Browsing by Author "Ramos, Fernando"
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- Assessment of fipronil toxicity to the freshwater midge Chironomus riparius: Molecular, biochemical, and organismal responsesPublication . Monteiro, Hugo R.; Pestana, João L. T.; Novais, Sara C.; Leston, Sara; Ramos, Fernando; Soares, Amadeu M. V. M.; Devreese, Bart; Lemos, Marco F. L.Fipronil is a phenylpyrazole insecticide that entered the market to replace organochlorides and organophosphates. Fipronil impairs the regular inhibition of nerve impulses that ultimately result in paralysis and death of insects. Because of its use as a pest control, and due to runoff events, fipronil has been detected in freshwater systems near agricultural areas, and therefore might represent a threat to non-target aquatic organisms. In this study, the toxicity of fipronil to the freshwater midge Chironomus riparius was investigated at biochemical, molecular, and whole organism (e.g. growth, emergence, and behavior) levels. At the individual level, chronic (28 days) exposure to fipronil resulted in reduced larval growth and emergence with a lowest observed effect concentration (LOEC) of 0.081 μg L−1. Adult weight, which is directly linked to the flying performance and fecundity of midges, was also affected (LOEC = 0.040 μg L−1). Additionally, behavioral changes such as irregular burrowing behavior of C. riparius larvae (EC50 = 0.084 μg L−1) and impairment of adult flying performance were observed. At a biochemical level, acute (48 h) exposure to fipronil increased cellular oxygen consumption (as indicated by the increase of electron transport system (ETS) activity) and decreased antioxidant and detoxification defenses (as suggested by the decrease in catalase (CAT) and glutathione S-transferase (GST) activities). Exposure to fipronil also caused alterations in the fatty acid profile of C. riparius, since high levels of stearidonic acid (SDA) were observed. A comparison between exposed and nonexposed larvae also revealed alterations in the expression of globins, cytoskeleton and motor proteins, and proteins involved in protein biosynthesis. These alterations may aid in the interpretation of potential mechanisms of action that lead to the effects observed at the organism level. Present results show that environmentally relevant concentrations of fipronil are toxic to chironomid populations which call for monitoring of phenylpyrazole insecticides and of their ecological effects in freshwaters. Present results also emphasize the importance of complementing ecotoxicological data with molecular approaches such as proteomics, for a better interpretation of the mode of action of insecticides in aquatic invertebrates.
- Fate and effects of two pesticide formulations in the invertebrate Folsomia candida using a natural agricultural soilPublication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Leston, Sara; Rosa, João; Ramos, Fernando; Pouca, Ana Sofia Vila; Freitas, Andreia; Barbosa, Jorge; Roelofs, Dick; Sousa, José P.; Van Straalen, Nico M.; Lemos, Marco F. L.Degradation rates of two widely used pesticides were assessed, and acute and chronic effects on a standard invertebrate species investigated. An herbicide (Montana®) and fungicide (Bravo500®) formulations were investigated and results were compared to the isolated active substances of each formulation (glyphosate and chlorothalonil, respectively). Tests were performed using the invertebrate Folsomia candida as test species and an agricultural natural soil. Degradation rate tests were determined under aerobic conditions at 20 ± 2 °C, using an ecologically relevant concentration of 5 mg (a.i.) kg−1 of soil for both chemicals. Results demonstrated degradation half-lives (DT50) of 2.2 days for Montana® and 2.8 days when pure glyphosate was tested. Values of 1.1 and 2.9 days were registered for Bravo500® and its active substance chlorothalonil, respectively. There were no effects on survival for the tested concentrations of both forms of the herbicide (up to 17.3 mg kg−1). However, reproduction was affected, but only by the herbicide formulation, with an estimated EC50 value of 4.63 mg (a.i.) kg−1. Effects were most unlikely related to glyphosate. For chlorothalonil, both tested forms affected survival and reproduction. The estimated LC50 values were 117 mg (a.i.) kg−1 and 73.5 mg (a.i.) kg−1, and the EC50 41.3 mg (a.i.) kg−1 and 14.9 mg kg−1 for the formulation and the active ingredient, respectively. The effects of the active ingredient were significantly stronger, indicating the major influence of the active substance in the effects caused also by the formulation. Overall results demonstrate the importance of evaluating the effects of the formulated chemicals, as they are applied in the field, and not only their isolated active ingredients.
