Laranjo Basin Research Articles

Mercury’s mitochondrial targeting with increasing age in Scrobicularia plana inhabiting a contaminated lagoon: Damage-protection dichotomy and organ specificities

This study aimed to understand bivalves’ (Scrobicularia plana) adaptive strategies, with emphasis on mitochondria as a target organelle, in response to mercury-contaminated environment. Inter-age and organ-specific approaches were applied using different annual size classes (2+, 3+, 4+ and 5+ years old) and assessing specific organs (gill, digestive gland), respectively. Bivalves were collected from moderately (M) and highly (H) contaminated sites at Laranjo basin - Ria de Aveiro (Portugal), where a mercury gradient was identified, and compared with those from a reference (R) site. Total antioxidants capacity (TAC) was measured in mitochondria, whereas the lipid peroxidation was assessed as a marker of damage. S. plana age determined the clams’ vulnerability towards mercury induced mitochondrial peroxidative damage depending upon the mercury accumulation: younger animals were more vulnerable than older. Clams showed a decreasing trend of TAC with increasing age. This decrease was found to be statistically significant in 4+ and 5+ years at M; whereas, at H, with depleted TAC, remained same until they have grown 5+ years. The organ specificity was evident for antioxidant response and peroxidative damage. A clear pattern of overall TAC increase in digestive gland (at M) and decrease in gill (at H) was observed, while only gills were found to be susceptible to peroxidative damage. Overall, mitochondria proved to be a sensitive fraction for the effects of mercury in S. plana inhabiting mercury contaminated area.

Morphological, compositional and ultrastructural changes in the Scrobicularia plana shell in response to environmental mercury – An indelible fingerprint of metal exposure?

The study aimed to assess morphological, structural and compositional alterations in Scrobicularia plana nacre environmentally exposed to mercury in order to seek out the possibility of the assessed alterations as a monitoring tool to handle complexity and interactions of metals in the environment involving a non-invasive methodology. Bivalves were collected from a mercury contaminated site (Laranjo basin – Ria de Aveiro, Portugal) and a reference site in the same aquatic system. The combination of scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDS) technique depicted a sheet like morphology of bivalve nacre collected from the reference site. Moreover, EDS plot exhibited the presence of potassium, oxygen, calcium, and carbon elements. Shells collected from the contaminated area depicted lamellar patches like structures with particle like morphology composition. SEM images corresponding to the elemental analysis by EDS plot clearly denoted the presence of mercury. SEM images from the other locations of the contaminated shells depicted large surface area, a broken or ruptured symmetry of organic matrix as well as crack-like gaps. The influence of environmental mercury affecting the surface morphology of S. plana nacre showed dimple like morphology (as proved by transmission electron microscopy, TEM). The possible explanation may be the replacement of calcium elements with other elements or alloys from the nacre composite collected from contaminated region. Therefore, the nacre fingerprint may be useful as innovative knowledge and applicable tool aiming at risk reduction from noxious mercury present in the environment. Overall results suggested the use of shell as an indelible fingerprint of metal exposure.

Mercury-Induced Chromosomal Damage in Wild Fish (Dicentrarchus labrax L.) Reflecting Aquatic Contamination in Contrasting Seasons

Ria de Aveiro (mainly Laranjo basin, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, currently presenting a well-described mercury gradient. This study aimed to assess mercury genotoxicity in this area by measuring the frequency of erythrocytic nuclear abnormalities (ENA) in the European sea bass (Dicentrarchus labrax), addressing the relation with total mercury concentration in the blood and the modulatory role of seasonal variables. Fish were collected, in warm and cold periods, at three locations differing in their distances to the main mercury source: reference (R), moderately (M), and highly (H) contaminated sites. Genotoxicity was detected in both degrees of contamination (M and H) and in both periods of the year (warm and cold), which is in line with the greater levels of mercury measured in fish blood. No significant seasonal variations were observed for mercury bioaccumulation or ENA frequency. The apparent low imperviousness of ENA frequency to seasonal factors reinforced its consistency as a genotoxicity biomarker, thus enabling a clearer identification of cause-and-effect relationships. Overall, the results reflected a serious environmental risk to native ichthyofauna at Laranjo basin due to mercury contamination, showing a potential of mercury to induce genetic damage in fish blood cells through clastogenic and/or aneugenic actions.

