Application of a robust analytical method for quantifying progestins in environmental samples from three Portuguese Estuaries.

In the last years, progestins have raised special concerns for their documented negative effects on aquatic species, yet little is known about their environmental levels in surface waters and bioaccumulation in the trophic web. This study aimed to 1) adapt an extraction method for quantifying progestins in freeze-dried matrices, 2) validate the analytical procedure for three matrices: bivalve, polychaete, and crustacean, and 3) characterize levels of the four most prescribed synthetic progestins in key species across three Portuguese estuaries. Through the validated method, progestins were only quantifiable for the crustacean. Values were generally low, peaking with drospirenone values in Ria de Aveiro (1.33 ± 0.26 ng/g ww) and Tagus estuary (1.42 ± 0.55 ng/g ww), while Ria Formosa exhibited the lowest progestin concentrations (< 1 ng/g ww). This study enabled the development of a precise extraction and analytical method for quantifying steroid hormones in three distinct biological matrices.

Baseline progestins characterization in surface waters of three main Portuguese estuaries.

Synthetic progestins are micropollutants of special concern, due to their growing use in human and veterinary therapies and their risks to aquatic life. Currently, there is a lack of environmental information on these compounds, worldwide. The main objective of this work was to characterize the levels of the most consumed progestins in Portugal. For that, Ria de Aveiro, Tagus estuary and Ria Formosa were sampled in a temporal perspective to evaluate levels of drospirenone (DRO), desogestrel (DSG), gestodene (GST) and levonorgestrel (LNG). Drospirenone and desogestrel were the most abundant progestins. In the North of Portugal, DSG was the most abundant (Aveiro: 193.9 ng L-1 in summer), while DRO was more representative in the South (Tagus: 178.9 ng L-1; Formosa: 125.7 ng L-1) and also in summer. These spatial differences can be associated with the hydrodynamics of each estuarine system as well as the distinct population and tourist levels associated with each site.

Influence of oceanic and climate conditions on the early life history of European seabass Dicentrarchus labrax.

Understanding how marine fish early-life history is affected in the long-term by environmental and oceanographic factors is fundamental given its importance to population dynamics and connectivity. This work aimed at determining the influence of these processes on the interannual variability in hatch day and early-life growth patterns of European seabass, over a seven-year period (2011-2017) in the Atlantic Iberian coast. To accomplish this, otolith microstructure analysis was used to determine seabass hatch day and to develop early-growth correlations. In most years, hatching occurred from February to April, with two exceptions: in 2012, hatching started in early-January, and in 2016 an exceptionally long hatching period was registered. Using generalized additive models (GAM), we observed that sea surface temperature (SST), the North Atlantic Oscillation index (NAOi) and Chlorophyll-a (Chla) were the main drivers behind the inter-annual variability in seabass hatch day. Analysis of correlations between growth increments allowed assessing important periods of seabass growth and how future growth is affected. Since seawater temperature is among the main drivers for seabass recruitment and growth, its life cycle may be hampered due to ocean warming and an increasingly unstable climate, with consequences for the natural marine stocks and their harvest.

A macroalgae-based biotechnology for water remediation: Simultaneous removal of Cd, Pb and Hg by living Ulva lactuca.

Metal uptake from contaminated waters by living Ulva lactuca was studied during 6 days, under different relevant contamination scenarios. In mono-metallic solutions, with concentrations ranging from 10 to 100 µg L-1 for Hg, 10-200 µg L-1 for Cd, and 50-1000 µg L-1 for Pb, macroalgae (500 mg L-1, d.w.) were able to remove, in most cases 93-99% of metal, allowing to achieve water quality criteria regarding both surface and drinking waters. In multi-metallic solutions, comprising simultaneously the three metals, living macroalgae still performed well, with Hg removal (c.a. 99%) not being significantly affected by the presence of Cd and Pb, even when those metals were in higher concentrations. Removal efficiencies for Cd and Pb varied between 57 and 96%, and 34-97%, respectively, revealing an affinity of U. lactuca toward metals: Hg > Cd > Pb. Chemical quantification in macroalgae, after bioaccumulation assays demonstrated that all Cd and Hg removed from solution was really bound in macroalgae biomass, while only half of Pb showed to be sorbed on the biomass. Overall, U. lactuca accumulated up to 209 µg g-1 of Hg, up to 347 µg g-1 of Cd and up to 1641 µg g-1 of Pb, which correspond to bioconcentration factors ranging from 500 to 2200, in a dose-dependent accumulation. Pseudo-first order, pseudo-second order and Elovich models showed a good performance in describing the kinetics of bioaccumulation, in the whole period of time. In the range of experimental conditions used, no mortality was observed and U. lactuca relative growth rate was not significantly affected by the presence of metals. Results represent an important contribution for developing a macroalgae-based biotechnology, applied for contaminated saline water remediation, more "green" and cost-effective than conventional treatment methods.

