Joana R. Almeida

Challenges in assessing the toxic effects of polycyclic aromatic hydrocarbons to marine organisms: A case study on the acute toxicity of pyrene to the European seabass (Dicentrarchus labrax L.)

The acute toxicity (96 h) of pyrene (PY) to European seabass (Dicentrachus labrax) juveniles assessed in a semi-static bioassay (SSB) with medium renewal at each 12h, and in a static bioassay (SB) without medium renewal was compared in laboratorial conditions (water PY concentrations: 0.07-10 mg L(-1)). Main findings in the SSB that assessed mainly the toxicity of PY and its metabolites were: increased levels of bile PY metabolites in good agreement with the profile of lipid peroxidation levels (LPO) in exposed fish relating PY exposure and oxidative damage; increased levels of PY-type compounds in the brain indicating their ability to cross the blood-brain barrier; increased levels of these substances in liver and muscle which are edible tissues for humans thus raising concern on potential adverse effects on consumers of fish from PY contaminated areas; a significant inhibition of glutathione S-transferase activity suggesting its involvement in PY detoxication as toxicant scavenger; finally, an almost complete impairment of the swimming velocity at all the PY concentrations linking sub-individual to higher population level effects. In the SB, where the overall toxicity of PY, its metabolites and environmental degradation products was evaluated, 19% and 79% of PY decay in test media was found at 12 and 96 h, respectively. In general, the effects were similar to those of SSB but with significant effects being induced at higher PY concentrations indicating that the parental compound is more toxic than its environmental degradation products. The other main differences relatively to the SSB were: increased levels of PY-type substances in the liver suggesting more accumulation in this organ. Therefore, these findings highlight the need of carefully considering experimental design options when assessing the toxicity of readily degradable substances to marine fish, and stress the importance of taking into consideration the toxicity of environmental degradation products in addition to toxic effects of the parental substance and its metabolites for marine ecological risk assessment.

Acute effects of deltamethrin on swimming velocity and biomarkers of the common prawn Palaemon serratus

The main purpose of the present study was to investigate the effects of deltamethrin on biomarkers and behavior of Palaemon serratus (common prawn), since this attempt to link different levels of biological organization will allow determining which biomarkers might be ecologically relevant and will be useful to complement the information about the effects of pesticides by using behavioral parameters. Therefore, parameters of liver antioxidant status, energy metabolism and neurotransmission were determined in different tissues of the common prawn and used to assess the effects at sub-individual level, whereas swimming velocity was used to assess the effects at the individual level. It was also investigated if the swimming velocity can be used as an endpoint in ecotoxicology bioassays and if it can be as sensitive as biomarker endpoints. Swimming velocity was significantly reduced in prawns exposed to deltamethrin, showing a lowest observed effect (LOEC) of 0.6 ng L(-1). Eye acetylcholinesterase (AChE) activity was significantly increased in prawns exposed to 0.6, 1.2 and 2.4 ng L(-1) deltamethrin, whereas muscle cholinesterase (ChE) activity was significantly increased in prawns exposed to 19 and 39 ng L(-1). On the other hand, lactate dehydrogenase (LDH) activity was significantly increased in muscle of prawns exposed to 0.6, 1.2, 2.4, 4.9 ng L(-1) deltamethrin, showing that organisms were requiring additional energy, but probably using it for detoxification processes rather than locomotion, since swimming velocity was inhibited. Glutathione S-transferase (GST) activity was significantly increased in the digestive gland of common prawn exposed to 19 and 39 ng L(-1) deltamethrin. Catalase (CAT) activity was significantly increased in digestive gland of prawn exposed to 19 ng L(-1) deltamethrin. However, CAT activity decreased in digestive gland of prawn exposed to 39 ng L(-1), suggesting an antioxidant defense system failure concomitant with high levels of lipid peroxidation. Thus, global results showed that decreased swimming velocity was not associated with cholinesterase inhibition. In fact, the impairment of swimming velocity may be due to allocation of energy for detoxification and antioxidant protection instead of swimming activity. The present study showed that swimming velocity could be used as an ecologically relevant tool and a sensitive endpoint to assess and complement the study of pesticide effects on marine organisms.

