Whole-transcriptome response to wastewater treatment plant and stormwater effluents in the Asian clam, Corbicula fluminea.

The increase in human population and urbanization are resulting in an increase in the volume of wastewater and urban runoff effluents entering natural ecosystems. These effluents may contain multiple pollutants to which the biological response of aquatic organisms is still poorly understood mainly due to mixture toxicity and interactions with other environmental factors. In this context, RNA sequencing was used to assess the impact of a chronic exposure to wastewater treatment plant and stormwater effluents at the whole-transcriptome level and evaluate the potential physiological outcomes in the Asian clam Corbicula fluminea. We de-novo assembled a transcriptome from C. fluminea digestive gland and identified a set of 3,181 transcripts with altered abundance in response to water quality. The largest differences in transcriptomic profiles were observed between C. fluminea from the reference site and those exposed to wastewater treatment plant effluents. On both anthropogenically impacted sites, most differentially expressed transcripts were involved in signaling pathways in relation to energy metabolism such as mTOR and FoxO, suggesting an energy/nutrient deficit and hypoxic conditions. These conditions were likely responsible for damages to proteins and transcripts in response to wastewater treatment effluents whereas exposure to urban runoff might result in immune and endocrine disruptions. In absence of comprehensive chemical characterization, the RNAseq approach could provide information regarding the mode of action of pollutants and then be useful for the identification of which parameters must be studied at higher integration level in order to diagnose sites where the presence of complex and variable mixtures of chemicals is suspected.

Long-Term Effectiveness, under a Mountain Environment, of a Novel Conservation Nanomaterial Applied on Limestone from a Roman Archaeological Site.

A novel alkoxysilane-based product was applied on limestone samples from a Roman archaeological site. The study consisted of an initial phase to evaluate site environmental conditions in order to choose the most suitable product type to be applied. The decay that was produced in the site is mainly caused by natural action, with water being the main vehicle for the decay agents. Thus, the effectiveness of an innovative product with hydrophobic/consolidant properties and two commercial products (consolidant and hydrophobic agent) were evaluated on limestone from Acinipo site, under laboratory conditions. Next, the long-term effectiveness of the three products under study was evaluated by the exposure of limestone samples in the archaeological site for a period of three years. Since the recognized incompatibility between alkoxysilanes and pure carbonate stones, the interaction between the products and the limestones was widely investigated. The results that were obtained allow for it to be concluded that the innovative product presents adequate compatibility and adherence to the limestone under study, producing a long-term effective, homogeneous, and continuous coating with a depth of penetration of up to 10 mm. However, the commercial products produced discontinuous aggregates on the limestone surface, did not penetrate into its porous structure and it did not produce long-lasting effects.

Transcriptomic approach for assessment of the impact on microalga and macrophyte of in-situ exposure in river sites contaminated by chlor-alkali plant effluents.

Water quality degradation is a worldwide problem, but risk evaluation of chronic pollution in-situ is still a challenge. The present study aimed to evaluate the potential of transcriptomic analyses in representative aquatic primary producers to assess the impact of environmental pollution in-situ: the microalga Chlamydomonas reinhardtii and the macrophyte Elodea nuttallii were exposed 2 h in the Babeni Reservoir of the Olt River impacted by chlor-alkali plant effluent release resulting in increased concentrations of Hg and NaCl in receiving water. The response at the transcriptomic level was strong, resulting in up to 5485, and 8700 dysregulated genes (DG) for the microalga and for the macrophyte exposed in the most contaminated site, respectively. Transcriptomic response was congruent with the concentrations of Hg and NaCl in the water of the impacted reservoir. Genes involved in development, energy metabolism, lipid metabolism, nutrition, and RedOx homeostasis were dysregulated during in-situ exposure of both organisms. In addition, genes involved in the cell motility of C. reinhardtii and development of the cell wall of E. nuttallii were affected. DG were in line with adverse outcome pathways and transcriptomic studies reported after exposure to high concentrations of Hg and NaCl under controlled conditions in the laboratory. Transcriptomic response provided a sensitive measurement of the exposure as well as hints on the tolerance mechanisms of environmental pollution, and is thus promising as an early-warning tool to assess water quality degradation.

