Optimal number of Fucus vesiculosus samples to differentiate between sites affected by distinct levels of heavy metal contamination.

The presence of trace elements in marine habitats is a serious environmental problem which increasingly affects ecosystem and human health. The use of macroalgae as contamination biomonitors represents a valuable alternative approach to traditional physicochemical methods. The present study was carried out to determine the optimal number of samples of Fucus vesiculosus needed to detect statistically significant differences in the mean concentrations of Al, As, Cd, Co Cr, Cu, Fe, Hg, Ni, Pb, Zn, N and δ15N between two sampling sites affected by different levels of contamination. For this purpose, we plotted the density distributions of the concentrations of the different elements and examined the local variability at three sites. For sites with mean concentrations differing by more than 30 %, 20 samples were sufficient to detect significant differences for all of the elements, except Cr. The proposed methodology could be used in other studies in the absence of specific research on each species and region.

Effects of a glyphosate-based herbicide on Fucus virsoides (Fucales, Ochrophyta) photosynthetic efficiency.

Herbicides are increasingly recognised as sources of water pollution. Glyphosate-based herbicides (GBHs) are widely used because of their low cost and high effectiveness. By measuring the photosynthetic efficiency of Fucus virsoides fronds exposed to a GBH (Roundup® Power 2.0), we investigated the effect of a continuous exposure (6 days) and the potential of recovery after a short exposure (24 h). Both experiments were carried out combining GBH with and without nutrient enrichment, simulating a runoff event. A factorial experimental design allowed us to assess the potential of interactions between GBH and nutrients, which are likely to co-occur in coastal areas. Our results show deleterious effects of GBH at low concentration on F. virsoides, independently from the duration of exposure and the presence of nutrients.

Bioaccumulation of Hg, Cd and Pb by Fucus vesiculosus in single and multi-metal contamination scenarios and its effect on growth rate.

Results of 7-days exposure to metals, using environmentally realistic conditions, evidenced the high potential of living Fucus vesiculosus to remove Pb, Hg and Cd from contaminated salt waters. For different contamination scenarios (single- and multi-contamination), ca 450 mg L-1 (dry weight), enable to reduce the concentrations of Pb in 65%, of Hg in 95% and of Cd between 25 and 76%. Overall, bioconcentration factors ranged from 600 to 2300. Elovich kinetic model described very well the bioaccumulation of Pb and Cd over time, while pseudo-second-order model adjusted better to experimental data regarding Hg. F. vesiculosus showed different affinity toward studied metals, following the sequence order: Hg > Pb > Cd. Analysis of metal content in the macroalgae after bioaccumulation, proved that all metal removed from solution was bound to the biomass. Depuration experiments reveled no significant loss of metal back to solution. Exposure to contaminants only adversely affected the organism's growth for the highest concentrations of Cd and Pb. Findings are an important contribute for the development of remediation biotechnologies for confined saline waters contaminated with trace metal contaminants, more efficient and with lower costs than the traditional treatment methods.

An evaluation of the toxicity and bioaccumulation of bismuth in the coastal environment using three species of macroalga.

Bismuth is a heavy metal whose biogeochemical behaviour in the marine environment is poorly defined. In this study, we exposed three different species of macroalga (the chlorophyte, Ulva lactuca, the phaeophyte, Fucus vesiculosus, and the rhodophyte, Chondrus crispus) to different concentrations of Bi (up to 50 µg L(-1)) under controlled, laboratory conditions. After a period of 48-h, the phytotoxicity of Bi was measured in terms of chlorophyll fluorescence quenching, and adsorption and internalisation of Bi determined by ICP after EDTA extraction and acid digestion, respectively. For all algae, both the internalisation and total accumulation of Bi were proportional to the concentration of aqueous metal. Total accumulation followed the order: F. vesiculosus > C. crispus > U. lactuca; with respective accumulation factors of about 4200, 1700 and 600 L kg(-1). Greatest internalisation (about 33% of total accumulated Bi) was exhibited by C. crispus, the only macroalga to display a phytotoxic response in the exposures. A comparison of the present results with those reported in the literature suggests that Bi accumulation by macroalgae is significantly lower than its accumulation by marine plankton (volume concentration factors of 10(5) to 10(7)), and that the phytotoxicity of Bi is low relative to other heavy metals like Ag and Tl.