Swimming velocity, avoidance behavior and biomarkers in Palaemon serratus exposed to fenitrothion

The aim of this study was to develop two behavioral tests (swimming velocity and avoidance behavior) specific for the common prawn, Palaemon serratus, and to investigate the effects of sublethal concentrations of fenitrothion on behavior and on several biomarkers. In a first bioassay, mortality was investigated in prawns exposed during 96 h to concentrations of fenitrothion ranging from 39 to 40000 ng L(-1). The effects on swimming velocity and biomarkers were determined in prawns exposed to the sub-lethal concentrations of fenitrothion (from 39 to 625 ng L(-1)). A specific newly bioassay was developed to assess the capability of prawns to avoid the toxicant (avoidance test). Finally, in order to determine the effects on biomarkers during the avoidance test, prawns were collected at different times of exposure (30, 60, 90 and 120 min). Results showed that prawns exposed to the sub-lethal concentrations of fenitrothion exhibited a significant inhibition of swimming velocity with a LOEC of 313 ng L(-1). A significant inhibition of both eye AChE (LOEC=78 ng L(-1)) and muscle ChE (LOEC=156 ng L(-1)) activities were also observed. Results from the avoidance test indicated that animals significantly avoided fenitrothion (78 ng L(-1)). However, this capability was not observed in prawns exposed to 156 ng L(-1) fenitrothion. Prawns exposed to fenitrothion showed alterations in enzymes involved in the production of energy (LDH and IDH) possibly in an attempt to cope with additional energetic demands. Impairment of locomotion and avoidance may lead to alterations at the population level. Thus, the present behavioral tests can be useful as ecologically relevant tools for ecological risk assessment.

Natural antifouling compounds: Effectiveness in preventing invertebrate settlement and adhesion.

Biofouling represents a major economic issue regarding maritime industries and also raise important environmental concern. International legislation is restricting the use of biocidal-based antifouling (AF) coatings, and increasing efforts have been applied in the search for environmentally friendly AF agents. A wide diversity of natural AF compounds has been described for their ability to inhibit the settlement of macrofouling species. However poor information on the specific AF targets was available before the application of different molecular approaches both on invertebrate settlement strategies and bioadhesive characterization and also on the mechanistic effects of natural AF compounds. This review focuses on the relevant information about the main invertebrate macrofouler species settlement and bioadhesive mechanisms, which might help in the understanding of the reported effects, attributed to effective and non-toxic natural AF compounds towards this macrofouling species. It also aims to contribute to the elucidation of promising biotechnological strategies in the development of natural effective environmentally friendly AF paints.

Behaviour and biomarkers as tools to assess the acute toxicity of benzo(a)pyrene in the common prawn Palaemon serratus

Benzo(a)pyrene (BaP) is considered an important marine environmental contaminant, given its recognised environmental persistence and toxicity. However, its effects in marine crustaceans are poorly studied, namely the accumulation and sub-cellular effects that might be linked to behavioural alterations and may lead to ecologically relevant consequences. This study aims to investigate behavioural and physiological responses of the common prawn (Palaemon serratus) after acute exposure to BaP and infer the potential effects for the population in the wild. The applied approach included the evaluation of swimming performance after exposure, and several biochemical biomarkers involved in biotransformation, oxidative damage, energy production and levels of BaP-type compounds in tissues (eye, digestive gland and muscle) in a 96 h acute bioassay with exposure to BaP (16-4096 μg/L). The objective was to establish a link between behaviour (swimming velocity) and biochemical responses in order to assess the ecological relevance of the effects induced by BaP in P. serratus and to select useful tools for environmental risk assessment. Results showed swimming velocity impairment (LOEC = 128 μg/L), lipid peroxidation (LPO) induction (LOEC = 4096 μg/L) and BaP-type compounds increase in eye (LOEC = 32 μg/L), digestive gland and muscle (LOEC = 512 μg/L) of prawn after exposure to BaP. This oxidative damage in lipids seems to be caused by the incapacity to activate detoxification and anti-oxidant enzymes, once glutathione-S-transferase (GST), catalase (CAT) and glutathione peroxidase (GPx) were not affected by the exposure. This could be also an explanation to the increased levels of BaP-type compounds observed in tissues. An inability to increase the activities of enzymes involved in the production of energy was also observed, which may help to explain the detoxification failure and consequent increased levels of lipid peroxidation. The inhibition of swimming velocity was negatively correlated with biochemical parameters, including the presence of BaP-type compounds in different tissues and LPO, and thus, these parameters might be used as ecologically relevant and early-warning tools to assess the effects of PAHs. This study also highlights the usefulness of the fixed wavelength fluorescence (FF) technique to quantify PAHs-type compounds in tissues as indicative of exposure of P. serratus to PAHs, namely the sensitivity of eyes, which might be used for monitoring purposes and in marine ecological risk assessment studies.

