The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time.

The present study validates the potential of the in vitro H9c2(2-1) cell-based sulforhodamine B (SRB) assay to evaluate the temporal variability of wastewater quality. The impact of effluent disposal on water quality and the efficiency of the wastewater treatment process were also assessed. To correlate standard analytical method results with in vitro results, a total of 16 physicochemical parameters, such as nutrients, pH, chemical oxygen demand, total suspended solids and metals, were determined in both raw and treated wastewater samples. Results revealed that the H9c2(2-1) cell-based SRB assay has an enormous potential to evaluate municipal wastewater quality over time and to discriminate influent and effluent toxic characteristics, as well as for water quality monitoring and surveillance of the efficacy of treatment processes. Finally, the gathered results alerted to the impact of phosphates in a biological system, leading us to recommend the selection of this parameter as a potential environmental health indicator.

Screening-level evaluation of marine benthic dinoflagellates toxicity using mammalian cell lines.

Complementary studies at different levels of the biological organization are fundamental to fully link environmental exposure to marine benthic dinoflagellate toxins and their effects. In order to contribute to this transdisciplinary evaluation, and for the first time, the present study aims to study the effects of Gambierdiscus excentricus, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and Prorocentrum lima extracts on seven functionally different mammalian cell lines: HEK 293, HepG2, HNDF, H9c2(2-1), MC3T3-E1, Raw 264.7 and SH-SY5Y. All the cell lines presented cell mass decrease in a concentration-dependence of dinoflagellate extracts, exhibiting marked differences in cell toxicity. Gambierdiscus excentricus presented the highest effect, at very low concentrations with EC50,24h (i.e., the concentration that gives half-maximal response after a 24-h exposure) between 1.3 and 13 cells mL-1, followed by O. cf. ovata (EC50,24h between 3.3 and 40 cells mL-1), and Prorocentrum species (P. lima: EC50,24h between 191 and 1027 cells mL-1 and P. hoffmannianum: EC50,24h between 152 and 783 cells mL-1). Cellular specificities were also detected and rat cardiomyoblast H9c2(2-1) cells were in general the most sensitive to dinoflagellate toxic compounds, suggesting that this cell line is an animal-free potential model for dinoflagellate toxin testing. Finally, the sensitivity of cells expressing distinct phenotypes to each dinoflagellate extract exhibited low relation to human poisoning symptoms.

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity.

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters, and thereby guarantee the protection of aquatic life and human health. Wastewater quality evaluation is conventionally based on physicochemical parameters, but increasing attention has been paid to integrate physicochemical and biological data. Nevertheless, the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns, and in vitro cell-based assays have thus become an important topic of interest. Hence, the present study intends: (a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells, and (b) maximizing the use of the effluent sample collected, to estimate the environmental condition of the receiving environment. The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity, and the best results were attained by lyophilising the sample as pre-treatment. Due to its response, the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents. The physicochemical status of the sample suggests a high potential for eutrophication, and iron exceeded the permissible level for wastewater discharge, possibly due to the addition of ferric chloride for wastewater treatment. In general, the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries, and both surpassed the aquatic protective values for long-term exposure.

Integron-associated genes are reliable indicators of antibiotic resistance in wastewater despite treatment- and seasonality-driven fluctuations.

The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n = 25) also presented seasonal differences, with an increase of the total ARGs' concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs' content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, blaGES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes blaGES and blaVIM, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.

Microplastics accumulate priority antibiotic-resistant pathogens: Evidence from the riverine plastisphere.

