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.

Combined effects of climate change and environmentally relevant mixtures of endocrine disrupting compounds on the fitness and gonads' maturation dynamics of Nucella lapillus (Gastropoda).

Coastal areas are affected by multiple stressors like climate change and endocrine disruptors (EDCs). In the laboratory, we investigated the combined effects of increased temperature and EDCs (drospirenone and mercury) on the fitness and gonads' maturation dynamics of the marine gastropod Nucella lapillus for 21 days. Survival was negatively affected by all the stressors alone, while, in combination, a synergistic negative effect was observed. Both chemicals, as single factors, did not cause any effect on the maturation stage of ovaries and testis. However, in the presence of a higher temperature, it was clear a delay in the maturation stage of the ovaries, but not in the testis, suggesting a higher negative impact of the stressors in females than in males. In summary, drospirenone caused a low negative impact in aquatic species, like gastropods, but in combination with other EDCs and/or increased temperature can be a matter of concern.

Benthic estuarine communities' contribution to bioturbation under the experimental effect of marine heatwaves.

Marine heatwaves are increasing worldwide, with several negative impacts on biological communities and ecosystems. This 24-day study tested heatwaves' effect with distinct duration and recovery periods on benthic estuarine communities' diversity and contribution to ecosystem functioning experimentally. The communities were obtained from a temperate estuary, usually subjected to high daily thermal amplitudes. Our goal was to understand the communities' response to the thermal change, including the community descriptors and behavioural changes expected during heat extremes. We measured community composition and structural changes and the bioturbation process and nutrient release as ecosystem functioning measurements. Overall, our findings highlight the potential tolerance of studied estuarine species to the temperature ranges tested in the study, as community composition and structure were similar, independently of the warming effect. We detected a slight trend for bioturbation and nutrient release increase in the communities under warming, yet these responses were not consistent with the heatwaves exposure duration. Overall, we conclude on the complexity of estuarine communities' contribution to functioning under warming, and the importance of scalable experiments with benthic organisms' responses to climate variability, accommodating longer time scales and replication. Such an approach would set more efficient expectations towards climate change mitigation or adaptation in temperate estuarine ecosystems.

Immunological and oxidative stress responses of the bivalve Scrobicularia plana to distinct patterns of heatwaves.

As a consequence of global warming, extreme events, such as marine heatwaves (MHW), have been increasing in frequency and intensity with negative effects on aquatic organisms. This innovative study evaluated for the first time, the immunological and physiological response of the estuarine edible bivalve Scrobicularia plana to different heatwaves, with distinct duration and recovery periods. So, extensive immune (total haemocyte count - THC, haemocyte viability, phagocytosis rate, respiratory oxidative burst of haemocytes, total protein, protease activity, nitric oxide and bactericidal activity of plasma) and oxidative stress (lipid peroxidation - LPO, superoxide dismutase - SOD, catalase - CAT and glutathione-S-Transferase - GST) analyses were performed in an experimental study that tested the impact of heatwaves during 25 days. The survival and condition of S. plana were not affected by the exposure to the extreme events. However, our data suggested that longer heatwaves with shorter recovery periods can be more challenging for the species, since THC and phagocytic activity were most affected under the temperature increase conditions. Regarding the oxidative status, the species increased its SOD activity while MDA production slightly declined to the increase of temperature, protecting the organism from cellular damage. These results indicate that S. plana has a great capacity to adapt to environmental temperature changes, however, the expected higher frequency/duration of heatwaves with climate change trends can cause some debility of the species face to other stressors, which can compromise its success in the future.

Combined effects of increased temperature and levonorgestrel exposure on zebrafish female liver, using stereology and immunohistochemistry against catalase, CYP1A, HSP90 and vitellogenin.

