Biofilm formation on polyethylene microplastics and their role as transfer vector of emerging organic pollutants.

Microplastic (MP)-colonizing microorganisms are important links for the potential impacts on environmental, health, and biochemical circulation in various ecosystems but are not yet well understood. In addition, biofilms serve as bioindicators for the evaluation of pollutant effects on ecosystems. This study describes the ability of three polyethylene-type microplastics, white (W-), blue (B-), and fluorescent blue (FB-) MPs, to support microbial colonization of Pseudomonas aeruginosa, the effect of mixed organic contaminants (OCs: amoxicillin, ibuprofen, sertraline, and simazine) on plastic-associated biofilms, and the role of biofilms as transfer vectors of such emerging pollutants. Our results showed that P. aeruginosa had a strong ability to produce biofilms on MPs, although the protein amount of biomass formed on FB-MP was 1.6- and 2.4-fold higher than that on B- and W-MP, respectively. When OCs were present in the culture medium, a decrease in cell viability was observed in the W-MP biofilm (65.0%), although a general impairing effect of OCs on biofilm formation was ruled out. Microbial colonization influenced the ability of MPs to accumulate OCs, which was higher for FB-MP. In particular, the sorption of amoxicillin was lower for all bacterial-colonized MPs than for the bare MPs. Moreover, we analysed oxidative stress production to assess the impact of MPs or MPs/OCs on biofilm development. The exposure of biofilms to OCs induced an adaptive stress response reflected in the upregulation of the katB gene and ROS production, particularly on B- and FB-MP. This study improves our understanding of MP biofilm formation, which modifies the ability of MPs to interact with some organic pollutants. However, such pollutants could hinder microbial colonization through oxidative stress production, and thus, considering the key role of biofilms in biogeochemical cycles or plastic degradation, the co-occurrence of MPs/OCs should be considered to assess the potential risks of MPs in the environment.

Transcriptomic and physiological effects of polyethylene microplastics on Zea mays seedlings and their role as a vector for organic pollutants.

The widespread employment of plastics in recent decades has resulted in the accumulation of plastic residues in all ecosystems. Their presence and degradation into small particles such as microplastics (MPs) may have a negative effect on plant development and therefore on crop production. In this study, the effects of two types of polyethylene MPs on Zea mays seedlings cultured in vitro were analysed. In addition, four organic pollutants (ibuprofen, simazine, sertraline, and amoxicillin) were adsorbed by the MPs to evaluate their capacity as other contaminant vectors. The development of the plants was negatively affected by MPs alone or with the organic compounds. The strongest effect was observed in the W-MPs treatments, with a reduction in leaf and root length near 70%. Chlorophyll content was also differentially affected depending on the treatment. Transcriptome analysis showed that MPs affected gene expression in the roots of maize seedlings. As observed in the physiological parameters analysed, some gene expression changes were associated with specific treatments, such as changes in sugar transport genes in the B-MIX treatment. These results contribute to a better understanding of the molecular mechanisms of plants in regard to plastic stress responses.

Copper and Chromium toxicity is mediated by oxidative stress in Caenorhabditis elegans: The use of nanoparticles as an immobilization strategy.

Environmental contamination by heavy metals (HMs) has impelled searching for stabilization strategies, where the use of zero-valent iron nanoparticles (nZVI) is considered a promising option. We have evaluated the combined effect of Cu(II)-Cr(VI) on two Caenorhabditis elegans strains (N2 and RB1072 sod-2 mutant) in aqueous solutions and in a standard soil, prior and after treatment with nZVI (5% w/w). The results showed that HMs aqueous solutions had an intense toxic effect on both strains. Production of reactive oxygen species and enhanced expression of the heat shock protein Hsp-16.2 was observed, indicating increased HM-mediated oxidative stress. Toxic effects of HM-polluted soil on worms were higher for sod-2 mutant than for N2 strain. However, nZVI treatment significantly diminished all these effects. Our findings highlighted C. elegans as a sensitive indicator for HMs pollution and its usefulness to assess the efficiency of the nanoremediation strategy to decrease the toxicity of Cu(II)-Cr(VI) polluted environments.

Assessing the role of polyethylene microplastics as a vector for organic pollutants in soil: Ecotoxicological and molecular approaches.