Effects of in situ exposure to tritiated natural environments: A multi-biomarker approach using the fathead minnow, Pimephales promelas.

Aquatic ecosystems are chronically exposed to radionuclides as well as other pollutants. Increased concentrations of pollutants in aquatic environments can present a risk to exposed organisms, including fish. The goal of this study was to characterize the effects of tritium, in the context of natural environments, on the health of fathead minnow, Pimephales promelas. Fish were exposed to tritium (activity concentrations ranging from 2 to 23,000Bq/L) and also to various concentrations of several metals to replicate multiple-stressor environments. Fish were exposed for 60days, then transferred to the tritium background site where they stayed for another 60days. Tritium, in the forms of tritiated water (HTO) and organically bound tritium (OBT), and a series of fish health indicators were measured in fish tissues at seven time points throughout the 120days required to complete the exposure and the depuration phases. Results showed effects of environmental exposure following the increase of tritium activity and metals concentrations in water. The internal dose rates of tritium, estimated from tissue HTO and OBT activity concentrations, were consistently low (maximum of 0.2µGy/h) compared to levels at which population effects may be expected (>100µGy/h) and no effects were observed on survival, fish condition, gonado-somatic, hepato-somatic, spleno-somatic and metabolic indices (RNA/DNA, proteins/DNA and protein carbonylation (in gonads and kidneys)). Using multivariate analyses, we showed that several biomarkers (DNA damages, MN frequency, gamma-H2AX, SFA/MUFA ratios, lysosomal membrane integrity, AChE, SOD, phagocytosis and esterase activities) were exclusively correlated with fish tritium internal dose rate, showing that tritium induced genotoxicity, DNA repair activity, changes in fatty acid composition, and immune, neural and antioxidant responses. Some biomarkers were responding to the presence of metals, but overall, more biomarkers were linked to internalized tritium. The results are discussed in the context of multiple stressors involving metals and tritium.

Evaluation of in vivo mutagenesis for assessing the health risk of air pollutants.

Various kind of chemical substances, including man-made chemical products and unintended products, are emitted to ambient air. Some of these substances have been shown to be mutagenic and therefore to act as a carcinogen in humans. National pollutant inventories (e.g., Pollutant Release and Transfer Registration in Japan) have estimated release amounts of man-made chemical products, but a major concern is the release of suspended particulate matter containing potent mutagens, for example, polycyclic aromatic hydrocarbons and related compounds generated by the combustion of fossil fuel, which are not estimated by PRTR system. In situ exposure studies have revealed that DNA adducts in the lung, and possibly mutations in germline cells are induced in rodents by inhalation of ambient air, indicating that evaluating in vivo mutations is important for assessing environmental health risks. Transgenic rodent systems (Muta, Big Blue, and gpt delta) are good tools for analyzing in vivo mutations induced by a mixture of chemical substances present in the environment. Following inhalation of diesel exhaust (used as a model mixture), mutation frequency was increased in the lung of gpt delta mice and base substitutions were induced at specific guanine residues (mutation hotspots) on the target transgenes. Mutation hotspots induced by diesel exhaust were different from those induced by benzo[a]pyrene, a typical mutagen in ambient air, but nearly identical to those induced by 1,6-dinitropyrene contained in diesel exhaust. Comparison between mutation hotspots in the TP53 (p53) gene in human lung cancer (data extracted from the IARC TP53 database) and mutations we identified in gpt delta mice showed that G to A transitions centered in CGT and CGG trinucleotides were mutation hotspots on both TP53 genes in human lung cancers and gpt genes in transgenic mice that inhaled diesel exhaust. The carcinogenic potency (TD50 value) of genotoxic carcinogen was shown to be correlated with the in vivo mutagenicity (total dose per increased mutant frequency). These results suggest that the mutations identified in transgenic rodents can help identify environmental mutagens that cause cancer.