Comparative study on metal biosorption by two macroalgae in saline waters: single and ternary systems.

The biosorption capability of two marine macroalgae (green Ulva lactuca and brown Fucus vesiculosus) was evaluated in the removal of toxic metals (Hg, Cd and Pb) from saline waters, under realistic conditions. Results showed that, independently of the contamination scenario tested, both macroalgae have a remarkable capacity to biosorb Hg and Pb. In single-contaminant systems, by using only c.a. 500 mg of non-pre-treated algae biomass (size <200 µm) per litter, it was possible to achieve removal efficiencies between 96 and 99 % for Hg and up to 86 % for Pb. Despite the higher removal of Hg, equilibrium was reached more quickly for Pb (after 8 h). In multi-contaminant systems, macroalgae exhibited a similar selectivity toward the target metals: Hg > Pb> > Cd, although Pb removal by U. lactuca was more inhibited than that achieved by F. vesiculosus. Under the experimental conditions used, none of the macroalgae was effective to remove Cd (maximum removal of 20 %). In all cases, the kinetics of biosorption was mathematically described with success. Globally, it became clear that the studied macroalgae may be part of simple, efficient, and cost-effective water treatment technologies. Nevertheless, Fucus vesiculosus has greater potential, since it always presented higher initial sorption rates and higher removal efficiencies.

Long-term functional changes in an estuarine fish assemblage.

The functional diversity of the fish assemblages of the Mondego estuary was studied for a discontinuous 30-year period (1988-2012). During this time, hydrological changes occurred due to man-induced alterations and weather extremes. These changes led to alterations in the structure and function of the fish community. Species richness and functional richness decreased over time and the fish community started to explore new micro-habitats and food resources. Before severe hydrological changes, the community was dominated by pelagic, detritivorous and species with wider salinity ranges. After, the community became dominated by demersal, benthic, piscivorous and marine species. During a drought, omnivorous became increasingly important, reflecting greater possibilities of using available feeding resources. We have also found an increase in sub-tropical species throughout the years, which might be related to gradual temperature increases at a global scale. This study also confirmed estuaries as extremely important for restocking several commercial species.

Field transplantation of the bivalve Scrobicularia plana along a mercury gradient in Ria de Aveiro (Portugal): uptake and depuration kinetics.

The bioaccumulation and depuration capabilities of mercury by the edible bivalve Scrobicularia plana was studied in a coastal lagoon (Ria de Aveiro, Portugal) through a transplantation experiment. Little information on this topic is available in the literature, especially concerning different tissues' responses to contaminant exposure, but the present study is one of the few works that can surpass this knowledge gap. Organisms from a reference area were transplanted to two different contaminated areas in the Ria de Aveiro. In both areas, the bivalves (i.e., entire organism, digestive gland and the rest of the organism) presented a similar saturation model of mercury accumulation, the digestive gland being the tissue that reached the highest concentrations after 25 days of exposure to the contaminant. During this short uptake period, the transplanted organisms reached 20-30% of the concentrations observed in resident contaminated organisms. After the exposure period, the organisms were transplanted to a clean area for more than 25 days of depuration. At the end of the transplantation period, organisms lost approximately 50% of their mercury body burden (60%: the entire organism and digestive gland; 35%: gills and 40%: the rest of the organism) and the ones from the least contaminated site almost reached the concentrations recorded in the reference area. So, the results suggest that S. plana is a promising biomonitoring species, since it accumulates the contaminant in a considerable extent quite rapidly and at the same time it has a low metal retention capacity (low biological half-life) when exposed to clean sediments.

Recovery trends of Scrobicularia plana populations after restoration measures, affected by extreme climate events.

