In Vitro Antiprotozoal Activity of Hibiscus sabdariffa Extract against a Ciliate Causing High Mortalities in Turbot Aquaculture.

Philasterides dicentrarchi is an histophagous parasite that infects flatfish, namely turbot (Scophthalmus maximus), and cause significant losses in aquaculture units. The available measures for P. dicentrarchi control have limited efficiency, and some cause harm to fish. Hence, sustainable and natural control strategies are urgently needed. This study evaluated the in vitro bioactivity of the ethanol extract of Hibiscus sabdariffa calyces on P. dicentrarchi population growth rate (PGR), oxidative stress biomarkers (glutathione-S-transferases (GST), glutathione reductase (GR), glutathione peroxidase (GPx), total glutathione (TG) and catalase (CAT), neurotoxicity (acetylcholinesterase, AChE), activity and gene expression of proteases as major virulence factors. H. sabdariffa extract inhibited parasite PGR (IC50 = 1.57 mg mL-1), and caused significant changes in the activity of antioxidant enzymes (LOEC = 0.22 mg mL-1), especially GPx, TG, and CAT. The activity of proteases was also severely inhibited (IC50 = 0.76 mg mL-1), and gene expression of catepsin 90 and leishmanolysin proteases was downregulated. Organic acids and phenolic phytochemicals in hibiscus extract are potentially responsible for the antiprotozoal bioactivity herein determined. Therefore, H. sabdariffa extract can be a promising disease-control alternative against the ciliate proliferation, cellular defense mechanisms and pathogenicity. Still, its applicability in aquaculture settings, and potential effects on farmed fish, should be further elucidated.

Inter-laboratory validation of an ISO test method for measuring enzyme activities in soil samples using colorimetric substrates.

The evaluation of soil quality requires the use of robust methods to assess biologically based indicators. Among them, enzyme activities are used for several decades, but there is a clear need to update their measurement methods for routine use, in combining feasibility, accuracy, and reliability. To this end, the platform Biochem-Env optimized a miniaturized method to measure enzyme activities in soils using colorimetric substrates in micro-well plates. The standardization of the method was carried out within the framework of ISO/TC 190/SC 4/WG 4 "Soil quality - Biological methods" workgroup, recommending an inter-laboratory evaluation for the publication of a full ISO standard. That evaluation, managed by the platform, was based on the measurement, in six soils of contrasted physicochemical properties, of the ten soil enzyme activities described in the standard. Eight laboratories were involved in the validation study. Only 2.7% of outliers were identified from the analyses of the whole dataset. The repeatability and reproducibility of the method were determined by computing, respectively, the intra-laboratory (CVr,) and inter-laboratory (CVR) coefficients of variation for each soil and enzyme. The mean CVr ranged from 4.5% (unbuffered phosphatase) to 9.9% (α-glucosidase), illustrating a reduced variability of enzyme activities within laboratories. The mean CVR ranged from 13.8% (alkaline phosphatase) to 30.9% (unbuffered phosphatase). Despite this large CVR noticed for unbuffered phosphatase, the method was repeatable, reproducible, and sensitive. It also proved to be applicable for measuring enzyme activities in different types of soils. These results have been found successful by ISO/TC 190/SC4 and resulted in the publication of ISO 20130:2018 standard.

Organic solvents alter photophysiological and oxidative stress profiles of the coral Zoanthus sp. - Towards an optimization of ecotoxicological protocols.

Coral reefs are declining, affected by climate change and escalating anthropogenic pressures, such as pollution or habitat alteration. Consequently, ecotoxicological assays with tropical corals have increased, specifically towards the study of emergent or persistent pollutants. However, standardized methodology to test for corals is non-existent, and their response to organic solvents, recurrently required in ecotoxicological appraisals, remains unknown. Therefore, we aimed to establish a threshold for the safe use of the selected solvents in ecotoxicological studies with these organisms. We assessed the oxidative stress response (antioxidant response and oxidative damage), cellular energy allocation and photophysiology of the photosynthetic coral Zoanthus sp. (Anthozoa, Hexacorallia) exposed to six doses of three different organic solvents (ethanol, methanol and dimethyl sulfoxide - DMSO). Our results suggest that the coral is more sensitive to methanol and DMSO than to ethanol. Methanol and DMSO LOEC were 0.01 mL L-1 affecting maximum quantum yield (Fv/Fm) and glutathione S-transferase (GST) activity, respectively, while for ethanol was 0.03 mL L-1, influencing Fv/Fm. Despite the higher tolerance of Zoanthus sp. to ethanol, 2.9 mL L-1 of this organic solvent was the only treatment causing mortality. Based on these findings, thresholds for the use of organic solvents with tropical corals can now be adopted. Nevertheless, species specificities should not be overlooked.