Microplastics (MPs) might accumulate and transport antibiotic-resistant bacteria (ARB) in aquatic systems. We determined the abundance and diversity of culturable ciprofloxacin- and cefotaxime-resistant bacteria in biofilms covering MPs placed in river water, and characterized priority pathogens from these biofilms. Our results showed that the abundance of ARB colonizing MPs tends to be higher compared to sand particles. Also, higher numbers were cultivated from a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET), compared to PP and PET alone. Aeromonas and Pseudomonas isolates were the most frequently retrieved from MPs placed before a WWTP discharge while Enterobacteriaceae dominated the culturable plastisphere 200 m after the WWTP discharge. Ciprofloxacin- and/or cefotaxime-resistant Enterobacteriaceae (n = 54 unique isolates) were identified as Escherichia coli (n = 37), Klebsiella pneumoniae (n = 3), Citrobacter spp. (n = 9), Enterobacter spp. (n = 4) and Shigella sp. (n = 1). All isolates presented at least one of the virulence features tested (i.e. biofilm formation, haemolytic activity and production of siderophores), 70% carried the intI1 gene and 85% exhibited a multi-drug resistance phenotype. Plasmid-mediated quinolone resistance genes were detected in ciprofloxacin-resistant Enterobacteriaceae [aacA4-cr (40% of the isolates), qnrS (30%), qnrB (25%), and qnrVC (8%)], along with mutations in gyrA (70%) and parC (72%). Cefotaxime-resistant strains (n = 23) harbored blaCTX-M (70%), blaTEM (61%) and blaSHV (39%). Among CTX-M producers, high-risk clones of E. coli (e.g. ST10 or ST131) and K. pneumoniae (ST17) were identified, most of which carrying blaCTX-M-15. Ten out of 16 CTX-M producers were able to transfer blaCTX-M to a recipient strain. Our results demonstrated the occurrence of multidrug resistant Enterobacteriaceae in the riverine plastisphere, harboring ARGs of clinical concern and exhibiting virulence traits, suggesting a contribution of MPs to the dissemination of antibiotic-resistant priority pathogens. The type of MPs and especially water contamination (e.g. by WWTPs discharges) seem to determine the resistome of the riverine plastisphere.

Are mercury levels in fishery products appropriate to ensure low risk to high fish-consumption populations?

The main goal of the present study is to determine the sources of methylmercury (MeHg) for high fish-consumption populations with the Portuguese population as showcase, as Portugal is the EU country with the highest fish consumption per capita (2019: 59.91 kg year-1). Since limited information is available on the effective levels of mercury after culinary treatments, cooked and raw codfish (Gadus morhua), hake (Merluccius merluccius), octopus (Octopus vulgaris), horse mackerel (Trachurus trachurus) and sardine (Sardina pilchardus) were considered. The mercury concentration ranking Hake > Horse mackerel > Codfish > Octopus > Sardine was observed in all situations (cooked and raw samples) for both MeHg and total mercury (T-Hg). The gathered results reinforce the general assumption that the loss of moisture during cooking increases MeHg and T-Hg concentrations in fish, but the idea that MeHg in fish muscle tissue represents the bulk of T-Hg cannot be generalised, as our study determined a MeHg/T-Hg ratio of 0.43 for grilled sardines.

Determination of intestinal absorption of the paralytic shellfish toxin GTX-5 using the Caco-2 human cell model.

Contributing to the human health risk assessment, the present study aims to evaluate the ability of paralytic shellfish toxins (PSTs) to cross the human intestinal epithelium by using the Caco-2 permeability assay. A crude extract prepared from the PST producer dinoflagellate Gymnodinium catenatum strain, GCAT1_L2_16, and the PST analogue gonyautoxin-5 (GTX-5) prepared from a certified reference material (CRM) were tested. In the conditions of the assay, none of the compounds altered Caco-2 viability, or the integrity of cell monolayers. The GTX-5 apparent permeability coefficients are 0.9×10-7 and 0.6×10-7 cm s-1 for the crude extract and CRM, respectively, thus, <10-6 cm s-1, which indicates that humans absorb this PST analogue poorly. The present study also reveals that, during a 90-min exposure, GTX-5 is not metabolised to a high extent by Caco-2 or retained in the Caco-2 cytoplasm. Since it is known that GTX-5 can be found in the spleen, liver or kidney of the victims, as well as in the urine samples of patients who consumed contaminated seafood, further research is needed to clarify the transport mechanisms involved, permeation time and dose-dependence, and the possible role of intestinal microflora.

H9c2(2-1)-based sulforhodamine B assay as a possible alternative in vitro platform to investigate effluent and metals toxicity on fish.

