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Pharmaceuticals can be considered a global threat to aquatic ecosystems due to their pseudo-persistence and their potential toxicity towards non-target species. Amoxicillin (AMX) and carbamazepine (CBZ) and their mixture (1:1) were investigated on the marine copepod Tigriopus fulvus (Fischer, 1860) considering both acute and chronic endpoints. While acute and chronic exposure did not directly affect survival, reproductive endpoints were affected like the mean egg hatching time that was significantly longer than the negative control for treatments with AMX (0.789 ± 0.079 μg/L), CBZ (8.88 ± 0.89 μg/L), and AMX and CMZ as a mixture (1.03 ± 0.10 μg/L and 0.941 ± 0.094 μg/L), in that order.

In recent years, the proliferation of Harmful Cyanobacterial Blooms (CyanoHABs) has increased with water eutrophication and climate change, impairing human health and the environment in relation to water supply. In drinking water treatment plants (DWTPs), the bio-coagulation based on natural coagulants has been studied as an eco-friendly alternative technology to conventional coagulants for both turbidity and CyanoHABs removal. Plant-based coagulants have demonstrated their coagulation efficiency in turbidity removal, as reported in several papers but its ability in cyanobacterial removal is still limited. This paper mainly reviewed the application of plant-based coagulants in DWTPs, with focus on turbidity removal, including cyanobacterial cells. The future potential uses of these green coagulants to reduce noxious effects of cyanobacterial proliferation are presented. Green coagulants advantages and limitations in DWTPs are reviewed and discussed summarizing more than 10 years of knowledge.

Nano-zero-valent iron (nZVI) and activated carbon (AC) addition are ongoing techniques for the remediation of hydrophobic organic compound-contaminated sediment and water, but with still unexplored eco(toxico)logical implications, especially when applied in situ. In this study, we investigated AC and nZVI as remediation methods for marine contaminated sediment and water, including chemical and toxicity (Artemia franciscana survival and genotoxicity) surveys. The removal efficiency of AC and nZVI (about 99%) was similar in both sediment and seawater, while the survival of nauplii and adults was mainly impacted by nZVI than AC. At the molecular level, the nZVI-addition induced down-regulation in the expression of two stress and one developmental genes, whereas AC was able to up-regulated only one gene involved in stress response. Results suggested that the use of AC is safer than nZVI that requires further investigation and potential optimization to reduce secondary undesired effects. Graphical abstract

Artemia spp. represent models species widely used in ecotoxicological studies due to its simple and fast manipulation in laboratory conditions that makes this crustacean well adaptable to several methodological approaches. Although cysts hatching, swimming behavior, reproductive success and mortality are the main endpoints used for the determination of toxicity, the detection of slight alterations induced by certain substances found at low concentrations in the environment may require more sensitive biomarkers. For this reason, the identification of DNA or chromosomal damages has been proposed as an additional and appreciable endpoint for the ecotoxicological assessment of environmental chemicals. Concerning Artemia models, only few studies indicated that the exposure to organic and inorganic compounds (i.e. pesticides, nanoparticles, bacterial products or heavy metals) can reduce the survival and fitness through the onset of DNA breaks or the dysregulation of key genes. In contrast, literature research revealed a lot of works primarily focusing on the mortality and hatching rates of Artemia nauplii and cysts despite the well-known low sensitivity of these species. The present review reports the current state of knowledge concerning the effects induced by various chemicals, including organic and inorganic compounds, on the common parameters and genotoxicity in both Artemia franciscana and Artemia salina. Advantages and limitations of Artemia spp. models in eco-toxicological investigations together with the most used classes of compounds are briefly discussed. Moreover, a mention is also addressed to scarce availability of literature data focusing on genotoxic effects and the great reliability of molecular approaches observed in this poorly sensitive model organism. Thus, the opportunity to take advantage of genotoxic analyses has also been highlighted, by suggesting this approach as a novel endpoint to be used for the eco-toxicological assessment of several stressors.