Potential of Eucalyptus globulus for the phytoremediation of metals in a Moroccan iron mine soil-a case study.

The contamination left by abandoned mines demands sustainable mitigation measures. Hence, the aim of this study was to examine the phytoremediator ability of Eucalyptus globulus Labill. to be used for cleaning up metal-contaminated soils from an African abandoned iron (Fe) mine (Ait Ammar, Oued Zem, Morocco). Plantlets of this species were exposed to a control (CTL), a reference (REF), and a mine-contaminated soil (CS). Morphological (growth, leaf area) and physiological stress biomarkers (photosynthetic efficiency, pigments content, leaf relative water, and malondialdehyde (MDA) levels) and metal bioaccumulation were assessed. The growth and leaf area of E. globulus increased overtime in all soils, although at a lower rate in the CS. Its photosynthetic efficiency was not markedly impaired, as well as MDA levels decreased throughout the experiment in CS. In this soil, higher metal contents were detected in E. globulus roots than in leaves, especially Fe (roots: 15.98-213.99 µg g-1; leaves: 5.97-15.98 µg g-1) and Zn (roots: 1.64-1.99 µg g-1; leaves: 0.67-1.19 µg g-1), indicating their reduced translocation. Additionally, though at low extent, the plants bioaccumulated some metals (Pb > Zn > Cu) from CS. Overall, E. globulus may be potentially used for the phytoremediation of metals in metal-contaminated soils.

Application of a standard risk assessment scheme to a North Africa contaminated site (Sfax, Tunisia) -Tier 1.

Phosphorus is a critical element to agriculture, consequently global phosphate rock demand will remain rising to feed a growing world population. The beneficiation of phosphorous ore gives rise to several tons of a waste by-product [phosphogypsum (PG)] which valorisation is limited, within other reasons, by the risks posed to environment and human health. Although threatening, the accumulation in stacks is the only procedure so far practiced by several countries as a means to get rid of this industrial externality. As part of a NATO Science for Peace Project (SfP 983311) this study describes the application of an environmental risk assessment (ERA) framework, to assess the risks posed by a PG stack to the surrounding soils, in Sfax, Republic of Tunisia. The ERA followed a weight of evidence approach, supported by two lines of evidence (LoE): the chemical (ChemLoE) and the ecotoxicological (EcotoxLoE). Integrated risks point for risk values greater than 0.5 in soils collected in PG stack surrounding area. Soil salinization, has likely contributed to the exacerbation of risks, as well as to the lack of consistency between both LoEs. This study highlights the need of rethinking the weight given to each LoE in ERA, in areas where soil salinization is a reality.

Bacterially assembled biopolyester nanobeads for removing cadmium from water.

Cadmium (Cd)-contaminated waterbodies are a worldwide concern for the environment, impacting human health. To address the need for efficient, sustainable and cost-effective remediation measures, we developed innovative Cd bioremediation agents by engineering Escherichia coli to assemble poly(3-hydroxybutyric acid) (PHB) beads densely coated with Cd-binding peptides. This was accomplished by translational fusion of Cd-binding peptides to the N- or C-terminus of a PHB synthase that catalyzes PHB synthesis and mediates assembly of Cd2 or Cd1 coated PHB beads, respectively. Cd1 beads showed greater Cd adsorption with 441 nmol Cd mg-1 bead mass when compared to Cd2 beads (334 nmol Cd mg-1 bead-mass) and plain beads (238 nmol Cd mg-1 bead-mass). The Cd beads were not ecotoxic and did attenuate Cd-spiked solutions toxicity. Overall, the bioengineered beads provide a means to remediate Cd-contaminated sites, can be cost-effectively produced at large scale, and offer a biodegradable and safe alternative to synthetic ecotoxic treatments.

Extremophilic Microfactories: Applications in Metal and Radionuclide Bioremediation.