The Mondego estuary (Portugal) went through different ecological scenarios over the last decades. An eutrophication process led to a decline in the ecosystem quality. The ensuing restoration plan resulted into a gradual ecological recovery, which was impaired by the occurrence of successive extreme climate events that affected dynamics and productivity of key species. In this study we assess the response of the bivalve Scrobicularia plana to the impacts of these events in a recovery scenario, by comparing populations in two different intertidal habitats: a seagrass bed and a sandflat area. As a general tendency, S. plana, which was negatively affected by eutrophication, responded positively to restoration. However, the occurrence of extreme climate events seemed to affect recruitment success, biomass and production, impairing the recovery process. In the seagrass bed, S. plana maintained a stable and structured population, while in the sandflat area recovery clearly reverted into a decline, mainly concerning biomass and production values. This sequence of multiple stressors might have reduced S. plana resilience to further impacts and therefore, understanding the behavior of biological populations following restoration initiatives requires acknowledgement that some changes may not be easily reversible.

Uptake and depuration of PCB-153 in edible shrimp Palaemonetes varians and human health risk assessment.

A medium-term mesocosm exposure study was conducted to elucidate bioaccumulation and depuration of polychlorinated biphenyl congener 153 (PCB-153) in edible shrimp Palaemonetes varians. Over the 15-day exposure period, shrimp under different exposure concentrations exhibited a significant increase in PCB-153 concentration compared with control organisms. Distinct bioaccumulation patterns and uptake rates were observed depending on the exposure concentrations. For low PCB-153 exposure levels (0.25µgL(-1)), accumulation followed a saturation model, reaching an apparent steady state after fifteen days exposure. For intermediate (2.5µgL(-1)) and high PCB-153 levels (25µgL(-1)), accumulation was faster and linear. In addition, the bioaccumulation rate was not proportional to PCB-153 concentration, and the bioaccumulation was higher at intermediate exposure concentrations. Regarding the depuration phase, P. varians lost up to 30% of PCB-153 after 72h and levels continued slowly to decrease until the end of the 30-d experimental period. However, PCB-153 levels in shrimp did not reach background values, and those exposed to moderate and high PCB-153 concentrations presented contamination levels much higher than the regulatory limit for human food consumption (75ngg(-1) ww for Σ6 PCB).

Kinetics of the PO4-P adsorption onto soils and sediments from the Mondego estuary (Portugal).

In order to assess the status of a North Atlantic Ocean Ecoregion (Mondego system, Portugal) after the implementation of a management programme, a study on PO4-P sorption was carried out. Considering that sorption velocity markedly determines the systems promptness to phosphorus external loading, the kinetics of the sorption of PO4-P onto estuarine sediments and the upstream agricultural soils was studied. The pseudo-second order kinetic equation gave an appropriate description of PO4-P sorption onto these soils and sediments, allowing for a quantitative comparison. For soils, the kinetic constants (k2*, µg(-1) g h(-1)) ranged between 0.007 and 0.017 µg(-1) g h(-1) while, in the case of sediments these ranged between 0.008 and 0.012 µg(-1) g h(-1). Results showed that the reduction of water residence time after the management action was relevant for the system status, affecting its reaction capacity to phosphorus loading.

Mercury bioaccumulation and the population dynamics of Mesopodopsis slabberi (Crustacea: Mysidacea) along a mercury contamination gradient.

The mercury bioaccumulation and population dynamics of the mysid Mesopodopsis slabberi was assessed along a mercury gradient in Ria de Aveiro (Portugal). M. slabberi is one of the most important mysid species in European temperate coastal shallow waters playing a key ecological role. Nevertheless, no references were found concerning the possible consequences of the Hg on the trophodynamics of these coastal ecosystems. M. slabberi showed a clear bioaccumulation along the Hg gradient and through life, with mature females reaching the highest concentrations. In terms of population structure, higher densities and biomasses of M. slabberi were assessed in the most contaminated areas contrarily to the least polluted areas. Despite the mercury accumulation in its tissues no strong negative effects on the structure and population dynamics of the species were observed. However, mysids might be important in the transfer of metals from the sediments and zooplankton to higher trophic levels such as fishes, most of them with commercial interest.

Influence of age, sex and breeding status on mercury accumulation patterns in the wandering albatross Diomedea exulans.

Although mercury bio-amplifies through the food chain and accumulates in top predators, mercury concentrations in tissues of the wandering albatross are greater than in any other vertebrate, including closely related species. In order to explore the alternative explanations for this pattern, we measured total mercury concentrations in feathers, plasma and blood cells of wandering albatrosses of known age, sex and breeding status sampled at South Georgia. Mercury concentrations were low in feathers and blood components of chicks, and higher in the feathers of young pre-breeders than in feathers or blood of older pre-breeders and breeding adults. There was no effect of sex on mercury concentrations in the feathers of pre-breeders or breeding adults, whereas levels were significantly higher in blood cells of breeding females than males. The high feather mercury concentrations of young pre-breeders compared with older birds suggest an increase in moult frequency as birds approach maturity.