Accumulation of silver by Fucus spp. (Phaeophyceae) and its toxicity to Fucus ceranoides under different salinity regimes.

Metals constitute an important group of abiotic stressors that elicit stress responses in marine algae that include the production of reactive oxygen species (ROS). Silver (Ag) is a highly toxic metal to organisms but despite this there are relatively few studies on how it affects marine macroalgae (seaweeds). In a landmark study published in 1977 the first information was provided on the accumulation of Ag in Fucus spp. (Phaeophyceae) from the Looe estuary, located in south-west England, an area with a long history of mining activity. In the present study, the estuary has been re-visited and the patterns of Ag accumulation in two Fucus spp. and sediment re-examined after 35 years. We conclude that Ag concentrations in sediment and macroalgae from specific sites within the catchment remain high, but more generally sediment concentrations have declined by approximately 65 % and the dissolved, bioavailable fraction by 24 % over this period. In addition, from laboratory studies we provide data on the speciation and toxic effects of Ag under different salinity regimes in the euryhaline brown seaweed, Fucus ceranoides. From these exposure experiments, it was found that with increasing Ag concentrations growth was inhibited and lipid peroxidation associated with ROS production increased. The magnitude of the toxic effects was greater at a salinity of 10 than 28 psu which reflects the greater bioavailability of the toxic species of Ag (Ag(+) and AgCl(0)) at reduced salinities. These findings emphasise the importance of investigating the effects of metal pollution in conjunction with other, natural, environmental stressors such as salinity.

A macroalgal germling bioassay to assess biocide concentrations in marine waters.

A bioassay method using the early life stages (germlings) of macroalgae was developed to detect toxicity of anti-fouling paint biocides. A laboratory based bioassay using Ulva intestinalis and Fucus spiralis germlings was performed with 4 common anti-fouling biocides (tributyltin (TBT), Irgarol 1051, Diuron and zinc sulphate), over a range of environmentally relevant concentrations (0.0033-10 µg l(-1)). Comparison between the two species showed that germlings of U. intestinalis were better adapted for in-situ monitoring, as germlings of F. spiralis appeared to be too robust to display sufficient growth differences. The response of U. intestinalis germling growth appeared to reflect environmental biocide concentrations. Overall the developed method showed potential for the assessment of the sub-lethal effects of anti-fouling biocides on the early developmental stages of U. intestinalis.

Box-Behnken methodology for Cr (VI) and leather dyes removal by an eco-friendly biosorbent: F. vesiculosus.

This study focused on leather industrial effluents treatment by biosorption using Fucus vesiculosus as low-cost adsorbent. These effluents are yellowish-brown color and high concentration of Cr (VI). Therefore, biosorption process was optimized using response surface methodology based on Box-Behnken design operating with a simulated leather effluent obtained by mixture of Cr (VI) solution and four leather dyes. The key variables selected were initial solution pH, biomass dosage and CaCl2 concentration in the pretreatment stage. The statistical analysis shows that pH has a negligible effect, being the biomass dosage and CaCl2 concentration the most significant variables. At optimal conditions, 98% of Cr (VI) and 88% of dyes removal can be achieved. Freundlich fitted better to the obtained equilibrium data for all studied systems than Temkin, Langmuir or D-R models. In addition, the use of the final biosorbent as support-substrate to grown of enzyme producer fungi, Pleurotus ostreatus, was also demonstrated.

Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.

A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

Induction of expression of a 14-3-3 gene in response to copper exposure in the marine alga, Fucus vesiculosus.