Nano Zero-Valent Iron (nZVI) and Activated Carbon (AC) addition is recently developed techniques applied for the remediation of sediments and aqueous solutions contaminated with hydrophobic organic compounds. However, an important point that must be underlined consists in the possible occurrence of eco(toxico)logical secondary effects, especially when applied in situ. In this study, we investigated AC and nZVI as remediation methods in contaminated water and sediment, evaluating also their possible negative impact on marine crustacean Artemia franciscana. The results showed that the AC and nZVI efficiency removal were similar for both sediment and seawater; and the survival of both nauplii and adults was mainly impacted by nZVI than by AC. At the molecular level, the nZVI-addition induced variations in the expression of stress and developmental genes. In conclusion, these data suggest potential ecotoxicological effects of nZVI and stress the need for a detailed further investigation of secondary effects of this technique.

Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated onMono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression ofThe limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm.These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

Microplastic debris from direct and indirect human activities is considered a major threat to the marine biodiversity mainly due to its abundance, durability, persistence, and ability to accumulate contaminants from the environment. Derelict tubular plastic nets of various colours (blue (BN), yellow (YN), green (GN), pink (PN), and white (WN) net), used to distinguish mussel farming owners, were collected by scuba-dive from the Mar Piccolo of Taranto (Ionian Sea). All nets were made of polypropylene. Investigations looked for potential acute (mortality) and sub-chronic (mortality, larval development and moult release number, and adult percentage after 5–9 days) effects on Tigriopus fulvus nauplii considering both whole plastics (microplastic (MP), 50 mg/L) and leachates (12.5–100%). Acute test determined a median lethal concentration (LC50) only for BN for both MPs (107 mg/L) and leachates (50.1%). The prolonged exposure (5 days) to microplastics did not affect the T. fulvus survival. After 9 days, YN and BN decreased of approximately 100% larval development. Graphical abstract

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non-albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 μM to >50 μM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis. The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1. Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non-albicans, providing fascinating scenarios for the identification of new potential antifungal targets.

Background: Biofilms have been found growing on implantable medical devices. This can lead to persistent clinical infections. The highly antibiotic-resistant property of biofilms necessitates the search for both potent antimicrobial agents and novel antibiofilm strategies. Natural product-based anti-biofilm agents were found to be as efficient as chemically synthesized counterparts with fewer side effects. In the present study, the effects of limonene as an antibiofilm agent were evaluated on Pseudomonas aeruginosa and Staphylococcus aureus biofilm formed on different surfaces using the CDC model system in continuous flow. The flgK gene and the pilA gene expression in P. aeruginosa, and the icaA gene and eno gene in S. aureus, which could be considered as efficient resistance markers, were studied. Methods: Mono- and dual-species biofilms were grown on polycarbonate, polypropylene, and stainless-steel coupons in a CDC biofilm reactor (Biosurface Technologies, Bozeman, MT, USA). To evaluate the ability of limonene to inhibit and eradicate biofilm, a sub-MIC concentration (10 mL/L) was tested. The gene expression of P. aeruginosa and S. aureus was detected by SYBR Green quantitative Real-Time PCR assay (Meridiana Bioline, Brisbane, Australia). Results: The limonene added during the formation of biofilms at sub-MIC concentrations works very well in inhibiting biofilms on all three materials, reducing their growth by about 2 logs. Of the same order of magnitude is the ability of limonene to eradicate both mono- and polymicrobial mature biofilms on all three materials. Greater efficacy was observed in the polymicrobial biofilm on steel coupons. The expression of some genes related to the virulence of the two microorganisms was differently detected in mono- and polymicrobial biofilm. Conclusions: These data showed that the limonene treatment expressed different levels of biofilm-forming genes, especially when both types of strains alone and together grew on different surfaces. Our findings showed that limonene treatment is also very efficient when biofilm has been grown under shear stress causing significant and irreversible damage to the biofilm structure. The effectiveness of the sanitation procedures can be optimized by applying antimicrobial combinations with natural compounds (e.g., limonene).