Metals and radionuclides (M&Rs) are a worldwide concern claiming for resilient, efficient, and sustainable clean-up measures aligned with environmental protection goals and global change constraints. The unique defense mechanisms of extremophilic bacteria and archaea have been proving usefulness towards M&Rs bioremediation. Hence, extremophiles can be viewed as microfactories capable of providing specific and controlled services (i.e., genetic/metabolic mechanisms) and/or products (e.g., biomolecules) for that purpose. However, the natural physiological plasticity of such extremophilic microfactories can be further explored to nourish different hallmarks of M&R bioremediation, which are scantly approached in the literature and were never integrated. Therefore, this review not only briefly describes major valuable extremophilic pathways for M&R bioremediation, as it highlights the advances, challenges and gaps from the interplay of 'omics' and biological engineering to improve extremophilic microfactories performance for M&R clean-up. Microfactories' potentialities are also envisaged to close the M&R bioremediation processes and shift the classical idea of never 'getting rid' of M&Rs into making them 'the belle of the ball' through bio-recycling and bio-recovering techniques.

A bacterium-based contact assay for evaluating the quality of solid samples-Results from an international ring-test.

The contact assay measuring the inhibition of Arthrobacter globiformis dehydrogenase activity as an endpoint to evaluate the toxicity of solid samples was tested in an international ring-test to validate its performance for ISO standardization (ISO/CD 18187). This work reports the results of the ring-test involving 9 laboratories from six countries. At least 8 valid data sets were obtained for each sample and more than three quarters of the participants attained the validity criteria defined in the standard. The coefficient of variation within (CVr) and between (CVR) laboratories was generally on average <15% and <30% for negative and positive controls, respectively. Regarding solid samples, the laboratories provided a similar ranking of the samples based on their toxicity, despite some variation in the LOEC values. The logarithmic within-lab standard deviation <0.50 for soils and <0.25 for wastes evidenced a good repeatability. The between-lab variability assessed by a CVR <30%, minimum-maximum factor <4 and a reproducibility standard deviation (SDR) <0.13 for a great part of the solid samples, confirmed the test reproducibility. Overall, this assay proved to be robust, sensitive and feasible for routine use towards the quality assessment of soils and wastes.

Combined effect of copper sulfate and water temperature on key freshwater trophic levels - Approaching potential climatic change scenarios.

This work relied on the use microcosms to evaluate the individual and the combined effects of different levels of copper sulfate (0.0, 0.013, 0.064 and 0.318mg Cu L-1) - a fungicide commonly exceeding allowable thresholds in agricultural areas - and a range of water temperature increase scenarios (15, 20 and 25°C) on freshwater species belonging to different functional groups. Hence, the growth inhibition of primary producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor), as well as the survival and feeding behavior of a shredder species (the Trichoptera Schizopelex sp.) were evaluated. The results revealed that copper was toxic to primary producers growth, as well as shredders growth and survival, being the growth of L. minor particularly affected. Higher water temperatures had generally enhanced the growth of primary producers under non-contaminated (microalgae and macrophytes) or low-contaminated (macrophytes) conditions. Despite the tendency for a more pronounced toxicity of copper under increasing water temperatures, a significant interaction between the two factors was only observed for microalgae. Since the test organisms represent relevant functional groups for sustaining freshwater systems functions, the present results may raise some concerns on the impacts caused by possible future climate change scenarios in aquatic habitats chronically exposed to the frequent or intensive use of the fungicide copper sulfate.

First study on oyster-shell-based phosphorous removal in saltwater - A proxy to effluent bioremediation of marine aquaculture.

The efficiency of oyster-shell waste for the removal of phosphorous (P) in saltwater was herein evaluated. For that, different factors were tested, being attained >56% and >98% P removal by natural oyster shell (NOS)-fraction <0.125mm and COS (calcined oyster shell)-fraction 0.5mm, respectively. Adsorption was the main mechanism suggested for NOS, whilst a co-mechanism of precipitation occurred with COS. NOS adsorption was consistent with Langmuir model and followed both the Elovich and Intraparticle Difusion kinetic models. COS followed only Pseudo-Second Order and, mainly, the Elovich model. Overall, optimal conditions for P removal from saltwater were established for NOS and COS, which will unquestionably allow to comply with regulated P levels for the discharge of wastewater from saltwater RAS.

Bio-rescue of marine environments: On the track of microbially-based metal/metalloid remediation.

The recent awareness of the huge relevance of marine resources and ecological services is driving regulatory demands for their protection from overwhelming contaminants, such as metals/metalloids. These contaminants enter and accumulate in different marine niches, hence deeply compromising their quality and integrity. Bioremediation has been flourishing to counteract metal/metalloid impacts, since it provides cost-effective and sustainable options by relying on ecology-based technologies. The potential of marine microbes for metal/metalloid bioremediation is the core of many studies, due to their high plasticity to overcome successive environmental hurdles. However, any thorough review on the advances of metal/metalloid bioremediation in marine environments was so far unveiled. This review is designed to (i) outline the characteristics and potential of marine microbes for metal/metalloid bioremediation, (ii) describe the underlying pathways of resistance and detoxification, as well as useful methodologies for their characterization, (iii) identify major bottlenecks on metal/metalloid bioremediation with marine microbes, (iv) present alternative strategies based on microbial consortia and engineered microbes for enhanced bioremediation, and (v) propose key research avenues to keep pace with a changing society, science and economy in a sustainable manner.