Microplastics (MPs), pharmaceuticals and pesticides are emerging pollutants with proposed negative impacts on the environment. Rising interest in investigations of MPs is likely related to their potential to accumulate in agricultural systems as the base of the food chain. We applied an integrated approach using classic bioassays and molecular methods to evaluate the impact associated with a mixture of three types of polyethylene (PE) microbeads, namely, white (W), blue (B), and fluorescent blue (FB), and their interactions with pollutants (OCs), including ibuprofen (IB), sertraline (STR), amoxicillin (AMX) and simazine (SZ), on different soil organisms. PE-MPs exhibited different abilities for the adsorption of each OC; W selectively adsorbed higher amounts of SZ, whereas B and FB preferably retained AMX. Standard soil was artificially contaminated with OCs and MPs (alone or combined with OCs) and incubated for 30 days. The presence of MPs or MPs and OCs (MIX) in soil did not produce any effect on Caenorhabditis elegans endpoint growth, reproduction, or survival. Inhibition of leaf growth in Zea mays was detected, but this negative effect declined over time, while the inhibition of root growth increased, especially when OCs (32%) or MIX (47%) were added. Moreover, the expression of the antioxidant genes CAT 1, SOD-1A and GST 1 on plants was affected by the treatments studied. The addition of MPs or MIX significantly affected the soil bacterial phylogenetic profile, which selectively enriched members of the bacterial community (particularly Proteobacteria). The predicted functional profiles of MP/MIX samples indicated a potential impact on the carbon and nitrogen cycle within the soil environment. Our results indicate that MPs and their capability to act as pollutant carriers affect soil biota; further studies should be carried out on the bioavailability of OCs adsorbed by microplastics and how long it takes to leach these OCs into different organisms and/or ecosystems.

Bioassays to assess the ecotoxicological impact of polyethylene microplastics and two organic pollutants, simazine and ibuprofen.

Research on the environmental impact of plastics, especially on the effect of microplastics (MPs), has become a priority issue in recent years, mainly in terrestrial ecosystems where there is a lack of studies. This work aims to assess the impact of two types of polyethylene MPs, white microbeads (W) and fluorescent blue microbeads (FB), and their interactions with two contaminants, ibuprofen (Ib) and simazine (Sz), on different organisms. A set of bioassays for Vibrio fischeri, Caenorhabditis elegans and Lactuca sativa was carried out, which helped to establish the ecotoxicological impact of those pollutants. C. elegans showed the least sensitivity, while V. fischeri and L. sativa showed a high toxicological response to MPs alone. We found that W and FB induced an inhibition of 27% and 5.79%, respectively, in V. fischeri, and the growth inhibition rates were near 70% in L. sativa for both MPs. MPs exhibited a potential role as contaminant vectors in V. fischeri since the inhibition caused by W-Ib or W-Sz complexes was near 39%. The W-Sz complex significantly reduced leaf development in L. sativa, and a reduction of 30% in seed germination was detected when the complex FB-Sz was tested. This study reveals the importance of designing a complete set of analyses with organisms from different trophic levels, considering the great variability in the effects of MPs and the high number of relevant factors.

Ecotoxicogenomic analysis of stress induced on Caenorhabditis elegans in heavy metal contaminated soil after nZVI treatment.

Soil contamination by heavy metals (HMs) is an environmental problem, and nanoremediation by using zero-valent iron nanoparticles (nZVI) has attracted increasing interest. We used ecotoxicological test and global transcriptome analysis with DNA microarrays to assess the suitability of C. elegans as a useful bioindicator to evaluate such strategy of nanoremediation in a highly polluted soil with Pb, Cd and Zn. The HMs produced devastating effect on C. elegans. nZVI treatment reversed this deleterious effect up to day 30 after application, but the reduction in the relative toxicity of HMs was lower at day 120. We stablished gene expression profile in C. elegans exposed to the polluted soil, treated and untreated with nZVI. The percentage of differentially expressed genes after treatment decreases with exposure time. After application of nZVI we found decreased toxicity, but increased biosynthesis of defensive enzymes responsive to oxidative stress. At day 14, when a decrease in toxicity has occurred, genes related to specific heavy metal detoxification mechanisms or to response to metal stress, were down regulated: gst-genes, encoding for glutathione-S-transferase, htm-1 (heavy metal tolerance factor), and pgp-5 and pgp-7, related to stress response to metals. At day 120, we found increased HMs toxicity compared to day 14, whereas the transcriptional oxidative and metal-induced responses were attenuated. These findings indicate that the profiled gene expression in C. elegans may be considered as an indicator of stress response that allows a reliable evaluation of the nanoremediation strategy.

Evaluation of nanoremediation strategy in a Pb, Zn and Cd contaminated soil.