Using a multibiomarker approach and behavioural responses to assess the effects of anthracene in Palaemon serratus

Polycyclic aromatic hydrocarbons (PAHs) are recognised as one of the main groups of contaminants that assume more importance in the marine environment, enhancing the need of studies concerning their adverse effects and more efficient and ecologically relevant tools for environmental monitoring purposes. This study aims to apply an integrated approach including several multi-level biological responses (accumulation levels, biochemical responses important for different physiological functions and behavioural alterations) to assess the ecological relevance of the effects induced by sub-lethal concentrations of anthracene (ANT) in Palaemon serratus (common prawn). ANT accumulation was assessed by measuring the levels of ANT-type compounds in prawn digestive gland, muscle and eye; biochemical responses were determined using biomarkers involved in biotransformation, oxidative damage, energy production and neurotransmission processes; and behavioural alterations through swimming performance after 96 h exposure bioassay (ANT:16-1,024 μg/L). The rationale behind this approach is to assess the ecologically relevant effects induced by ANT in prawn, given by the association between behavioural alterations with biochemical responses, in search for more efficient tools for environmental risk assessment. Results show a significant decrease of swimming velocity (LOEC=128 μg/L) along with increased levels of ANT-type compounds in digestive gland (LOEC=128 μg/L), muscle (LOEC=256 μg/L) and eye (LOEC=32 μg/L) in prawn exposed to ANT. Increased activities of glutathione peroxidase (GPx) and catalase (CAT), involved in anti-oxidant defence system, were also observed (LOEC=256 μg/L; 1024μg/L, respectively) in the digestive gland of prawn, induction of oxidative damage in lipids (LPO) also occurred (LOEC=32 μg/L). The inhibition of swimming velocity showed a correlation with some biochemical parameters measured, including the levels of ANT-type compounds in tissues and LPO, and thus these may be considered sensitive and ecologically relevant criteria as well as early warning endpoints for assessing polycyclic aromatic compounds exposure effects on marine organisms.

Antifouling potential of Nature-inspired sulfated compounds

Natural products with a sulfated scaffold have emerged as antifouling agents with low or nontoxic effects to the environment. In this study 13 sulfated polyphenols were synthesized and tested for antifouling potential using the anti-settlement activity of mussel (Mytilus galloprovincialis) plantigrade post-larvae and bacterial growth inhibition towards four biofilm-forming bacterial strains. Results show that some of these Nature-inspired compounds were bioactive, particularly rutin persulfate (2), 3,6-bis(β-D-glucopyranosyl) xanthone persulfate (6), and gallic acid persulfate (12) against the settlement of plantigrades. The chemical precursors of sulfated compounds 2 and 12 were also tested for anti-settlement activity and it was possible to conclude that bioactivity is associated with sulfation. While compound 12 showed the most promising anti-settlement activity (EC50 = 8.95 μg.mL−1), compound 2 also caused the higher level of growth inhibition in bacteria Vibrio harveyi (EC20 = 12.5 μg.mL−1). All the three bioactive compounds 2, 6, and 12 were also found to be nontoxic to the non target species Artemia salina (<10% mortality at 250 μM) and Vibrio fischeri (LC50 > 1000 μg.mL−1). This study put forward the relevance of synthesizing non-natural sulfated small molecules to generate new nontoxic antifouling agents.

Acetylcholinesterase in Biofouling Species: Characterization and Mode of Action of Cyanobacteria-Derived Antifouling Agents

Effective and ecofriendly antifouling (AF) compounds have been arising from naturally produced chemicals. The objective of this study is to use cyanobacteria-derived agents to investigate the role of acetylcholinesterase (AChE) activity as an effect and/or mode of action of promising AF compounds, since AChE inhibitors were found to inhibit invertebrate larval settlement. To pursue this objective, in vitro quantification of AChE activity under the effect of several cyanobacterial strain extracts as potential AF agents was performed along with in vivo AF (anti-settlement) screening tests. Pre-characterization of different cholinesterases (ChEs) forms present in selected tissues of important biofouling species was performed to confirm the predominance of AChE, and an in vitro AF test using pure AChE activity was developed. Eighteen cyanobacteria strains were tested as source of potential AF and AChE inhibitor agents. Results showed effectiveness in selecting promising eco-friendly AF agents, allowing the understanding of the AF biochemical mode of action induced by different compounds. This study also highlights the potential of cyanobacteria as source of AF agents towards invertebrate macrofouling species.