Mercury contaminated systems under recovery can represent an increased risk to seafood human consumers – A paradox depicted in bivalves’ body burdens

The present study, carried out in a mercury (Hg) contaminated estuary, aimed to investigate: (i) the longterm evolution of the Hg bioavailability in the environmental matrices, in a period of 5 years (2003– 2008), without new anthropogenic inputs; (ii) the temporal evolution of Hg load (organic and inorganic forms) in the native bivalve Scrobicularia plana, inferring the progression of human health risk associated to its consumption and the dependence on the animals’ size. The area selected was Laranjo basin of Ria de Aveiro (Portugal), where a Hg gradient was identified because of past discharges from a chlor-alkali plant. Two sites termed M (moderately contaminated) and H (highly contaminated) were compared with an uncontaminated reference (R) site. Results displayed a persistence of Hg in the environment, though the levels in sediment decreased at site H, confirming the ecosystem recovery. The risk associated to clam consumption remained stable in 2008 considering their total Hg (T-Hg) load and the limits established by public health authorities, though T-Hg levels significantly decreased at H site for size classes C3 (2 year) and C4 (4 year). Organic Hg (O-Hg) accumulation increased from 2003 to 2008, reaching threatening levels and suggesting an increased bioavailability of this Hg form. This evolution towards an increase of OHg accumulation was particularly prominent under a moderate contamination scenario (site M). Overall, it was demonstrated that a period of 5 years of ecosystem recovery was not enough to eliminate the risk to human consumers, highlighting Hg contamination in estuaries as a long-lasting legacy. Paradoxically, it was pointed out that in a given step of the long-term restoration process, occurring naturally in aquatic systems, the risk associated to bivalves’ consumption can appear augmented due to O-Hg accumulation increments.

Impact of Seasonal Fluctuations on the Sediment-Mercury, its Accumulation and Partitioning in Halimione portulacoides and Juncus maritimus Collected from Ria de Aveiro Coastal Lagoon (Portugal)

The availability of metals to plants is a complex function of numerous environmental factors. Many of these factors are interrelated, and vary seasonally and temporally. The current study intended to understand the influence of seasonal fluctuations and the vegetation of salt marsh plants (SMPs; Halimione portulacoides, Juncus maritimus) on sediment’s mercury (Hg) and its pH and redox potential (Eh), as well as their cumulative effect on the plant’s Hg-accumulation and Hg-partitioning potential. The area selected for the study was Laranjo Basin at Ria de Aveiro lagoon (Portugal) where a known Hg gradient was existed due to chlor-alkali plant discharge. Three sampling sites (L1, L2 and L3) were selected along a transect defined by the distance from the main Hg source. Samples were also collected from the Hg-free site (R). Irrespective of the plant vegetation, Hg in sediments gradually increased with a decreasing distance towards Hg-point source. The sediment colonised by J. maritimus showed more Hg concentration compared with H. portulacoides irrespective of the season. As a whole, J. maritimus accumulated Hg more than H. portulacoides at all the sampling sites, whereas in root, stem and leaf, the concentration was ranked as: L1 > L2 > L3 in both the plant species and was differentially influenced by seasonal changes. Moreover, root of both plants exhibited highest Hg concentration compared with stem and leaf. In addition, the leaf of H. portulacoides exhibited more Hg than leaves of J. maritimus. Bioaccumulation and translocation factors and dry weight were differentially influenced by seasonal changes. Taking together the results, the physico-chemical properties of sediment especially the sediment-Eh seems to be influnced by the type of plant vegetation and seasonal changes which in turn may have influenced the chemistry of sediments; thus, it enfluences the bioavalability of Hg and the Hg-retention capacity of both salt marsh sediments (SMSs) and SMPs (bioaccumulation factor). Moreover, SMSs vegetated by J. maritimus exhibited a stronger capacity for the retention and phytostabilization of Hg belowground (in sediments and/or roots) than those dominated by H. portulacoides. Conversely, those SMSs extensively vegetated by H. portulacoides are expected to translocate more Hg to aboveground parts, acting as a potential source of this metal to the marsh ecosystem. Therefore, J. maritimus and H. portulacoides may be used repectively for phytostabilization (in rhizosediments) and phytoextraction (by accumulation in aboveground plant tissue for subsequent plant removal).