Unraveling the interactive effects of climate change and oil contamination on laboratory-simulated estuarine benthic communities.

There is growing concern that modifications to the global environment such as ocean acidification and increased ultraviolet radiation may interact with anthropogenic pollutants to adversely affect the future marine environment. Despite this, little is known about the nature of the potential risks posed by such interactions. Here, we performed a multifactorial microcosm experiment to assess the impact of ocean acidification, ultraviolet B (UV-B) radiation and oil hydrocarbon contamination on sediment chemistry, the microbial community (composition and function) and biochemical marker response of selected indicator species. We found that increased ocean acidification and oil contamination in the absence of UV-B will significantly alter bacterial composition by, among other things, greatly reducing the relative abundance of Desulfobacterales, known to be important oil hydrocarbon degraders. Along with changes in bacterial composition, we identified concomitant shifts in the composition of oil hydrocarbons in the sediment and an increase in oxidative stress effects on our indicator species. Interestingly, our study identifies UV-B as a critical component in the interaction between these factors, as its presence alleviates harmful effects caused by the combination of reduced pH and oil pollution. The model system used here shows that the interactive effect of reduced pH and oil contamination can adversely affect the structure and functioning of sediment benthic communities, with the potential to exacerbate the toxicity of oil hydrocarbons in marine ecosystems.

Contribution for the derivation of a soil screening value (SSV) for uranium, using a natural reference soil.

In order to regulate the management of contaminated land, many countries have been deriving soil screening values (SSV). However, the ecotoxicological data available for uranium is still insufficient and incapable to generate SSVs for European soils. In this sense, and so as to make up for this shortcoming, a battery of ecotoxicological assays focusing on soil functions and organisms, and a wide range of endpoints was carried out, using a natural soil artificially spiked with uranium. In terrestrial ecotoxicology, it is widely recognized that soils have different properties that can influence the bioavailability and the toxicity of chemicals. In this context, SSVs derived for artificial soils or for other types of natural soils, may lead to unfeasible environmental risk assessment. Hence, the use of natural regional representative soils is of great importance in the derivation of SSVs. A Portuguese natural reference soil PTRS1, from a granitic region, was thereby applied as test substrate. This study allowed the determination of NOEC, LOEC, EC20 and EC50 values for uranium. Dehydrogenase and urease enzymes displayed the lowest values (34.9 and <134.5 mg U Kg, respectively). Eisenia andrei and Enchytraeus crypticus revealed to be more sensitive to uranium than Folsomia candida. EC50 values of 631.00, 518.65 and 851.64 mg U Kg were recorded for the three species, respectively. Concerning plants, only Lactuca sativa was affected by U at concentrations up to 1000 mg U kg(1). The outcomes of the study may in part be constrained by physical and chemical characteristics of soils, hence contributing to the discrepancy between the toxicity data generated in this study and that available in the literature. Following the assessment factor method, a predicted no effect concentration (PNEC) value of 15.5 mg kg-1dw was obtained for U. This PNEC value is proposed as a SSV for soils similar to the PTRS1.

Toxicity screening of soils from different mine areas--a contribution to track the sensitivity and variability of Arthrobacter globiformis assay.

This study used the Arthrobacter globiformis solid-contact test for assessing the quality of soils collected in areas subjected to past and present mine activities in Europe (uranium mine, Portugal) and North Africa (phosphogypsum pile, Tunisia; iron mine, Morocco). As to discriminate the influence of soils natural variability from the effect of contaminants, toxicity thresholds were derived for this test, based on the dataset of each study area. Furthermore, the test sensitivity and variability was also evaluated. As a result, soils that inhibited A. globiformis dehydrogenase activity above 45% or 50% relatively to the control, were considered to be toxic. Despite the soil metal content determined, the properties of soils seemed to influence dehydrogenase activity. Overall, the contact test provided a coherent outcome comparing to other more time-consuming and effort-demanding ecotoxicological assays. Our results strengthened the feasibility and ecological relevance of this assay, which variability was quite reduced hence suggesting its potential integration within the test battery of tier 1 of soil risk assessment schemes.