High salt intake is associated with a higher risk of cardiovascular events: a 7.2-year evaluation of a cohort of hypertensive patients.

BackgroundIt is controversial whether high salt intake is directly associated with cardiovascular (CV) events and how far this relation is independent of blood pressure (BP). As Portugal has higher salt consumption and higher mortality by stroke than other European countries, we examined whether salt intake could predict the development of stroke and CV events in a hypertensive population. MethodsIn a longitudinal retrospective study of a cohort of 608 adult treated hypertensive patients 54.1±14.3 years of age, BMI 29.3±8.3 kg/m2, 56.3% women and 17.1% diabetics, we evaluate the long-term prognostic significance of urinary sodium (UNa) excretion measured in 24 h valid samples within the first 3 months after admission along with 24 h ambulatory blood pressure monitoring and pulse wave velocity [(PWV), complior)] measurements. ResultsThe mean follow-up duration was 7.2 years (0.5–11.1 years), during which 122 CV events occurred including 80 strokes and 36 coronary events. In 608 patients (group A=507 without events and group B=101 with events: 69 strokes, 26 coronary events, six others), the mean 24 h UNa was 208±79 mmol/day, corresponding to a salt intake of 12.1±4.6 g/day. Twenty-four hours UNa correlated positively with BMI, PWV and systolic blood pressure (SBP) particularly with night-time SBP. Group B versus A showed higher UNa (260+98 vs. 198+71 mmol/day, P<0.001) and higher PWV, BP office, 24 h, daytime and night-time SBP. Logistic regression analysis identified age, night-time SBP and 24 h UNa+ [HR=1.09 (95% CI, 1.06–1.12, P<0.001)] for each 10 mmol increase of UNa+ as the only independent predictors of CV events. UNa+ above the median (189 mmol sodium/day) predicted CV events with HR=2.99 (95% CI, 1.75–5.13, P<0.001) with worse CV event-free survival rates (log rank statistics of 17.44, P<0.001). ConclusionIn a cohort of hypertensive patients, high salt intake independently predicts the occurrence of CV events, particularly of stroke.

Os valores da pressão arterial aórtica e índice de aumentação central em indivíduos com hipertensão da bata branca são mais próximos dos indivíduos normotensos do que dos hipertensos tratados para idênticas idades, género e pressão noturna

INTRODUCTION There is disagreement whether white coat hypertensives (WCH) have different hemodynamic and structural characteristics compared to normotensives (NT) and hypertensives (HT). METHODS We compared cardiovascular prognostic markers (pulse wave velocity [PWV] and aortic stiffness index [ASI]) and data on central hemodynamics and central pressures (augmentation index [AIx], augmentation pressure [AugP] and pulse pressure amplification [PPA]) from aortic pulse wave analysis between NT (n=175), WCH (n=315) and treated HT (n=691), all with 24-h blood pressure (BP) <130/80 and nocturnal BP <120/70 mmHg after matching for age, gender, body mass index (BMI) and and nocturnal BP. The groups were also compared separately in terms of 24-h systolic BP <120 mmHg and 120-129 mmHg. RESULTS The percentage of non-dippers was 40.1% in NT, 34.5% in WCH and 38.3 in HT. For similar 24-h and nocturnal systolic BP (NT 109/64±7/5, WCH 110/66±7/6, HT 109/64±7/5 mmHg), aortic stiffness was greater in HT (n=691, PWV 10.8±2.6 m/s and ASI 0.33±0.16, p<0.01) than in WCH (n=316, PWV 9.7±2.4 m/s and ASI 0.28±0.17) and NT (n=175, PWV 9.5±2.0 m/s and ASI 0.29±0.15); AugP and AIx were higher (p<0.01) in HT (13.9±8.2 and 29.6±12.6 mmHg) than in WCH (11.5±8.5 mmHg and 24.9±15.2) and NT (11.0±6.4 mmHg and 26.6±11.5). PPA was lower (p<0.01) in HT (11.3±5.5 mmHg) than in WCH (13.2±7.1 mmHg) and in NT (12.4±4.9 mmHg). The findings were similar when the 24-h systolic BP <120 mmHg and 120-129 mmHg subgroups were analyzed separately. CONCLUSION Our data suggest that for similar age, gender distribution, BMI, and 24-h and nocturnal BP, aortic stiffness, central aortic pressures and wave reflection in WCH are closer to those of NT than to those with treated HT. This supports the idea that white coat hypertension may be a more benign condition than treated hypertension for similar 24-h and particularly nocturnal BP levels.