Brain as a critical target of mercury in environmentally exposed fish ( Dicentrarchus labrax)—Bioaccumulation and oxidative stress profiles

Although mercury is recognized as a potent neurotoxicant, information regarding its threat to fish brain and underlying mechanisms is still scarce. In accordance, the objective of this work was to assess vulnerability of fish to mercury neurotoxicity by evaluating brain pro-oxidant status in wild European sea bass ( Dicentrarchus labrax) captured in an estuarine area affected by chlor-alkali industry discharges (Laranjo Basin, Ria de Aveiro, Portugal). To achieve this goal, brain antioxidant responses such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) activities and total glutathione (GSHt) content were measured. Additionally, damage was determined as lipid peroxidation. To ascertain the influence of seasonal variables on both mercury accumulation and oxidative stress profiles, surveys were conducted in contrasting conditions—warm and cold periods. In the warm period, brain of fish from mercury contaminated sites exhibited ambivalent antioxidant responses, viz. higher GR activity and lower CAT activity regarded, respectively, as possible signs of protective adaptation and increased susceptibility to oxidative stress challenge. Though the risk of an overwhelming ROS production cannot be excluded, brain appeared to possess compensatory mechanisms and was able to avoid lipid peroxidative damage. The warm period was the most critical for the appearance of oxidative damage as no inter-site alterations on oxidative stress endpoints were detected in the cold period. Since seasonal differences were found in oxidative stress responses and not in mercury bioaccumulation, environmental factors affected the former more than the latter. This work increases the knowledge on mercury neurotoxicity in feral fish, highlighting that the definition of critical tissue concentrations depends on environmental variables.

Lipid peroxidation vs. antioxidant modulation in the bivalve Scrobicularia plana in response to environmental mercury—Organ specificities and age effect

This study aimed at the assessment of mercury burden and its association to damage vs. antioxidant protection in the bivalve Scrobicularia plana environmentally exposed to mercury. Inter-age and organ-specific approaches were applied by using different annual size classes (2+, 3+, 4+ and 5+ year old) and assessing specific organs (gills, digestive gland), respectively. Bivalves were collected from moderately and highly contaminated sites at Laranjo basin – Ria de Aveiro (Portugal), where a mercury gradient was identified, and compared with those from a reference site. Besides total and organic mercury accumulation, endpoints combining lipid peroxidation (LPO), as a damage sign, and antioxidant protection (catalase, CAT; glutathione peroxidase, GPX; glutathione S-transferase, GST, activities) were determined. The whole-body accumulation as total mercury changed according to the environmental gradient for all age classes, while in terms of organic mercury, only 5+-year-old animals showed increased accumulation with increased environmental mercury level. Mercury induced peroxidative damage, showing that antioxidative mechanisms were insufficient. The adaptive capacity to pro-oxidant challenge, expressed as antioxidant induction and lesser vulnerability to enzyme inhibition, increased with age. The specific analyses of gills and digestive gland revealed that both organs were able to mirror external levels of exposure in the accumulation of total and organic mercury. Nevertheless, gills displayed higher potential to accumulate organic forms. The organ specificity was evident for antioxidant response with a clear pattern of overall increase in gills and decrease in digestive gland. In addition, only gills displayed statistical correlations between oxidative stress responses and mercury accumulation. Besides the contribution to understand mercury toxicodynamics, specific organs approach is strongly recommended in order to avoid misinterpretations. The use of whole-body analyses can be particularly compromising when oxidative stress responses (rather than mercury accumulation) are addressed.