To overcome restrictions on the use of fish in toxicity testing, the present study proposes to compare the 50% growth inhibition potential (EC50) of four types of effluents on the rat cardiomyoblast H9c2(2-1) cell line by using the sulforhodamine B (SRB) cell mass colorimetric assay, with the corresponding fish lethal test results. Our objective was to evaluate if H9c2(2-1) cells shows comparable sensitivities, in both relative and absolute terms, to those provided by fish. In parallel, this study also compared the results of the chemical characterization with the legislation in force for environmental protection against effluent release into the receiving environment. Moreover, we tested the H9c2(2-1)-based SRB assays with the metals of concern found in the effluent samples. Both fish and cell assays showed the same toxicity rank for effluents: Metal > Oil > Municipal > Paper, and it should be stressed that the complementarity of using chemical and biological data represents a step forward to guarantee both environmental and human safety, since the chemical characterization showed a different toxicity rank: Metal > Municipal > Oil > Paper. Regarding metal elements, the short-term fish results showed a toxicity rank non-comparable with the rank obtained for cells. Nevertheless, the gathered results reveal the potentiality of the in vitro H9c2(2-1) platform as an alternative for fish lethal testing to assess, in absolute terms, the toxicity of effluents, particularly municipal effluents, and metals.

Exposure to marine benthic dinoflagellate toxins may lead to mitochondrial dysfunction.

Even though marine dinoflagellates are important primary producers, many toxic species may alter the natural equilibrium of aquatic ecosystems and even generate human intoxication incidents, as they are the major causative agents of harmful algal blooms. In order to deepen the knowledge regarding benthic dinoflagellate adverse effects, the present study aims to clarify the influence of Gambierdiscus excentricus strain UNR-08, Ostreopsis cf. ovata strain UNR-03 and Prorocentrum lima strain UNR-01 crude extracts on rat mitochondrial energetic function and permeability transition pore (mPTP) induction. Our results, expressed in number of dinoflagellate cell toxic compounds tested in a milligram of mitochondrial protein, revealed that 934 cells mg prot-1 of G. excentricus, and 7143 cells mg prot-1 of both O. cf. ovata and P. lima negatively affect mitochondrial function, including by decreasing ATP synthesis-related membrane potential variations. Moreover, considerably much lower concentrations of dinoflagellate extracts (117 cells mg prot-1 of G. excentricus, 1429 cells mg prot-1 of O. cf. ovata and 714 cells mg prot-1 of P. lima) produced mPTP-induced swelling in Ca2+-loaded isolated mitochondria. The present study clearly demonstrates the toxicity of G. excentricus, O. cf. ovata and P. lima extracts at the mitochondrial level, which may lead to mitochondrial failure and consequent cell toxicity, and that G. excentricus always provide much more severe effects than O. cf. ovata and P. lima.

Cell-based assays as an alternative for the study of aquatic toxicity of pharmaceuticals.

An increasing number and amount of pharmaceuticals for human and veterinary use currently reach the aquatic environment, and the determination of their effects on aquatic organisms becomes of major importance. The 96-h fish lethal test is one of the conventional assays required for environmental hazardous assessment, but it is extremely time-consuming and costly, and it raises ethical concerns. In a broad study, we compared the ability of cell-based assays to detect, in absolute terms, lethal toxicity in fish due to pharmaceuticals in order to select sensitive cell lines to be posteriorly used as an alternative to fish testing. This study also explored the sensitivity of the rat cardiomyoblast H9c2(2-1) cell line and the suitability of the sulforhodamine B colorimetric assay regarding 15 pharmaceuticals belonging to 9 different therapeutic classes. The relation between in vivo and in vitro data was expressed as LC50,96h/EC50 ratios, and 66% of concordant data were attained. Accordingly, it was possible to conclude that cell-based assays could be considered a suitable alternative to fish lethal testing for pharmaceuticals, which, after validation, may dramatically reduce the number of fish required for environmental hazardous assessment. Several cell lines were selected as promising alternatives, but H9c2(2-1), HepG2, PLHC-1, and RTG-2 could be considered suitable starting cell types for further studies, as relevant results were obtained with low exposure times.

High sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds.

