higher than those found for group B. Samples of group A presented Cs, Ni, Mn, Pb, U and Zn concentrations higher than values reported for exposed workers assessed by other studies. Forty-four samples from group A exceed As-reference limits. Group A had approximately seven times more inorganic As (as arsenite) and 14 times more organic As (as dimethyl As) than group B. A statistically significant difference was observed in the elemental concentration in the workers' urine by the time in the function. Moreover, alcohol consumption is probably influencing the urine concentration of As, Ba, Cd, Co, Cu, Fe, I, Se and Zn.ConclusionThe monitored foundry workers are exposed to potentially toxic elements and more attention must be given to their health. Therefore, workplace safety conditions must be improved, and constant biomonitoring is necessary to ensure workers' health.     

Seasonal accumulation of metals by red alga Gracilaria verrucosa (Huds.) Papens. from Thermaikos Gulf, Greece

Concentrations of Fe, Pb, Cu, Zn and Cd were determined during one season in the red alga Gracilaria verrucosa, sediment and seawater from the Thermaikos Gulf, Greece. This region has been subject to change due to increases in industrial and domestic activities. The relative abundance of metals in G. verrucosa and seawater decreased in the order: Fe>Zn>Pb>Cu>Cd and in the sediment: Pb>Fe>Zn>Cu>Cd. Cadmium concentration in the alga correlated positively with that in seawater. There was positive correlation between Fe concentrations in the alga and those of the Zn and Cu. The concentrations of metals in the alga showed no significant differences between the stations. Lead, Zn and Cu concentrations in the alga were slightly higher at Biamyl, whereas Cd was higher at Perea and Fe at Nea Krini. Seasonal variation of metal concentrations in the alga was significant for Cd and Fe. Copper and Fe increased from winter to summer, whereas Cd was the opposite. Zinc concentrations were minimum and Pb concentrations were maximum during spring. These variations are discussed in relation to tissue age, life cycle, ambient concentrations of metals and other environmental conditions. Cd and Pb concentrations inG. verrucosa in the Thermaikos Gulf were higher and those of Cu and Zn were lower than in other species of the genus. This revised version was published online in August 2006 with corrections to the Cover Date.