Climate change and pharmaceuticals contamination constitute two of the most relevant stressors on the aquatic ecosystems, however, there is a huge lack of information regarding the interactive effects of both stressors. For that, a mesocosm experiment was implemented where adult zebrafish were exposed to combined temperature and the progestin levonorgestrel (LNG) for 21 days. Considering that the liver is one of the organs where there is a greater metabolization and accumulation of toxicants, the main objective of this work was to assess the effects of both stressors on the female zebrafish hepatocytes morphology and functioning, through stereological and immunohistochemical techniques. Our results revealed an increase of coefficient of variation of the number distribution of hepatocytes volume (CVN(υ)) for individuals exposed to LNG, which denotes an increase of the hepatocytes size variability and is suggestive of functional impacts. This was corroborated by the signs of increased glycogen content with the exposure to increased LNG concentrations and temperature, indicating modified hepatocyte glycogen metabolism. Such disturbances can be considered indicators that the fish had to deal with impacts caused by the stress factors. Regarding the immunoreactivity, from the four proteins selected (catalase, CYP1A, HSP90 and Vtg), just in two of them (catalase and Vtg) were observed some responses to both stressors. For catalase there was a hormetic response, in which exposure to lower LNG concentrations caused a significant higher positive immunostaining than under higher LNG concentrations. While, for Vtg, significant effects of temperature and LNG existed, in which a decline in Vtg immunostaining was observed with exposure to higher temperature and lower LNG concentrations. These results should be seen as a warning sign about fine impacts of multiple stressors, such as temperature and progestogens, on the structure and functioning of zebrafish liver and potentially in other aquatic organisms, and on their health implications.

Lecanicillium aphanocladii: snake venom phospholipases A2 and proteases as tools to prospect enzymatic inhibitors.

Natural enzyme inhibitors have been widely described in literature because of its pharmacological and cosmetic applications. Fungi found in caves represent a promising source of bioactive substances that are still little explored scientifically. Thus, the present work evaluated the presence of enzymatic modulators in a filtrate obtained from the cultivation of the cave fungus Lecanicillium aphanocladii (Family: Cordycipitaceae). Snake venoms from Bothrops alternatus and Bothrops atrox were used as an enzymatic source for the induction of the phospholipase, proteolytic, thrombolytic, cytotoxic and coagulant activities. Compounds present in the fungal filtrate inhibited 50, 23·8, 26·6, 50·9 and 52·5% of the proteolytic, phospholipase, haemolytic, thrombolytic and coagulant activities respectively. The filtrate was not cytotoxic on erythrocytes, but induced partial dissolution of thrombi. Fungal enzyme inhibitors that have low or no toxicity and can be obtained on a large scale and at low cost are relevant in the medical-scientific context. Therefore, the inhibition of phospholipases A2 and proteases observed in the present work highlights the potential of fungal metabolites for the development of drugs that can be used in the treatment of haemostasis and inflammation-related disorders. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, secondary metabolites synthesized by Lecanicillium aphanocladii, a fungus isolated from caves, demonstrated modulating action on proteases and phospholipases A2 present in snake venoms of the Bothrops genus, widely used as tools for the study of pathophysiology processes related to haemostasis and inflammation. The results suggest the possibility of future applications for these metabolites in the development of pharmaceuticals of medical-scientific interest.

Genotoxic effects of combined multiple stressors on Gammarus locusta haemocytes: Interactions between temperature, pCO2 and the synthetic progestin levonorgestrel.

Climate change and pharmaceutical contamination are two priority research topics due to their impacts in the aquatic ecosystems and in the food chain structure. In the bottom of many food chains are the invertebrates, like the amphipods, which are important environmental and ecotoxicological models. In this study, we combined the increase of temperature [ambient and warming temperature], pCO2 [normocapnia and hypercapnia] and the synthetic progestin levonorgestrel (LNG) [environmentally relevant concentration (10 ng L-1) and 100-fold higher (1000 ng L-1)] to evaluate the genotoxic effects on the amphipod Gammarus locusta haemocytes, using the comet assay technique. Additionally, the study examined protective/potentiating effects of the three tested factors against hydrogen peroxide (H2O2)-induced DNA damage in haemocytes after ex vivo exposure. Our data revealed no significant effects of any of the three stressors on DNA damage of G. locusta haemocytes or protection against H2O2-induced DNA damage after twenty-one days exposure. Only a significant effect of the solvent was visible, since it was able to induce higher DNA damage (i.e. strand breaks) on exposed individuals. On the other hand, LNG exposure seemed to induce a slight increase of DNA damage after H2O2 exposure. Our findings suggest that more short-term studies to conclude about the genotoxicity and/or protective effects of the stress factors in G. locusta should be made, attending to the fast turnover rate of repairing cells that could have masked impacts seen only after the end of the experiment.