Immunosuppression in the infaunal bivalve Scrobicularia plana environmentally exposed to mercury and association with its accumulation

This study aimed to test the hypothesis whether mercury (Hg) activates or suppresses inappropriately the immunity of the bivalve Scrobicularia plana inhabiting a Hg contaminated area (Laranjo basin, Ria de Aveiro, Portugal). Immunity endpoints, as well as lipid peroxidation (LPO) as a sign of damage, were evaluated in parallel with total Hg burden. Bivalves from both moderately (MO) and highly (HI) contaminated sites displayed higher haemolymph Hg load and reduced plasma agglutination. Increased haemocytes density and decreased phagocytosis were observed at HI, whereas increased oxidative burst activity (OBA) was observed at MO, pointing out that the immunotoxicity is a result of Hg direct contact involving no ROS intervention. OBA observed at MO was concomitantly associated to peroxidative damage as depicted by LPO increase in haemocytes and haemolymph plasma. Thus, S. plana can be suggested as a suitable bioindicator of metal pollution in coastal areas on the basis of Hg bioaccumulation and immunotoxicity responses.

Evaluation of DNA Damage Induced by Environmental Exposure to Mercury in Liza aurata Using the Comet Assay

Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer approximately autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems.

Antioxidant and biotransformation responses in Liza aurata under environmental mercury exposure – Relationship with mercury accumulation and implications for public health

This study was carried out in the Laranjo basin (Ria de Aveiro, Portugal), an area impacted by mercury discharges. Liza aurata oxidative stress and biotransformation responses were assessed in the liver and related to total mercury (Hg t) concentrations. A seasonal fish survey revealed a sporadic increase in total glutathione (GSH t) and elevated muscle Hg t levels, although Hg levels did not exceed the EU regulatory limit. As a complement study, fish were caged for three days both close to the bottom and on the water surface at three locations, and displayed higher Hg t levels accompanied by increased GSH t content and catalase activity as well as EROD activity inhibition. The bottom group displayed higher hepatic Hg t and GSH t contents compared with the surface group. Globally, both wild and caged fish revealed that the liver accumulates higher Hg t concentrations than muscle and, thus, better reflects environmental contamination levels. The absence of peroxidative damage in the liver can be attributed to effective detoxification and antioxidant defense.

Erythrocytic nuclear abnormalities in wild and caged fish ( Liza aurata) along an environmental mercury contamination gradient

Laranjo basin (Aveiro, Portugal) has been subjected to mercury contamination from a chlor-alkali plant, presenting a well-described mercury gradient. This study aims the assessment of mercury genotoxicity in this area by measuring erythrocytic nuclear abnormalities (ENA) frequency in the mullet Liza aurata, and its relation with total mercury concentration (Hg t) in blood. Wild fish were seasonally analysed, and, complementarily, fish were caged for 3 days at three locations differing on their distances to the mercury source. The results from Laranjo were compared with those from a reference area (S. Jacinto). Wild fish from Laranjo showed elevated ENA frequency in summer and autumn in concomitance with increased blood Hg t. Surprisingly, no ENA induction was found in winter, despite the highest blood Hg t, which may be explained by haematological dynamics alterations, as supported by a decreased immature erythrocytes frequency. Caged fish displayed ENA induction only at the closest site to the contamination source, also showing a correlation with blood Hg t.

Distribution of mercury in the upper sediments from a polluted area (Ria de aveiro, Portugal)

It is well known that chlor-alkali industries using mercury as a cathode are an important source of this heavy metal to aquatic environments. Once discharged into rivers, lakes or coastal waters, mercury accumulates in sediments and in the aquatic food web (Gonzalez, 1991). Both elemental mercury and its compounds are noxious to all living organisms, and are bioaccumulative and persistent. Metal concentrations in sediments usually exceed those of the overlying water column. As a consequence, metals originating from human activities can often be identified more readily by analysis of sediments than by the quantification of metal concentrations present in water (Haynes et al., 1995). Sediments also integrate the temporal variability that characterizes metals originating from human sources. However, metals may be remobilized during the early stages of post-depositional transformations of the sediment (Gobeil and Cossa, 1993). Ria de Aveiro is a long, narrow, coastal lagoon, of 43 km wet area with a single, artificially maintained connection to the sea (Fig. 1). The coastal plain around the lagoon supports an intensive, diversified agriculture, a variety of heavy and light industries and a population of about half a million people, a part of which discharge their untreated or partially treated sewage into the lagoon (Lucas et al., 1986). An extensive web of islands and channels make water circulation inside the lagoon difficult and complex, thus allowing the spread of any conservative contaminants before they are discharged into the costal waters through the single sea mouth. One such contaminant is the mercury, released by a chlor-alkali plant into one of the remotest branches of the lagoon—Estarreja Channel (Fig. 1) (Hall et al., 1988). The Channel ends in a basin (Laranjo Basin) where most of the discharged mercury settles. Laranjo Basin has an area of about 1.5 km and is totally emptied in most ebb tides. The average volume of water exported with each tidal cycle may be around 2 km (Lucas et al., 1986). This work reports levels of Hg, Fe and Mn and sulphides in the upper sediments of the Laranjo Basin, and examines the influence of these elements on the distribution of mercury. Short sediment cores (10 cm) were collected by hand at sites A, B and C of Ria de Aveiro (Fig. 1). Cores were sampled in March, May, June 1996 and April 1997. The sediments were sliced in situ at the following depths: 0– 2, 2–4, 8–10, 14–16, 24–26, 34–36, 44–46, 54–56, 64–66 and 94–96 mm. The samples were rapidly transferred to vials which were placed under nitrogen atmosphere and frozen. In the laboratory, part of the sediments samples Edited by Bruce J. Richardson