Polycyclic aromatic hydrocarbons (PAHs) consist of a group of over 100 different organic compounds mainly generated by anthropogenic activities. Because of their low water solubility, they tend to be accumulated in sediment, where their degradation rate is very low. Few studies have been carried out so far to investigate the effects of PAHs on Artemia franciscana. Artemia is easy to manage at laboratory scale, but it is not a sensitive biological model considering the traditional endpoints (i.e., mortality). In addition to evaluating the lethality on nauplii and adults of A. franciscana after 24 and 48 h, we focused on the genotoxicity to investigate the potential effects of phenanthrene (PHE), naphthalene (NAP), fluoranthene (FLT), and benzo(k)fluoranthene (BkF). Results showed that FLT was the most toxic both for nauplii and adults after 48 h of exposure. Real-time qPCR showed that all toxicants, including BkF, which had no negative effects on the survival of the crustacean, were able to switch the gene expression of all nine genes. This work has important ecological implications, especially on contaminated sediment assessment considering that PAHs represent the most abundant organic group of compounds in marine environment, opening new perspectives in understanding the molecular pathways activated by crustaceans.

Sediment pollution is a major issue in coastal areas, potentially endangering human health and the marine environments. We investigated the short-term sublethal effects of sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) on the sea urchin Paracentrotus lividus for two months. Spiking occurred at concentrations below threshold limit values permitted by the law (TLVPAHs = 900 µg/L, TLVPCBs = 8 µg/L, Legislative Italian Decree 173/2016). A multi-endpoint approach was adopted, considering both adults (mortality, bioaccumulation and gonadal index) and embryos (embryotoxicity, genotoxicity and de novo transcriptome assembly). The slight concentrations of PAHs and PCBs added to the mesocosms were observed to readily compartmentalize in adults, resulting below the detection limits just one week after their addition. Reconstructed sediment and seawater, as negative controls, did not affect sea urchins. PAH- and PCB-spiked mesocosms were observed to impair P. lividus at various endpoints, including bioaccumulation and embryo development (mainly PAHs) and genotoxicity (PAHs and PCBs). In particular, genotoxicity tests revealed that PAHs and PCBs affected the development of P. lividus embryos deriving from exposed adults. Negative effects were also detected by generating a de novo transcriptome assembly and its annotation, as well as by real-time qPCR performed to identify genes differentially expressed in adults exposed to the two contaminants. The effects on sea urchins (both adults and embryos) at background concentrations of PAHs and PCBs below TLV suggest a need for further investigations on the impact of slight concentrations of such contaminants on marine biota.

Adult sea urchins and their embryos are ideal targets to investigate the medium- and long-term effects of various toxic agents, such as organic and inorganic pollutants, to forecast and mitigate their environmental effects. Similarly, small colonial tunicates such as Botryllid ascidians may reveal acute toxicity processes and permit quick responses for the management of contaminants impacting coastal waters, to preserve the functional integrity of marine ecosystems. This investigation compares the functional responses of two model invertebrates, the sea urchin Paracentrotus lividus and the sea squirt Botryllus schlosseri, to chronic and acute exposures to organic and inorganic toxic agents. Such heavy metals as copper produce both acute and chronic effects on marine biota, while polycyclic aromatic hydrocarbons (PAHs) mainly produce chronic effects at the concentrations ordinarily measured in marine coastal waters. Both models were tested over a range of concentrations of copper and PAHs. Copper triggered a clear effect in both species, producing a delay in the embryo development of P. lividus and a rapid death of sea squirts. B. schlosseri was less sensitive to PAHs than P. lividus. The results on both species may synergistically contribute to assess the toxicity of organic and inorganic compounds at various concentrations and different physiologic levels.

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) represent the most common pollutants in the marine sediments. Previous investigations demonstrated short-term sublethal effects of sediments polluted with both contaminants on the sea urchin Paracentrotus lividus after 2 months of exposure in mesocosms. In particular, morphological malformations observed in P. lividus embryos deriving from adults exposed to PAHs and PCBs were explained at molecular levels by de novo transcriptome assembly and real-time qPCR, leading to the identification of several differentially expressed genes involved in key physiological processes. Here, we extensively explored the genes involved in the response of the sea urchin P. lividus to PAHs and PCBs. Firstly, 25 new genes were identified and interactomic analysis revealed that they were functionally connected among them and to several genes previously defined as molecular targets of response to the two pollutants under analysis. The expression levels of these 25 genes were followed by Real Time qPCR, showing that almost all genes analyzed were affected by PAHs and PCBs. These findings represent an important further step in defining the impacts of slight concentrations of such contaminants on sea urchins and, more in general, on marine biota, increasing our knowledge of molecular targets involved in responses to environmental stressors.