- From laboratory to the field: Validating molecular markers of effect in Folsomia candida exposed to a fungicide-based formulationPublication . Simões, Tiago; Novais, Sara C.; Natal-da-Luz, Tiago; Renaud, Mathieu; Leston, Sara; Ramos, Fernando; Römbke, Jörg; Dick, Roelofs; Van Straalen, Nico M.; Sousa, José P.; Lemos, Marco F. L.Under controlled laboratory conditions, toxicity data tend to be less variable than in more realistic in-field studies and responses may thus differ from those in the natural environment, creating uncertainty. The validation of data under environmental conditions is therefore a major asset in environmental risk assessment of chemicals. The present study aimed to validate the mode of action of a commercial fungicide formulation in the soil invertebrate F. candida, under more realistic exposure scenarios (in-field bioassay), by targeting specific molecular biomarkers retrieved from laboratory experiments. Organisms were exposed in soil cores under minimally controlled field conditions for 4 days to a chlorothalonil fungicide dosage causing 75% reduction of reproduction in a previous laboratory experiment (127 mg a.i. kg−1 ) and half this concentration (60 mg a.i. kg−1 ). After exposure, organisms were retrieved and RNA was extracted from each pool of organisms. According to previous laboratorial omics results with the same formulation, ten genes were selected for gene expression analysis by qRT-PCR, corresponding to key genes of affected biological pathways including glutathione metabolism, oxidation-reduction, body morphogenesis, and reproduction. Six of these genes presented a dose-response trend with higher up- or down-regulation with increasing pesticide concentrations. Highly significant correlations between their expression patterns in laboratory and in-field experiments were observed. This work shows that effects of toxicants can be clearly demonstrated in more realistic conditions using validated biomarkers. Our work outlines a set of genes that can be used to assess the early effects of pesticides in a realistic agricultural scenario.
- Integrated multitrophic aquaculture systems: potential risks for food safetyPublication . Rosa, João; Lemos, Marco F.L.; Crespo, Daniel; Nunes, Margarida; Freitas, Andreia; Ramos, Fernando; Pardal, Miguel Ângelo; Leston, SaraBackground: The demand for fish and fish products is now higher than ever. However, several problems such as nutrient loading or excessive use of resources can be associated with the intensification of aquaculture systems. Integrated multitrophic aquaculture systems (IMTAs) refer to the co-culture of different species belonging to different trophic levels, and offer a sustainable approach to aquaculture development. In these systems, organic and inorganic extractive species will feed on other species waste or on uneaten feed nutrients, acting as bioremediators. Scope and approach: The extractive capacity that these organisms have to take up nutrients from the water also means they will accumulate chemicals that are often administered in intensive productions. The present review describes a vast number of substances that can be found in IMTAs, either intentionally administered or resulting from contamination, and subsequently accumulated in species reared afterwards in these systems. The presence of such chemicals in organisms produced in IMTAs raises several food safety and human health concerns, which need to be addressed. Key findings and conclusions: Although IMTAs still face many challenges in terms of large scale production, legislations are not yet ready to comprise co-cultivation of multiple species in proximity. Also, maximum residue limits already existent for fish must be set for other organisms also produced in IMTAs in order to protect consumer's health. An increase in extractive species consumption (e.g. seaweeds) has been noticed during the past few years, and as IMTAs gain importance as a sustainable production method, food safety issues must be tackled.
- Novel active food packaging films based on whey protein incorporated with seaweed extract: development, characterization, and application in fresh poultry meatPublication . Andrade, Mariana A.; Barbosa, Cássia H.; Souza, Victor G. L.; Coelhoso, Isabel M.; Reboleira, João; Bernardino, Susana; Ganhão, Rui; Mendes, Susana; Fernando, Ana Luisa; Vilarinho, Fernanda; Silva, Ana Sanches; Ramos, FernandoAlgae and seaweeds are used in cookery since the beginnings of human civilization, particularly in several Asian cultures. Phenolic compounds are secondary metabolites produced by aquatic and terrestrial plants for their natural defense against external stimuli, which possess powerful antimicrobial and antioxidant properties that can be very important for the food industry. The main objective of this study was to develop a whey protein concentrate active coating, incorporated with a Fucus vesiculosus extract in order to delay the lipid oxidation of chicken breasts. Ten hydroethanolic extracts from F. vesiculosus were obtained and their antioxidant capacity was evaluated through two antioxidant activity assays: the DPPH radical scavenging activity and -carotene leaching assay. The total content in phenolics compounds was also determined by Folin-Ciocalteu method. The chosen extract was the one obtained from the freeze-dried F. vesiculosus using 75% (v/v) ethanol as extraction solvent. The extract was successfully incorporated into a whey protein film and successfully strengthened the thickness, tensile strength, and elastic modulus. The active film also was able to inhibit the chicken breasts lipid oxidation for 25 days of storage.