Ciguatera fish poisoning is a frequently reported non-bacterial food-borne illness related to the consumption of seafood contaminated with ciguatoxins, and possibly maitotoxins. These toxins are synthesized by marine dinoflagellate species of Gambierdiscus and Fukuyoa genera, and their abundance is a matter of great concern due to their adverse effects to aquatic life and human health. The present study aims to assess the sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds using concentration- and time-dependent sulforhodamine B (SRB) colorimetric assays. Low concentrations of Gambierdiscus extracts (corresponding to 1.3-2.3 cells mL-1) induced a concentration-dependent response. Specificity in time-dependent response of H9c2(2-1) cells was demonstrated for G. excentricus after a 180 min exposure compared to both G. cf. belizeanus and G. silvae species, with EC50s obtained after 720 and 360 min, respectively. The sensitivity of H9c2(2-1) cells to dinoflagellate toxic compounds was also tested with other genera from benthic (Coolia malayensis, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and P. lima) and planktonic (Amphidinium carterae and Lingulodinium polyedrum) habitats. Amphidinium, Coolia and Lingulodinium data did not present any concentration-response relationships, and EC50 values could only be obtained after 720 and 1440 min of exposure to both Prorocentrum species and O. cf. ovata, respectively. This study demonstrated that the H9c2(2-1) SRB assay represents a promising and sensitive tool for the detection of Gambierdiscus toxic compounds present in water samples, particularly of G. excentricus at very low cell abundances.

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis.

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2-1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.

Cell-based assays seem not to accurately predict fish short-term toxicity of pesticides.

A key action of current aquatic environmental sciences is the determination of concentration-response assessments to quantitatively measure the risk of pollutants, and fish lethal tests are still a regulatory requirement for assessing the environmental risk of both new and existing substances in the aquatic compartment. However, animal health and welfare aspects, as well as the time and resources required to support fish lethal testing, stimulate research for alternative in vitro methods, and cell-based assays are considered as one such alternative. The first goal of the present study was to compile existing fish short-term toxicity and cell toxicity data of pesticides through a scientific literature search, and relate in vivo and in vitro results to identify sensitive cell models, mammalian-, fish- or arthropod-derived. Discovering cell models which are sensitive is of great importance, since the risk of false negatives, believed to be the main limitation of cell-based assays as an alternative to fish tests, may decrease. As to the second goal, this study also determined and compared cell toxicity results for 12 pesticides using rat cardiomyoblast H9c2(2-1) cells with the corresponding fish LC50,96h (50% lethal concentration at 96 h of exposure) values collected in literature. On the whole, the in vivo/in vitro ratio was obtained for 50 pesticides belonging to 23 groups, and presented only 27% of positive results, thus confirming the low sensitivity of cell-based assays in relation to fish lethal data for pesticides. In general, cell-based assays still do not seem to be an alternative to the regulatory short-term fish assay for pesticides, making further studies necessary.

The environmental condition of an estuarine ecosystem disturbed by pesticides.

Knowledge regarding the concentration levels resulting from the use of agricultural pesticides may indicate the nature of the controls necessary to reduce environmental and human health risks to an acceptable level. Therefore, the main goal of the present work was to assess the spatial and temporal occurrence of 35 pesticides in the River Sado estuary (Portugal) in 2017 and evaluate its environmental condition, as data for estuarine ecosystems is scarce. Since pesticides are very susceptible to matrix effects promoted by environmental samples, to attain the main goal, we developed a fast and almost solvent-free environmentally friendly method with a good performance for both estuarine surface water and sediment samples. Quantified residues were determined mostly during summer, in line with the pesticide application period. Five herbicides (alachlor, bentazon, metobromuron, metribuzin and triclopyr) were measured in the water before and after the production season, suggesting a long-term aquatic exposure. Sediment samples were less contaminated, since a lower number of quantified pesticides were found in the study area, in lower frequencies and lower concentrations. No potential high adverse effects of the use of agricultural pesticides were expected on the aquatic organisms of the Sado estuary, even considering the potential combination effect of pesticide mixtures.

Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin.

The present study aims at identifying, among six mammalian and fish cell lines, a sensitive cell line whose in vitro median inhibitory concentration (IC50) better matches the in vivo short-term Sparus aurata median lethal concentration (LC50). IC50s and LC50 were assessed after exposure to the widely used fungicide azoxystrobin (AZX). Statistical results were relevant for most cell lines after 48 h of AZX exposure, being H9c2 the most sensitive cells, as well as the ones which provided the best prediction of fish toxicity, with a LC50,96h/IC50,48h = 0.581. H9c2 cell proliferation upon 72 h of AZX exposure revealed a LC50,96h/IC50,72h = 0.998. Therefore, identical absolute sensitivities were attained for both in vitro and in vivo assays. To conclude, the H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for AZX, and could be used to get valuable insights into the toxic effects of other pesticides.