Tidal export of particulate mercury from the most contaminated area of Aveiro's Lagoon, Portugal

Mercury pollution is a regional problem in the Aveiro's Lagoon. During 4 decades, a remote branch (Estarreja Channel) has received a discharge of mercury from a chlor-alkali plant, that is spread in the Laranjo Basin. Of special interest is the dispersion of mercury from these areas to the Lagoon. This work is a first attempt to quantify the net flux of particulate mercury from the most contaminated basin (Laranjo). Measurements of current intensity, suspended particulate matter and particulate mercury were carried out at two sampling stations over a spring tidal cycle, in April 1994. Based on a mass balance calculation at two stations, at the end of Estarreja Channel (F 1) and near the outlet of Laranjo Basin (F 2), the net tidal fluxes of particulate anthropogenic mercury were estimated: 0.4 g of mercury enter the Laranjo Basin and 94.5 g of particulate mercury escape the Basin. These results indicate that mercury from the most contaminated area continues to be dispersed, predominantly through the particulate fraction, to the rest of the Lagoon.

An estimation of industrial mercury stored in sediments of a confined area of the lagoon of aveiro (Portugal)

In the period from 1950 to 1994, most of the mercury discharged to the Lagoon of Aveiro was retained in the sediments of a small area of around 1.7 km2 (25.4 × 103 kg, about 77% of the total), which corresponds to the Estarreja Channel plus a basin named Laranjo Basin located downstream. In 1994, the mercury concentration in surface sediments of the area ranged from 2.3 to 343 μg g−1, with the highest values recorded near the industrial source of the metal. In places of the basin where the sedimentation rate could be considered uniform, the highest levels of Hg (35 μg g−1) were recorded at depths around 40 cm, as result of reduction of the anthropogenic mercury input to the lagoon. Assuming an uniform sedimentation rate and a continuously low industrial input of mercury for the near future, the levels of Hg in the surface sediments would be less than 1 μg g−1 in 20 years. This may be considered the expected restoration time. The feasibility and consequences of several management options for this pollution problems is briefly presented.

An estimation of industrial mercury stored in sediments of a confined area of the Lagoon of Aveiro (Portugal)

In the period from 1950 to 1994, most of the mercury discharged to the Lagoon of Aveiro was retained in the sediments of a small area of around 1.7 km2 (25.4 x 103 kg, about 77% of the total), which corresponds to the Estarreja Channel plus a basin named Laranjo Basin located downstream. In 1994, the mercury concentration in surface sediments of the area ranged from 2.3 to 343 μg g−1, with the highest values recorded near the industrial source of the metal. In places of the basin where the sedimentation rate could be considered uniform, the highest levels of Hg (35μg g−1) were recorded at depths around 40 cm, as result of reduction of the anthropogenic mercury input to the lagoon. Assuming an uniform sedimentation rate and a continuously low industrial input of mercury for the near future, the levels of Hg in the surface sediments would be less than 1 μg g−1 in 20 years. This may be considered the expected restoration time. The feasibility and consequences of several management options for this pollution problems is briefly presented.