Toxicity evaluation of natural samples from the vicinity of rice fields using two trophic levels.

An ecotoxicological screening of environmental samples collected in the vicinity of rice fields followed a combination of physical and chemical measurements and chronic bioassays with two freshwater trophic levels (microalgae: Pseudokirchneriella subcapitata and Chlorella vulgaris; daphnids: Daphnia longispina and Daphnia magna). As so, water and sediment/soil elutriate samples were obtained from three sites: (1) in a canal reach crossing a protected wetland upstream, (2) in a canal reach surrounded by rice fields and (3) in a rice paddy. The sampling was performed before and during the rice culture. During the rice cropping, the whole system quality decreased comparatively to the situation before that period (e.g. nutrient overload, the presence of pesticides in elutriates from sites L2 and L3). This was reinforced by a significant inhibition of both microalgae growth, especially under elutriates. Contrary, the life-history traits of daphnids were significantly stimulated with increasing concentrations of water and elutriates, for both sampling periods.

Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulation versus active ingredient.

The Ecological Risk Assessment of pesticides requires data regarding their toxicity to aquatic and terrestrial non-target species. Such requirements concern active ingredient(s), generally not considering the noxious potential of commercial formulations. This work intends to contribute with novel information on the effects of short-term exposures to two herbicides, with different modes of action (Spasor, Stam Novel Flo 480), and an insecticide (Lannate), as well as to corresponding active ingredients (Glyphosate, Propanil and Methomyl, respectively). The microalga Pseudokirchneriella subcapitata (growth inhibition), the cladoceran Daphnia magna (immobilisation), and the earthworm Eisenia andrei (avoidance behaviour) were used as test species. Both herbicides were innocuous to all test organisms at environmentally realistic concentrations, except for Stam and Propanil (highly toxic for Pseudokirchneriella; moderately toxic to Daphnia). Lannate and Methomyl were highly toxic to Daphnia and caused Eisenia to significantly avoid the spiked soil at realistic application rates. The toxicity of formulations either overestimated (e.g. Stam/Propanil for P. subcapitata) or underestimated (e.g. Stam/Propanil for D. magna) that of the active ingredient.

Life-history traits of standard and autochthonous cladocerans: I. Acute and chronic effects of acetylsalicylic acid.

Pharmaceuticals have been recognized as an important group of aquatic micropollutants, mainly because of their biologically active nature. Acetylsalicylic acid (ASA), which is the active compound of Aspirin and many other pharmaceuticals, is consumed in large quantities every year. Therefore, its acute and chronic effects on standard (Daphnia magna) and autochthonous (Daphnia longispina) daphnids were investigated. The results showed that ASA impaired the survivorship, reproduction, and growth of the cladoceran species. The standard daphnid was the more tolerant species in acute assays (48-h EC(50) = 1293.05 mg/L; D. longispina: 48-h EC(50) = 647.31 mg/L); whereas the autochthonous daphnid seemed to be more resistant under chronic exposure to ASA, mainly its population-level traits. Despite this, the observed effect concentrations were much higher than the environmental concentrations of ASA. Notwithstanding this, the impairment of individual-level traits is likely to occur at environmental levels as an ultimate response to long-term exposure.

Life-history traits of standard and autochthonous cladocerans: II. Acute and chronic effects of acetylsalicylic acid metabolites.

Metabolic products are often more toxic than their pharmacological parent compounds. Therefore, the acute and chronic effects of the main metabolites--salicylic acid (SAL), gentisic acid (GEN), and o-hydroxyhippuric acid (HDP)--of acetylsalicylic acid (ASA), the active ingredient in Aspirin and many other pharmaceuticals, were assessed using standard (Daphnia magna) and autochthonous (Daphnia longispina) cladocerans. The sequence of decreasing levels of acute and chronic toxicity of ASA metabolites to daphnids was GEN > SAL > HDP. HDP did not present acute toxicity, but chronic exposures enabled the production of abnormal neonates and, in particular, egg abortion. Thus, reproduction was the end point most susceptible to HDP. On the other hand, SAL and GEN induced changes in the normal patterns of reproduction and growth of both species. In general, D. longispina was more sensitive than was D. magna, although the population growth of the autochthonous species was superior under SAL exposures than that of the standard test species. Although the concentrations that were determined to have a toxic effect were above the levels detected in aquatic environmental samples, exposure to low levels of pharmacologically active substances for a duration longer than the test period may induce changes in nontarget organisms.