The ability to form biofilms is a common feature of microorganisms, which can colonize a variety of surfaces, such as host tissues and medical devices, resulting in infections highly resistant to conventional drugs. This aspect is particularly critical in polymicrobial biofilms involving both fungi and bacteria, therefore, to eradicate such severe infections, new and effective anti-biofilm strategies are needed. The efficacy of pentadecanal and pentadecanoic acid as anti-biofilm agents has been recently reported against different bacterial strains. Their chemical similarity with diffusible signal factors (DSFs), plus the already known ability of fatty acids to act as anti-biofilm agents, suggested to explore their use against Candida albicans and Klebsiella pneumoniae mixed biofilm. In this work, we demonstrated the ability of both molecules to prevent the formation and destabilize the structure of the dual-species biofilm. Moreover, the pentadecanoic acid anti-biofilm coating, previously developed through the adsorption of the fatty acid on polydimethylsiloxane, was proved to prevent the polymicrobial biofilm formation in dynamic conditions by confocal laser scanning microscopy analysis. Finally, the evaluation of the expression levels of some biofilm-related genes of Candida albicans and Klebsiella pneumoniae treated with pentadecanoic acid provided some insights into the molecular mechanisms underpinning its anti-biofilm effect.

Contaminated sediment is a major issue for aquatic environments, but attention must be kept even during remediation activities that can negatively affect resident biota especially when applied in situ. For the first time, the species sensitivity distribution (SSD) approach was applied to amendments used for in situ sediment remediation considering 39 papers including both freshwater (F) and saltwater (S) effect data (i.e. n = 17 only F, n = 19 only S, and n = 3 both F and S). Toxicity data related to the application of activated carbon (AC), nano-Zero-Valent-Iron (nZVI), apatite (A), organoclay (OC) and zeolite (Z) were collected and analyzed. SSD curves were constructed by lognormal model providing comprehensive comparisons of the sensitivities of different species to the relative testing methods. Results indicated that Bacteria were the most sensitive group of testing organisms, while Crustaceans were the less sensitive. The hazardous concentration for 5% of the affected species (HC5) were derived to determine the concentration protecting 95% of the species. OC, A and Z presented both acute and chronic toxicity. The HC5 values in descending order are: AC (4.79 g/L) > nZVI (0.02 g/L) > OC, A and Z (1.77E-04 g/L). AC and nZVI can be considered safer than OC, A and Z in sediment remediation activities, even if in situ long-term effects remained still underexplored.

Emergence of Candida tropicalis, which causes potential life-threatening invasive candidiasis, is often associated with colonization of medical devices as biofilm. Biofilm plays an important role in the virulence of the pathogen because of its complex structure, which provides resistance to conventional antimicrobials. In this study, the metabolic response of a clinical strain of C. tropicalis colonizing three distinct surfaces (polytetrafluoroethylene (PTFE), polystyrene, and polycarbonate) as well as the expression of virulence and stress related genes (ALS3, Hsp21, SAP1, SAP2, SAP3, and CYR1), were explored. Our results showed that lesser biofilm was developed on PTFE compared to polystyrene and polycarbonate. GS-MS metabolic analysis identified a total of 36 metabolites in the intracellular extract of cells grown on polystyrene, polycarbonate, and PTFE, essentially belonging to central carbon metabolism, amino acids, and lipids metabolism. The metabolic analysis showed that saturated and unsaturated fatty acids are preferentially produced during biofilm development on polycarbonate, whereas trehalose and vitamin B6, known as cellular protectors against a variety of stressors, were characteristic of biofilm on PTFE. The results of the transcriptomic analysis consider the different degrees of colonization of the three substrates, being CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA, downregulated in PTFE biofilm compared to polycarbonate or polystyrene biofilms, while Hsp21 was upregulated in concomitance with the potential unfavorable conditions for biofilm formation on PTFE. Overall, this work provides new insights into the knowledge of C. tropicalis biofilm development on surfaces of medical relevance in the perspective of improving the management of Candida infections.