Histopathological Evaluation of Combined Impacts of the Synthetic Progestin Levonorgestrel and Temperature on the Female Zebrafish Maturation Using a Semi-quantitative Grading Analysis-Is it Enough?

Pharmaceuticals contamination (e.g., synthetic progestins), and global climate change, represent two of the most stressful factors affecting aquatic species. To our knowledge, there is huge gap of data regarding the combined effects of both stressors on vertebrates' reproduction. Thus, it is crucial to implement rapid screenings of measurable histopathological alterations in fish gonads. For that, we propose: (1) an evaluation of the combined effects of progestin (levonorgestrel) and temperature on maturation of zebrafish female gonads, using a semi-quantitative method (i.e., grading) and (2) testing the robustness of the grading analysis comparatively to a quantitative method (i.e., stereology). Grading analysis showed a decrease on maturation stage of ovaries exposed to both stressors. Although grading is less robust than stereological analysis, it is recommended for a preliminary approach, since it gives a correct idea on trends and it is fast and cost-effective. For a detailed histological assessment, we recommend a stereological study.

Interactive effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on the fitness and breeding of the amphipod Gammarus locusta.

Given the lack of knowledge regarding climate change-chemical exposure interactions, it is vital to evaluate how these two drivers jointly impact aquatic species. Thus, for the first time, we aimed at investigating the combined effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on survival, growth, consumption rate and reproduction of the amphipod Gammarus locusta. For that, a full factorial design manipulating temperature [ambient temperature and warming (+4 °C)], pCO2 [normocapnia and hypercapnia (Δ pH 0.5 units)] and the progestin levonorgestrel (LNG: L1 - 10 ngLL-1 and L2 - 1000 ngLL-1, control - no progestin and solvent control - vehicle ethanol (0.01%)) was implemented for 21 days. G. locusta was strongly negatively affected by warming, experiencing higher mortality rates (50-80%) than in any other treatments. Instead, growth rates were significantly affected by interactions of LNG with temperature and pCO2. It was observed, in the short-term (7d) that under ambient temperature (18 °C) and hypercapnic conditions (pH 7.6), the LNG presence promoted the amphipod's growth, while in the medium-term (21d) this response was not observed. Relative consumption rates (RCRs), during the first week were higher than in the third week. Furthermore, in the first week, RCRs were negatively affected by higher temperature while in the third week, RCRs were negatively affected by acidification. Furthermore, it was observed a negative effect of higher temperature and acidification on G. locusta fecundity, contrarily to LNG. Concluding, the impact of increased temperature and pCO2 was clearly more adverse for the species than exposure to the synthetic progestin, however, some interactions between the progestin and the climate factors were observed. Thus, in a future scenario of global change, the presence of LNG (and other progestins alike) may modulate to a certain level the effects of climate drivers (and vice-versa) on the gammarids fitness and reproduction.

Warming modulates the effects of the endocrine disruptor progestin levonorgestrel on the zebrafish fitness, ovary maturation kinetics and reproduction success.

Interactive effects between multiple stressors, namely climate drivers (e.g., temperature) and chemical pollution (e.g., endocrine disruptors) are poorly studied. Here, it was for the first time evaluated the combinatory effects of temperature and a synthetic progestin, levonorgestrel (LNG), on the fitness and reproductive-related endpoints of zebrafish (Danio rerio). A multi-factorial design was implemented by manipulating both temperature [setting as baseline an ambient temperature of 27 °C, against warming (+3 °C)] and LNG levels (10 ngL-1 and 1000 ngL-1). Groups of males and females were exposed sub-acutely, for 21-days. Increased temperature caused an overall decrease in the females' gonadosomatic index (GSI), during the pre-reproduction phase, LNG did not affect GSI. In addition, fecundity (number of ovulated eggs) was negatively affected by both temperature and LNG, being the effect of the latter more intense. Fish exposed to the highest LNG concentration (at both temperatures) did not reproduce, but also in those exposed to the lowest dose of progestin at a higher temperature, a complete reproductive failure occurred. These results reflect what was observed in the stereological analysis of the ovary maturation stages prior to reproduction. Accordingly, the higher the LNG concentration, the lower the degree of maturation of the ovary. This was exacerbated by the higher temperature. As to embryonated eggs, they hatched significantly faster at higher temperatures, but exposure to 10 ngL-1 of LNG (at 27 °C) reduced significantly the hatching rate, comparing to control. Further, the recrudescence of the ovary 48 h after spawning seems to be not affected by both stressors. Our data suggest that in a future scenario of global warming and synthetic hormones exposure, the reproduction of fish species, such as the zebrafish, can be endangered, which can put at risk their success, and consequently affect the structure and functioning of associated aquatic ecosystems.