The macro-alga Fucus vesiculosus has a broad global and estuarine distribution and exhibits exceptional resistance to toxic metals, the molecular basis of which is poorly understood. To address this issue a cDNA library was constructed from an environmental isolate of F. vesiculosus growing in an area with chronic copper pollution. Characterisation of this library led to the identification of a cDNA encoding a protein known to be synthesised in response to toxicity, a full length 14-3-3 exhibiting a 71% identity to human/mouse epsilon isoform, 70-71% identity to yeast BMH1/2 and 95 and 71% identity to the Ectocarpus siliculosus 14-3-3 isoforms 1 and 2 respectively. Preliminary characterisation of the expression profile of the 14-3-3 indicated concentration- and time-dependent inductions on acute exposure of F. vesiculosus of copper (3-30 µg/l). Higher concentrations of copper (≥150 µg/l) did not elicit significant induction of the 14-3-3 gene compared with the control even though levels of both intracellular copper and the expression of a cytosolic metal chaperone, metallothionein, continued to rise. Analysis of gene expression within environmental isolates demonstrated up-regulation of the 14-3-3 gene associated with the known copper pollution gradient. Here we report for the first time, identification of a gene encoding a putative 14-3-3 protein in a multicellular alga and provide preliminary evidence to link the induction of this 14-3-3 gene to copper exposure in this alga. Interestingly, the threshold exposure profile may be associated with a decrease in the organism's ability to control copper influx so that it perceives copper as a toxic response.

Effect studies of human pharmaceuticals on Fucus vesiculosus and Gammarus spp.

In two experiments, the human pharmaceutical propranolol negatively affected the physiology of two test organisms, Fucus vesiculosus and Gammarus spp. from a Baltic Sea littoral community in a concentration of 1000 µg l⁻¹. Some effects were also observed for the lower, more ecologically relevant concentrations (10 µg l⁻¹ and 100 µg l⁻¹). The effects on F. vesiculosus not only increased with increasing concentration, but also with exposure time; while the effects on Gammarus spp. were more inconsistent over time. No clear effects of the pharmaceuticals diclofenac and ibuprofen were observed for any of the organisms. Physiological parameters measured were GP:R-ratio, chlorophyll fluorescence and release of coloured dissolved organic matter, respiration and ammonium excretion. Pharmaceutical substances are repeatedly detected in the Baltic Sea which is the recipient for STP effluents from more than 85 million people living in the catchment area, but the knowledge of their effects on non-target organisms is still very limited.

Stress ecology in fucus: abiotic, biotic and genetic interactions.

Stress regimes defined as the synchronous or sequential action of abiotic and biotic stresses determine the performance and distribution of species. The natural patterns of stress to which species are more or less well adapted have recently started to shift and alter under the influence of global change. This was the motivation to review our knowledge on the stress ecology of a benthic key player, the macroalgal genus Fucus. We first provide a comprehensive review of the genus as an ecological model including what is currently known about the major lineages of Fucus species with respect to hybridization, ecotypic differentiation and speciation; as well as life history, population structure and geographic distribution. We then review our current understanding of both extrinsic (abiotic/biotic) and intrinsic (genetic) stress(es) on Fucus species and how they interact with each other. It is concluded that (i) interactive stress effects appear to be equally distributed over additive, antagonistic and synergistic categories at the level of single experiments, but are predominantly additive when averaged over all studies in a meta-analysis of 41 experiments; (ii) juvenile and adult responses to stress frequently differ and (iii) several species or particular populations of Fucus may be relatively unaffected by climate change as a consequence of pre-adapted ecotypes that collectively express wide physiological tolerences. Future research on Fucus should (i) include additional species, (ii) include marginal populations as models for responses to environmental stress; (iii) assess a wider range of stress combinations, including their temporal fluctuations; (iv) better differentiate between stress sensitivity of juvenile versus adult stages; (v) include a functional genomic component in order to better integrate Fucus' ecological and evolutionary responses to stress regimes and (vi) utilize a multivariate modelling approach in order to develop and understand interaction networks.

Impacts of ambient salinity and copper on brown algae: 2. Interactive effects on phenolic pool and assessment of metal binding capacity of phlorotannin.

The aim of this study was to establish in laboratory experiments a quantitative link between phenolic pool (production, composition and exudation) in Ascophyllum nodosum and Fucus vesiculosus and their potential to bind metals. Additionally, the copper binding capacity of purified phlorotannin was investigated. A reduction in salinity decreased total phenolic contents, altered phenolic composition by increasing proportion of cell-wall phenolics, and also increased phenolic exudation of the two seaweed species. After 15 days at a salinity of 5, the inhibition of photosynthesis observed previously for A. nodosum coincided with the high exudation of phenolic compounds into the surrounding water of the seaweed tips which resulted in a significant reduction of phenolic contents. Increased copper concentration also reduced total phenolic contents, changed phenolic composition (increase in proportion and level of cell-wall phenolics), and positively affected phenolic exudation of A. nodosum and F. vesiculosus. A decrease in salinity enhanced the copper toxicity and caused the earlier impact on the physiology of seaweed tips. An involvement of phlorotannins in copper binding is also demonstrated; purified phlorotannins from A. nodosum collected from a site with little anthropogenic activity contained all four metals tested. When placed in copper-enriched water, as for the seaweed material, copper contents of the phenolics increased, zinc and cadmium contents decreased, but no change in chromium content was observed. The use of cell-wall phenolic content as biomarker of copper contamination seems promising but needs further investigation.