We addressed the efficiency of a nanoremediation strategy using zero-valent iron nanoparticles (nZVI), in a case of co-mingled heavy metals (HM) pollution (Pb, Cd and Zn). We applied a combined set of physical-chemical, toxicological and molecular analyses to assess the effectiveness and ecosafety of nZVI (5% w/w) for environmental restoration. After 120 days, nZVI showed immobilization capacity for Pb (20%), it was scarcely effective for Zn (8%) and negligibly effective for Cd. The HMs immobilization in the nZVI treated soils (compared to control soil), reaches its maximum after 15 days (T3) as reflected in the decrease of HM toxicity towards V. fischeri. The overall abundance of the microbial community was similar in both sets of samples during all experiment, although an increase in the number of metabolically active bacteria was recorded 15 days post treatment. We studied the induced impact of nanoremediation on the soil microbial community structure by Next Generation Sequencing (NGS). Even when higher HM immobilization was recorded, no significant recovery of the microbial community structure was found in nZVI-treated soil. The most marked nZVI-induced structural shifts were observed at T3 (increase in the Firmicutes population with a decrease in Gram-negative bacteria). Predictive metagenomic analysis using PICRUSt showed differences among the predicted metagenomes of nZVI-treated and control soils. At T3 we found decrease in detoxification-related proteins or over-representation of germination-related proteins; after 120 days of nZVI exposure, higher abundance of proteins involved in regulation of cellular processes or sporulation-related proteins was detected. This study highlights the partial effectiveness of nanoremediation in multiple-metal contaminated soil in the short term. The apparent lack of recovery of biodiversity after application of nZVI and the decreased effectiveness of nanoremediation over time must be carefully considered to validate this technology when assurance of medium- to long-term immobilization of HMs is required.

New insights into the impact of nZVI on soil microbial biodiversity and functionality.

Nanoscale zero-valent iron (nZVI) is a strong reducing agent used for in situ remediation of soil. The impacts of nZVI (5-10% w/w) on the soil microbial biodiversity and functionality of two soils (Lufa 2.2 and 2.4) were assessed. Illumina MiSeq technology was used to evaluate the structure of soil microbiomes after 21 days of exposure. Proteobacteria, Verrucomicrobia, Firmicutes and Actinobacteria were the most abundant phyla in both soils. However, the dynamics of bacterial community composition following nZVI addition differed. nZVI exposure induced pronounced shifts in the microbial composition of soil 2.4, but not in soil 2.2; an increase in Verrucomicrobia abundance was the unique common taxonomic pattern observed in both soils. The PICRUSt approach was applied to predict the functional composition of each metagenome. Environmental information processing function (membrane transport) was decreased in both nZVI-spiked soils, although soil 2.4 samples were enriched in functions involved in cellular processes and metabolism. The effects of nZVI on autochthonous bacterial communities clearly varied with the soil type assessed; changes at the phylogenetic level appeared to be more abundant than those observed at the functional level, and thus, the overall effort of the soil ecosystem might involve the maintenance of functionality following nZVI exposure.

Molecular stress responses to nano-sized zero-valent iron (nZVI) particles in the soil bacterium Pseudomonas stutzeri.

Nanotoxicological studies were performed in vitro using the common soil bacterium Pseudomonas stutzeri to assess the potentially toxic impact of commercial nano-sized zero-valent iron (nZVI) particles, which are currently used for environmental remediation projects. The phenotypic response of P. stutzeri to nZVI toxicity includes an initial insult to the cell wall, as evidenced by TEM micrographs. Transcriptional analyses using genes of particular relevance in cellular activity revealed that no significant changes occurred among the relative expression ratios of narG, nirS, pykA or gyrA following nZVI exposure; however, a significant increase in katB expression was indicative of nZVI-induced oxidative stress in P. stutzeri. A proteomic approach identified two major defence mechanisms that occurred in response to nZVI exposure: a downregulation of membrane proteins and an upregulation of proteins involved in reducing intracellular oxidative stress. These biomarkers served as early indicators of nZVI response in this soil bacterium, and may provide relevant information for environmental hazard assessment.

Integrating classical and molecular approaches to evaluate the impact of nanosized zero-valent iron (nZVI) on soil organisms.

Nanosized zero-valent iron (nZVI) is a new option for the remediation of contaminated soil and groundwater, but the effect of nZVI on soil biota is mostly unknown. In this work, nanotoxicological studies were performed in vitro and in two different standard soils to assess the effect of nZVI on autochthonous soil organisms by integrating classical and molecular analysis. Standardised ecotoxicity testing methods using Caenorhabditis elegans were applied in vitro and in soil experiments and changes in microbial biodiversity and biomarker gene expression were used to assess the responses of the microbial community to nZVI. The classical tests conducted in soil ruled out a toxic impact of nZVI on the soil nematode C. elegans in the test soils. The molecular analysis applied to soil microorganisms, however, revealed significant changes in the expression of the proposed biomarkers of exposure. These changes were related not only to the nZVI treatment but also to the soil characteristics, highlighting the importance of considering the soil matrix on a case by case basis. Furthermore, due to the temporal shift between transcriptional responses and the development of the corresponding phenotype, the molecular approach could anticipate adverse effects on environmental biota.