Organochlorine accumulation on a highly consumed bivalve (Scrobicularia plana) and its main implications for human health.

Contamination by polychlorinated biphenyls (PCBs) and hexachlorobenzene (HCB) was investigated along a spatial gradient in water, sediments and in commercially important bivalve species Scrobicularia plana, from Ria de Aveiro (Portugal). Organochlorines dissolved in water were below detection limit and concerning suspended particulate matter, only PCBs were quantified, ranging from 3.8 to 5.8 ng∙g(-1) DW (Σ13PCBs). There was a distinct spatial gradient regarding PCB accumulation in sediments. The highest concentrations were found in deeper layers and closest to the pollution source, decreasing gradually along a 3 km area. Contamination in sediments exceeded the Canadian and Norwegian sediment quality guidelines, inducing potential toxic effects in related biota. PCBs tended to bioaccumulate throughout S. plana lifespan but with different annual rates along the spatial gradient. The maximum values were found in older individuals up to 3+ years old, reaching 19.4 ng∙g(-1) DW. HCB concentrations were residual and no bioaccumulation pattern was evident. Congeners 138, 153 and 180 were the most accumulated due to their abundance and long-term persistence in the environment. In the inner area of the Laranjo Bay (0.6 km(2)), the species was able to remove up to 0.4 g of PCBs annually from sediments into their own tissues, which is consequently free for trophic transfer (biomagnification). Concerning human health, and despite the high concentrations found in sediments, PCB levels in bivalves do not exceed the limit established by the European Union for fishery products and are largely below tolerable daily intake. Although PCBs in Scrobicularia plana are present at low levels, their impact to human health after consumption over many years might be harmful and should be monitored in future studies.

Mercury biomagnification in a contaminated estuary food web: effects of age and trophic position using stable isotope analyses.

The main aim of this study was to ascertain the biomagnification processes in a mercury-contaminated estuary, by clarifying the trophic web structure through stable isotope ratios. For this purpose, primary producers (seagrasses and macroalgae), invertebrates (detritivores and benthic predators) and fish were analysed for total and organic mercury and for stable carbon and nitrogen isotopic signatures. Trophic structure was accurately described by δ(15)N, while δ(13)C reflected the carbon source for each species. An increase of mercury levels was observed with trophic level, particularly for organic mercury. Results confirm mercury biomagnification to occur in this estuarine food web, especially in the organic form, both in absolute concentrations and fraction of total mercury load. Age can be considered an important variable in mercury biomagnification studies, and data adjustments to account for the different exposure periods may be necessary for a correct assessment of trophic magnification rates and ecological risk.

Mercury bioaccumulation and decontamination kinetics in the edible cockle Cerastoderma edule.

Mercury bioaccumulation and decontamination kinetics in the edible cockle Cerastoderma edule were studied through a mesocosms experiment after a medium-term exposure to the metal. The results revealed that the bivalve presented distinct bioaccumulation kinetics according to the different tissues. While the gills showed a linear accumulation pattern, the digestive gland and the entire organism presented a saturation model, with higher accumulation during the first 7d of exposure and lower during the rest of the time. In addition, the bioaccumulation rate was not proportional to the Hg concentration, since the organisms under lower contamination presented higher bioconcentration factors than the ones under higher contamination. Gills were the tissues with higher mercury accumulation capability. Concerning the decontamination phase, C. edule lost approximately 80% of the mercury after 24h exposure in clean seawater. Nevertheless, never reached the original condition, showing in the final (20 d detox), Hg levels (>0.5 ppm) higher than those allowed by the legislation regulating human food consumption. This represents a matter of concern for Human health.

Implications of Zostera noltii recolonization on Hydrobia ulvae population structure success.

Over 1990-1998, the Mondego estuary, Portugal, experienced profound modifications due to eutrophication, culminating in the disappearance and replacement of Zostera noltii by opportunistic macroalgae in the inner most disturbed areas. A decade after restoration measures implementation, Z. noltii started to gradually recolonize the inner parts, following 20 years of absence. This work explores the factors underlying successful Z. noltii recolonization and its subsequent implications on a mud snail Hydrobia ulvae population. During the macroalgal bloom, highest values in H. ulvae abundance, biomass and production were recorded, strongly declining afterwards. Three recovery attempts characterized the post-restoration phase, with considerably increases in H. ulvae abundance, biomass and production since Z. noltii reappearance. The seagrass provided long-term protection and abundant food resources for reproductive adults, contrarily to the ephemeral macroalgae. Through time, large size individuals increased, becoming the population more stable, structured and similar to the one inhabiting the "original"Z. noltii meadows.