Adaptation to high light irradiances enhances the photosynthetic Cu2+ resistance in Cu2+ tolerant and non-tolerant populations of the brown macroalgae Fucus serratus.

The relationship between light acclimation and Cu(2+) tolerance was studied in two populations of Fucus serratus known to be naturally non-tolerant and tolerant to Cu(2+). Acclimation to high irradiances increased the photosynthetic tolerance to Cu(2+). The xanthophyll cycle was apparently not involved in protecting the photosynthetic apparatus against Cu(2+) toxicity, as results showed that Cu(2+) did not induce dynamic photoinhibition. The higher photosynthetic Cu(2+) resistance of high light algae did not result in increased growth. The excess energy acquired by high light-adapted algae appeared to be utilized in Cu(2+) defense mechanisms in the Cu(2+) non-tolerant population. The polyphenol content of the algae was reciprocal to the Cu(T) content, suggesting that polyphenol may be the primary Cu(2+) defense of non-tolerant low light algae, acting through secretion and extracellular chelating of Cu(2+), while the compounds do not seem to be involved in the primary Cu(2+) tolerance mechanism in Cu(2+) tolerant algae.

Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein.

The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.

Cadmium tolerance and adsorption by the marine brown alga Fucus vesiculosus from the Irish Sea and the Bothnian Sea.

Cadmium (Cd) uptake capacities and Cd tolerance of the marine alga Fucus vesiculosus from the Irish Sea (salinity 35 psu) and from the Bothnian Sea (northern Baltic, 5 psu) were quantified. These data were complemented by measurements of changes in maximal photosynthetic rate (P(max)), dark respiration rate and variable fluorescence vs. maximal fluorescence (F(v):F(m)). At concentrations between 0.01 and 1 mmol Cd l(-1), F. vesiculosus from the Bothnian Sea adsorbed significantly more (about 98%) Cd compared with F. vesiculosus from the Irish Sea. The photosynthetic measurements showed that the Bothnian Sea F. vesiculosus were more sensitive to Cd exposure than the Irish Sea algae. The algae from the Irish Sea showed negative photosynthetic effects only at 1 mmol Cd l(-1), which was expressed as a decreased P(max) (-12.3%) and F(v):F(m) (-4.6%). On the contrary, the algae from the Bothnian Sea were negatively affected already at Cd concentrations as low at 0.1 mmol Cd l(-1). They exhibited increased dark respiration (+11.1%) and decreased F(v):F(m) (-13.9%). The results show that F. vesiculosus from the Bothnian Sea may be an efficient sorption substrate for Cd removal from Cd contaminated seawater and this algae type may also have applications for wastewater treatment.

Dissolved organic carbon reduces the toxicity of copper to germlings of the macroalgae, Fucus vesiculosus.

This study investigates the effects of waterborne copper exposure on germling growth in chemically defined seawater. Germlings of the macroalgae, Fucus vesiculosus were exposed to a range of copper and dissolved organic carbon (DOC as humic acid) concentrations over 14 days. Germling growth was found to be a sensitive indicator of copper exposure with total copper (TCu) and labile copper (LCu) EC(50) values of approximately 40 and 20 microg/L, respectively, in the absence of added DOC. The addition of DOC into the exposure media provided germlings with protection against copper toxicity, with an increased TCu EC(50) value of 117.3 microg/L at a corrected DOC (cDOC from humic acid only) concentration of 2.03 mg/L. The LCu EC(50) was not affected by a cDOC concentration of 1.65 mg/L or less, suggesting that the LCu concentration not the TCu concentration was responsible for inhibiting germling growth. However, at a cDOC concentration of approximately 2mg/L an increase in the LCu EC(50) suggests that the LCu concentration may play a role in the overall toxicity to the germlings. This is contrary to current understanding of aquatic copper toxicity and possible explanations for this are discussed.