- Oxytetracycline accumulation in the macroalgae Ulva: Potential risks for IMTA systemsPublication . Rosa, João; Leston, Sara; Freitas, Andreia; Pouca, Ana Sofia Vila; Barbosa, Jorge; Lemos, Marco F. L.; Pardal, Miguel A.; Ramos, FernandoOxytetracycline (OTC) is one of the most used antibiotics in aquaculture. With the development of Integrated Multitrophic Aquaculture (IMTA) systems in order to mitigate some aquacultures' adverse effects, attention needs to be shifted to other co-cultured species that can also accumulate such pharmaceuticals and pose a risk to human consumption. Therefore, the present work evaluated the exposure of the seaweed Ulva to OTC at two realistic concentrations (0.040 and 0.120 mg L1 ). Oxytetracycline degradation rates in seawater were dependent on the initial concentration but were not influenced by the presence of Ulva. The macroalgae presented good assimilation rates of OTC, with internal concentrations reaching 40.9934 ng g1 WW for the lowest concentration tested and 108.6787 ng g1 WW for the highest, with a steep decrease after 48 and 24 h, respectively. Nonetheless, concentrations were still half of the Maximum Residue Limit set for fish (100 mg kg1) 48 h after C2 treatment. The highest dosage tested stimulated growth 96 h after the beginning of the trial, although some signs of decay could also be found in Ulva's fronds.
- Tissue depletion of five antibiotic residues in farmed European seabass (Dicentrarchus labrax)Publication . Rosa, João; Leston, Sara; Freitas, Andreia; Barbosa, Jorge; Rema, Paulo; Dias, Jorge; Lemos, Marco F. L.; Pardal, Miguel Ângelo; Ramos, FernandoConcerns about antibiotic use led to stricter legislations and overall better practices in aquaculture production. However, depletion time periods in tissues vary greatly with different antimicrobials, fish species, or experimental setup. In the present work, five drugs (sulfadiazine (SDZ), trimethoprim (TRI), flumequine (FLU), oxolinic acid (OXO), and oxytetracycline (OTC)) were incorporated into medicated feed, and their retention in European seabass muscle tissues assessed. Juveniles were placed in 300 L tanks at 18 °C and were manually fed with medicated feeds for a 7 day period (two concentrations per antimicrobial, ranging from 6 to 220 mg kg−1 , which were based on previous studies on the occurrence of these antimicrobials). Residues were analyzed through a multi-class quantification method (UHPLC-MS/MS). Data on residues concentration through time followed a one-compartment model, with TRI concentrations above the established Maximum Residue Level (MRL) throughout the experiment. Similarly, OTC concentrations at the highest dose were also above 100 μg kg−1 up to 14 days after the medication period. Results obtained for TRI may indicate the presence of a dose dependency for this antimicrobial. FLU concentrations in muscle samples were the lowest through time (Cday7 of 279.70 and 386.63 μg kg−1 , for prophylactic and therapeutic treatments, respectively). Half-life values of 14.37, 10.87, 5.36, 7.20, and 27.22 h (prophylactic treatment), and 20.95, 8.41, 5.61, 11.22, and 17.99 h (therapeutic treatment), were determined for SDZ, TRI, FLU, OXO, and OTC, respectively. Withdrawal times of 0, 2, and 5 days were determined for FLU, OXO, and SDZ, but sampling times for OTC and TRI should be longer, since antimicrobial concentrations were above the MRL 14 days after the end of the feeding period with medicated feed. Therefore, special attention should be given since they are the most used antibiotics in aquaculture and European seabass plays a major part in human nutrition in the Mediterranean region.
- Uptake of enrofloxacin from seawater to the macroalgae Ulva and its use in IMTA systemsPublication . Rosa, João; Leston, Sara; Crespo, Daniel; Freitas, Andreia; Pouca, Ana Sofia Vila; Barbosa, Jorge; Lemos, Marco F.L.; Pardal, Miguel Ângelo; Ramos, FernandoIntegrated multi-trophic aquaculture systems can minimize the environmental impacts of aquaculture, while delivering economical benefits. However, the use of extractive species such as seaweeds can accumulate pharmaceuticals commonly used in these systems. Therefore, this work evaluated the exposure of the seaweed Ulva to enrofloxacin (ENR), a vastly used antimicrobial in aquaculture, at two dosages (C1, 7.5 μg L−1 and C2, 15 μg L−1), and concentrations in water and in Ulva were measured through time. Traditional endpoints such as growth and mortality were assessed as ENR effects in the macroalgae. Enrofloxacin presented good stability in seawater, and degradation rates were influenced by the presence of seaweed at the lowest concentration tested. The seaweed was able to assimilate the antibiotic, reaching internal concentrations of 7.76 ± 1.11 ng g−1 WW after 30 min of exposure for C1, and 14.51 ± 1.22 ng g−1 WW, after 15 min for C2. Lowest concentrations detected at the end of experimental time were 4.08 ± 0.42 ng g−1 WW and 5.09 ± 1.57 ng g−1 WW for C1 and C2, respectively, which nonetheless, corresponds to ∼5% of the maximum residue limit established for fish for ENR by the European regulation. The presence of ENR stimulated Ulva growth, with differences observed 96 h after the beginning of the trial.