Compensatory response of fathead minnow larvae following a pulsed in-situ exposure to a seasonal agricultural runoff event.

Agriculturally-dominated waterways such as those found throughout the Midwestern United States often experience seasonal pulses of agrichemical contaminants which pose a potential hazard to aquatic organisms at varying life stages. The objective of this study was to characterize the developmental plasticity of fathead minnow larvae in a natural environment subject to a seasonal episodic perturbation in the form of a complex mixture of agricultural stressors. Fathead minnow larvae were maintained at the Elkhorn River Research Station for a 28-d in situ exposure to an agrichemical pulse event. Minnow larvae were sampled after 14 and 28days to characterize developmental plasticity through growth measures and relative gene expression. Concentrations of agrichemical contaminants measured in water using polar organic chemical integrative samplers and composite sediment samples throughout the 28-d exposure were quantified using gas chromatography-mass spectrometry. Elevated concentrations of acetochlor, atrazine, and metolachlor were indicative of inputs from agricultural sources and were associated with reductions in body mass, condition factor, and androgenic gene expression in river exposed fathead minnow larvae. However, following a 14-d in situ depuration during the post-pulse period, river exposed larvae overcompensated in previously suppressed biological endpoints. These results indicate that fathead minnow larvae are capable of compensatory responses following episodic exposure to agrichemical stressors.

A subchronic in situ exposure method for evaluating effects in small-bodied fish at contaminated sites.

In situ fish-caging studies at contaminated sites can provide information that is more realistic compared to traditional laboratory-based studies. However, few methods have been developed for exposing sentinel fish species for subchronic durations, and fewer still are optimized for exposing small-bodied fish while maintaining fish health and growth throughout the caging trial. Those methods typically lack a feeding regimen during the fish caging trial. While that may be acceptable or even appropriate for typical short-term toxicity testing, it does limit the duration of the exposure, and may not be suitable when post-caging trials or observations are necessary. Returning healthy fish to the lab following the in situ exposure would be important, for example, in studies designed to examine long-term or multigenerational effects following an in situ exposure. In this article we describe a subchronic method for caging small fish at contaminated sites while maintaining growth and reproductive development. Fathead minnow (Pimephales promelas) were caged in situ for 6 weeks, after which time they were returned to the lab where they were evaluated for health and reproductive performance. Growth and reproductive endpoints revealed no adverse effect on fish due to fish caging and related handling, demonstrating the suitability of our caging and feeding method for long-term caging studies.

Experimental in situ exposure of the seagrass Posidonia oceanica (L.) Delile to 15 trace elements.

The Mediterranean seagrass Posidonia oceanica (L.) Delile has been used for trace element (TE) biomonitoring since decades ago. However, present informations for this bioindicator are limited mainly to plant TE levels, while virtually nothing is known about their fluxes through P. oceanica meadows. We therefore contaminated seagrass bed portions in situ at two experimental TE levels with a mix of 15 TEs (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Pb and Bi) to study their uptake and loss kinetics in P. oceanica. Shoots immediately accumulated pollutants from the beginning of exposures. Once contaminations ended, TE concentrations came back to their original levels within two weeks, or at least showed a clear decrease. P. oceanica leaves exhibited different uptake kinetics depending on elements and leaf age: the younger growing leaves forming new tissues incorporated TEs more rapidly than the older senescent leaves. Leaf epiphytes also exhibited a net uptake of most TEs, partly similar to that of P. oceanica shoots. The principal route of TE uptake was through the water column, as no contamination of superficial sediments was observed. However, rhizomes indirectly accumulated many TEs during the overall experiments through leaf to rhizome translocation processes. This study thus experimentally confirmed that P. oceanica shoots are undoubtedly an excellent short-term bioindicator and that long-term accumulations could be recorded in P. oceanica rhizomes.