Fucus spp. as a mercury contamination bioindicator in costal areas (Northwestern Portugal).

Mercury has been considered as one of the most important pollutants in coastal and estuarine areas. Efforts have been made to detect, as early as possible, the effects of this and other metals in several species. Macroalgae, particularly Fucus spp., have been widely used as biomonitors of metal pollution. In this study, three Fucus species (F. spiralis, F. vesiculosus and F. ceranoides) were collected from several sampling sites in Portugal. The concentrations of mercury were determined in three structural parts (holdfast, stipe and receptacles). Two different techniques were used to determine mercury concentrations. Almost all mercury concentrations (in sediments and in water) were below national and international standards. Mercury concentration in the specimens (0.012-0.061 microg g(-1) for receptacles, 0.028-0.221 microg g(-1) for stipe and 0.029-0.287 microg g(-1) for holdfast) was always higher that those obtained for the sediment (0.001-0.112 microg g(-1)). With few exceptions the contrary was found for receptacles. In general, a good agreement between concentrations of mercury in sediment and Fucus was found. The results indicate that Fucus accumulate mercury and may be a suitable species for use in risk assessment for coast and estuarine areas, by providing valuable information regarding the levels of mercury that will be available for the consumers of Fucus spp.

The influence of halide-mediated oxidation on algae-born adhesives.

Adhesive materials extracted from the brown algae Fucus Serratus were studied. These adhesives are composed of cross-linked alginate and polyphenols oxidized in the presence of KI or KBr. All formulations were capable of adhering to a variety of surfaces, however the adhesion properties were influenced by the halide used. SAXS and TEM experiments revealed that oxidized polyphenols self-assemble into chain-like objects, irrespective of the oxidation conditions. Yet, slight differences in the aggregate size were detected. QCM-D results showed that the kinetics of the oxidation was faster with iodide than with bromide. Moreover, oxidation with iodide generates stiffer networks, suggesting that the interaction between the alginate and the polyphenol could be the cause of the reduced adhesion.

Nodularin induces oxidative stress in the Baltic Sea brown alga Fucus vesiculosus (Phaeophyceae).

In the Baltic Sea regular, intensive cyanobacterial blooms rich in the cyanobacterium Nodularia spumigena occur during the summer season. N. spumigena is known to produce the cyclic pentapeptide nodularin (NOD) in high concentrations. Marine macroalgae, together with sea-grass meadows, are an extremely important habitat for life in the sea. In addition to this, the decaying macroalgae substantially contribute to the substrate for the microbial loop in coastal food webs. Uptake of nodularin into the brown macroalga Fucus vesiculosus was assessed using an ELISA technique resulting in an uptake of up to 45.1 microg kg(-1) fresh weight (fw). Nodularin was also detected in the reproductive part of the algae (receptacle) at 14.1 microg kg(-1) fw. The induction of oxidative stress in F. vesiculosus, after exposure to NOD, was also shown by monitoring cellular damage as changes in lipid peroxidation and the activation of antioxidative defence systems (antioxidative capacity, superoxide dismutase and soluble glutathione S-transferase).

Photosynthetic responses to Cu2+ exposure are independent of light acclimation and uncoupled from growth inhibition in Fucus serratus (Phaeophyceae).

We have studied the effect of light acclimation on photosynthetic responses and growth during Cu2+ exposure (0-0.84 microM) in the brown seaweed Fucus serratus. Measurements of chlorophyll fluorescence parameters showed that Cu2+ exposure amplified ETR, reduced the chlorophyll content at the cellular level and that there was no effect of light adaptation on the Cu2+ resistance of the algae. In contrast to the inhibitory effects of Cu2+ on chlorophyll fluorescence, O2 evolution and the total content of chlorophyll and carotenoid of the algae was unaffected by Cu2+. We conclude that photoinhibition and perhaps pigment degradation in the meristoderm was compensated for by cells deeper in the thallus with the result that the overall photosynthetic fitness of the algae was maintained. The pronounced inhibitory effects of Cu2+ on algae growth was not a consequence of photoinhibition and could be attributed to direct inhibitory effects on the growth process.