Effects of extreme climate events on the macrobenthic communities' structure and functioning of a temperate estuary.

The Mondego estuary (Portugal) experienced profound structural and functional modifications due to eutrophication, which was exacerbated by consecutive weather extremes that compromised a previous restoration project. This work explores multiple climate impacts on macrobenthic communities' structure and functioning and its implications on ecosystem's recovery. Floods and heat waves had a stronger negative effect on macrobenthic assemblages than the droughts, imposing a total abundance decline. Contrarily, biomass was not so affected by climate events, being stable and even increased in a mudflat area, where seagrass is re-colonizing. Bivalves and oligochaetes decreased with the flooding episodes, likewise subsurface-deposit feeders and suspension feeders, while crustaceans were particularly sensitive to heat waves. Species richness declined with the floods and heat waves, whilst evenness increased in sandflat area, constituting a positive sign towards recovery. Succession of different climate extremes affected ecosystem structure and functioning, delaying its recovery with possible consequent effects at higher trophic levels.

Mercury bioaccumulation in the spotted dogfish (Scyliorhinus canicula) from the Atlantic Ocean.

The annual and life-cycle mercury bioaccumulation pattern in selected tissues of the economically relevant Elasmobranchii species Scyliorhinus canicula was studied, and the risks associated with its consumption evaluated. Preferential mercury bioaccumulation occurred in muscle tissue, and followed the order muscle>heart>liver>gills>pancreas. Total mercury in muscle tissue ranged from 0.13mgkg(-1) (wwt) in 1+year old males to 0.8mgkg(-1) (wwt) in 8+ year old mature females, with no significant differences found between genders, and no clear lifespan bioaccumulation pattern observed, except for mature females. Organic mercury in the muscle ranged from 0.05mgkg(-1) (wwt) to 0.52mgkg(-1) (wwt), corresponding to an average of 70% of total mercury content. In mature females, a significant correlation (R=0.99, P=0.01) was found between size and organic mercury fraction, suggesting reproduction as an important factor controlling organic mercury bioaccumulation in the spotted dogfish.

Implications of nutrient decline in the seagrass ecosystem success.

The Mondego estuary (Portugal) suffered major changes in environmental quality due to eutrophication, however, in the late 1990 s a restoration project was implemented in order to return the system to its original condition. The main goal of this paper is to evaluate the ecosystem response to the restoration measures applied at three different levels: water quality, primary producers and primary consumers. In post-restoration period a clear decline was observed in dissolved inorganic nitrogen which was reflected in the gradual recovery of Zostera noltii and a concomitant decline in green macroalgae. Macrobenthic assemblages responded variably to the recovery process. In the seagrass bed and intermediate area, there was a large increase in total biomass, but in the eutrophic area species diversity increased. Despite improvement in the ecological status of the system, full recovery has not been achieved yet, possible due to hysteresis in the dynamics of this system.

Different mercury bioaccumulation kinetics by two macrobenthic species: the bivalve Scrobicularia plana and the polychaete Hediste diversicolor.

Mercury is a global priority pollutant and given its huge relevance in terms of environmental damage and a threat to human health, it is important to study the bioaccumulation processes at the level of macrobenthic organisms and evaluate possible consequences for the trophic chain. In this work we evaluate the mercury accumulation rates of two distinct and economically important macrobenthic species, the polychaete Hediste diversicolor and the bivalve Scrobicularia plana, through a mesocosms laboratory experiment. The present experiment demonstrated different bioaccumulation kinetics for each species: while S. plana has a rapid accumulation after 48 h of exposure to contaminated sediments and then reaches a steady state, H. diversicolor has a linear mercury accumulation throughout the experiment time (31 days). The different patterns observed can be related to different feeding strategies and assimilation efficiencies. In addition, we can conclude that independently of the mercury concentration in the sediments, the two studied species in both contaminated sediments reached the same percentage (25%) of mercury accumulated at the end of the experiment when compared to the maximum concentrations recorded in the field. This experiment highlights that the mercury bioaccumulation rate by these two economically and ecologically important macrobenthic species is a reasonably rapid process which can promote serious consequences for the higher trophic levels, constituting a severe risk to the natural environment and ultimately to human health.