Effects of nano zero-valent iron on Klebsiella oxytoca and stress response.

Nano zero-valent iron (NZVI) is a new option for contaminated soil and groundwater treatment, despite little is known on their impact on environmental microorganisms. Klebsiella oxytoca K5 strain, isolated from the NZVI-treated soil, was used to investigate the bacterial, phenotypical and molecular response to commercial NZVI exposure. Cytotoxicity assays at three NZVI concentrations (1, 5 and 10 mg mL(-1)) suggested a negligible bacteriostatic effect and the lack of bactericidal effect. Structural changes were analysed by electronic microscopy. Scanning electron microscopy revealed the presence of NZVI around some bacterial cells, but no apparent morphological changes were seen. NZVI attachment to the cell surface was confirmed by transmission electron microscopy, although most of them were not affected. A proteomic approach (two-dimensional electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry) was used to investigate NZVI impact. For the first time to our knowledge, results revealed that exposure of a soil bacterium to NZVI resulted in the overproduction of tryptophanase, associated with oxidative stress response. K5 may set up an adaptative stress response involving indole as a signal molecule to inform the bacterial population about environmental changes. These findings would improve knowledge on the molecular mechanisms underlying bacterial response to NZVI exposure.

A new fluorescent oligonucleotide probe for in situ detection of s-triazine-degrading Rhodococcus wratislaviensis in contaminated groundwater and soil samples.

A bacterial strain (FPA1) capable of using terbuthylazine, simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine or ethylamine as its sole carbon source was isolated from a shallow aquifer chronically contaminated with s-triazine herbicides. Based on its 16S rDNA sequence analysis, the strain FPA1 was identified as Rhodococcus wratislaviensis. The disappearance time of 50% of the initial terbuthylazine concentration in the presence of this strain (DT(50)) was 62days. This strain was also able to mineralise the [U-ring (14)C] triazine-ring, albeit at a slow rate. A 16S rRNA target oligonucleotide probe (RhLu) was designed, and the FISH protocol was optimised, in order to detect R. wratislaviensis in s-triazine-contaminated sites. The RhLu probe gave a positive signal (expressed as % of total DAPI-positive cells) in both the groundwater (2.19+/-0.41%) and soil (2.10+/-0.96%) samples analysed. Using the RhLu probe, R. wratislaviensis can be readily detected, and its population dynamics can be easily monitored, in soil and in water ecosystems contaminated with s-triazine. To the best of our knowledge, this is the first report showing the isolation, from groundwater, of a bacterial strain able to degrade s-triazines.

Fluorescent in situ hybridization and flow cytometry as tools to evaluate the treatments for the control of slime-forming enterobacteria in paper mills.

Slime formation is a serious problem nowadays in the paper industry. Some enterobacteria are associated with the formation of slime deposits in paper and board mills. Detection and characterization of slime forming bacteria, belonging to the genus Enterobacter, Raoultella, and Klebsiella have been achieved by fluorescence in situ hybridization (FISH), using one probe based on the enterobacterial repetitive intergenic consensus sequence and other two rRNA targeted oligonucleotide probes. The effects of three kinds of antimicrobiological products (biocides, dispersants, and enzymes) on these enterobacterial cells were analyzed by flow cytometry (FC). Biocides B: utrol 1009 and 1072 were the most effective microbiocides against all enterobacterial cells analyzed, reaching 90% of dead bacteria after 24 h. However, the enzymatic treatment (Buzyme) was not equally efficient on enterobacteria and its microbiocide capacity varied depending on the type of microorganism. FISH and FC were effective tools to detect important slime forming enterobacteria and to select specific treatments to control microbial problems in the paper industry.

Application of fluorescence in situ hybridization technique to detect simazine-degrading bacteria in soil samples.

We propose a new approach to evaluate the natural attenuation capacity of soil by using fluorescence in situ hybridization (FISH). A specific oligonucleotide probe AtzB1 was designed based on the sequence data of the atzB gene involved in the hydrolytic deamination of s-triazines; this gene, located in a multiple copy plasmid was detected by the optimized FISH protocol. Two agricultural soils (Lodi and Henares) with a history of simazine treatments, and two natural soils (Soto and Monza), without previous exposure to simazine, were studied. AtzB1 probe-target cells were found only in the agricultural soils and, in a greater percentage, in the Lodi soil, compared to the Henares one. Moreover, the greatest percentage of AtzB1 probe-target cells in Lodi was accompanied by a greater mineralization rate, compared to the Henares soil. The FISH method used in this study was suitable for the detection of simazine-degrading bacteria and could be a useful indicator of the potential of soil bioremediation.