Oxylipins are diatom-derived secondary metabolites, deriving from the oxidation of polyunsatured fatty acids that are released from cell membranes after cell damage or senescence of these single-celled algae. Previous results revealed harmful toxic effects of polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) on sea urchin Paracentrotus lividus embryonic development by testing individual compounds and mixtures of the same chemical group. Here, we investigated the combined effects of these compounds on sea urchin development at the morphological and molecular level for the first time. Our results demonstrated that oxylipin mixtures had stronger effects on sea urchin embryos compared with individual compounds, confirming that PUAs induce malformations and HEPEs cause developmental delay. This harmful effect was also confirmed by molecular analysis. Twelve new genes, involved in stress response and embryonic developmental processes, were isolated from the sea urchin P. lividus, these genes were found to be functionally interconnected with 11 genes already identified as a stress response of P. lividus embryos to single oxylipins. The expression levels of most of the analyzed genes targeted by oxylipin mixtures were involved in stress, skeletogenesis, development/differentiation, and detoxification processes. This work has important ecological implications, considering that PUAs and HEPEs represent the most abundant oxylipins in bloom-forming diatoms, opening new perspectives in understanding the molecular pathways activated by sea urchins exposed to diatom oxylipins.

Oxylipins are diatom-derived secondary metabolites, deriving from the oxidation of polyunsatured fatty acids that are released from cell membranes after cell damage or senescence of these single-celled algae. Previous results revealed harmful toxic effects of polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) on sea urchin Paracentrotus lividus embryonic development by testing individual compounds and mixtures of the same chemical group. Here, we investigated the combined effects of these compounds on sea urchin development at the morphological and molecular level for the first time. Our results demonstrated that oxylipin mixtures had stronger effects on sea urchin embryos compared with individual compounds, confirming that PUAs induce malformations and HEPEs cause developmental delay. This harmful effect was also confirmed by molecular analysis. Twelve new genes, involved in stress response and embryonic developmental processes, were isolated from the sea urchin P. lividus; these genes were found to be functionally interconnected with 11 genes already identified as a stress response of P. lividus embryos to single oxylipins. The expression levels of most of the analyzed genes targeted by oxylipin mixtures were involved in stress, skeletogenesis, development/differentiation, and detoxification processes. This work has important ecological implications, considering that PUAs and HEPEs represent the most abundant oxylipins in bloom-forming diatoms, opening new perspectives in understanding the molecular pathways activated by sea urchins exposed to diatom oxylipins.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

Micro- and/or mesocosms are experimental tools bringing ecologically relevant components of the natural environment under controlled conditions closest to the real world, without losing the advantage of reliable reference conditions and replications, providing a link between laboratory studies and filed studies in natural environments. Here, for the first time, a formal comparison of different types of mesocosm applied to the study of marine contaminants is offered, considering that pollution of coastal areas represented a major concern in the last decades because of the abundance of discharged toxic substances. In particular, the structural characteristics of micro- and mesocosms (m-cosms) used to study marine contaminated sediments were reviewed, focusing on their advantages/disadvantages. Their potentiality to investigate sediment remediation have been discussed, offering new perspective on how the use of m-cosms can be useful for the development of practical application in the development of solutions for contaminated sediment management in the contaminated marine environment.

The trophic ecology of the sea urchin Paracentrotus lividus, a key species in several shallow benthic communities, has been intensively studied, but the role of various foods in the processes of growth and gonadal maturation is still scarcely understood. This research assessed the effects of two fundamental food items for wild specimens of the sea urchin Paracentrotus lividus, the tissues of the seagrass Posidonia oceanica and of the green alga Ulva rigida, compared to the effect of a commercial compound feed on the somatic growth, gonad development, fertilization success and post-embryonic development. Consumption rates along with the C/N ratios were measured in the feeds and in the faecal pellets. We demonstrated that feeding for three months on U. rigida and P. oceanica did not affect growth and gonadal index of adults, fertilization processes and first cleavage and development, as well as field-collected animals. In contrast, a diet based on formulated pellets triggered a significant increase of gonadal index, but lack of gamete production, due to a follicular hyperthrophy. Our work will be useful for the definition of optimal diets for the production of mature broodstocks of an ecologically important marine model organism.