Pollen limitation may be a common Allee effect in marine hydrophilous plants: implications for decline and recovery in seagrasses.

Pollen limitation may be an important factor in accelerated decline of sparse or fragmented populations. Little is known whether hydrophilous plants (pollen transport by water) suffer from an Allee effect due to pollen limitation or not. Hydrophilous pollination is a typical trait of marine angiosperms or seagrasses. Although seagrass flowers usually have high pollen production, floral densities are highly variable. We evaluated pollen limitation for intertidal populations of the seagrass Zostera noltei in The Netherlands and found a significant positive relation between flowering spathe density and fruit-set, which was suboptimal at <1200 flowering spathes m(-2) (corresponding to <600 reproductive shoots m(-2)). A fragmented population had ≈35 % lower fruit-set at similar reproductive density than a continuous population. 75 % of all European populations studied over a large latitudinal gradient had flowering spathe densities below that required for optimal fruit-set, particularly in Southern countries. Literature review of the reproductive output of hydrophilous pollinated plants revealed that seed- or fruit-set of marine hydrophilous plants is generally low, as compared to hydrophilous freshwater and wind-pollinated plants. We conclude that pollen limitation as found in Z. noltei may be a common Allee effect for seagrasses, potentially accelerating decline and impairing recovery even after environmental conditions have improved substantially.

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.

Temporal characterization of mercury accumulation at different trophic levels and implications for metal biomagnification along a coastal food web.

The main goal of this study was to assess temporal mercury variations along an estuarine food web to evaluate the mercury contamination level of the system and the risks that humans are exposed to, due to mercury biomagnification. The highest mercury concentrations in the sediments and primary producers (macrophytes) were observed during winter sampling. Instead, the highest mercury concentrations in the water, suspended particulate matter as well as in the zooplanktonic and suprabenthic communities were observed during summer sampling. Evidences of mercury biomagnification along the food web were corroborated by the positive biomagnification factors, particularly for omnivorous macrobenthic species. Comparing the mercury levels at distinct components with several environmental quality criteria it suggests that sediments, water and edible species (e.g., bivalve Scrobicularia plana and the crustacean Carcinus maenas) presented higher mercury levels than the values accepted by legislation which represent a matter of concern for the environment and human health.

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).

Identification and assessment of kefir yeast potential for sugar/ethanol-resistance.

Biochemical and molecular analysis was used for identification of different kefir yeasts species from Brazil, Canada and the United States of America. The sugar/ethanol-resistant activity of the yeasts was evaluated. Saccharomyces cerevisiae and Kluyveromyces marxianus had the highest growth rates, suggesting biotechnological applications possible for these strains.

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.

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.

The effects of mercury on the dynamics of the peracarida community in a temperate coastal lagoon (Ria de Aveiro, Portugal).

This study focuses on the impact of mercury on the dynamics and bioaccumulation patterns of suprabenthic peracarida community along a mercury gradient. Suprabenthic peracarida was negatively affected in terms of biomass, diversity and productivity. Mysids including Mesopodopsis slabberi dominated the community, presenting a regular distribution along the Hg gradient, so are considered tolerant species to mercury. Then, isopods, the second most abundant group, dominated in the most contaminated areas being considered also a tolerant group. Contrarily, amphipods were negatively correlated with higher Hg concentrations, dominating in the intermediate areas, thus are considered more sensitive species. In addition, isopods and amphipods were the taxa with higher mercury body burden, followed by mysids. This paper highlights the importance of suprabenthic peracarida as a vehicle of mercury transference through the estuarine trophic web since it is a main link between benthic and pelagic levels and an essential food resource for numerous fish species.