Oil spills offshore can cause long-term ecological effects on coastal marine ecosystems. Despite their important ecological roles in the cycling of energy and nutrients in food webs, effects on bacteria, protists or arthropods are often neglected. Environmental DNA (eDNA) metabarcoding was applied to characterize changes in the structure of micro- and macro-biota communities of surface sediments over a 7-year period since the occurrence of Hebei Spirit oil spill on December 7, 2007. Alterations in diversities and structures of micro- and macro-biota were observed in the contaminated area where concentrations of polycyclic aromatic hydrocarbons were greater. Successions of bacterial, protists and metazoan communities revealed long-term ecological effects of residual oil. Residual oil dominated the largest cluster of the community-environment association network. Presence of bacterial families (Aerococcaceae and Carnobacteriaceae) and the protozoan family (Platyophryidae) might have conferred sensitivity of communities to oil pollution. Hydrocarbon-degrading bacterial families (Anaerolinaceae, Desulfobacteraceae, Helicobacteraceae and Piscirickettsiaceae) and algal family (Araphid pennate) were resistant to adverse effects of spilt oil. The protistan family (Subulatomonas) and arthropod families (Folsomia, Sarcophagidae Opomyzoidea, and Anomura) appeared to be positively associated with residual oil pollution. eDNA metabarcoding can provide a powerful tool for assessing effects of anthropogenic pollution, such as oil spills on sediment communities and its long-term trends in coastal marine environments.

The ubiquity of pollutants, such as agrochemicals and heavy metals, constitute a serious risk to human health. To evaluate the induction of DNA damage and programmed cell death (PCD), root cells of Allium cepa and Vicia faba were treated with two organophosphate insecticides (OI), fenthion and malathion, and with two heavy metal (HM) salts, nickel nitrate and potassium dichromate. An alkaline variant of the comet assay was performed to identify DNA breaks; the results showed comets in a dose-dependent manner, while higher concentrations induced clouds following exposure to OIs and HMs. Similarly, treatments with higher concentrations of OIs and HMs were analyzed by immunocytochemistry, and several structural characteristics of PCD were observed, including chromatin condensation, cytoplasmic vacuolization, nuclear shrinkage, condensation of the protoplast away from the cell wall, and nuclei fragmentation with apoptotic-like corpse formation. Abiotic stress also caused other features associated with PCD, such as an increase of active caspase-3-like protein, changes in the location of cytochrome C (Cyt C) toward the cytoplasm, and decreases in extracellular signal-regulated protein kinase (ERK) expression. Genotoxicity results setting out an oxidative via of DNA damage and evidence the role of the high affinity of HM and OI by DNA molecule as underlying cause of genotoxic effect. The PCD features observed in root cells of A. cepa and V. faba suggest that PCD takes place through a process that involves ERK inactivation, culminating in Cyt C release and caspase-3-like activation. The sensitivity of both plant models to abiotic stress was clearly demonstrated, validating their role as good biosensors of DNA breakage and PCD induced by environmental stressors.

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR.The limits of quantification (LOQ) of the ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Atrazine (ATR), a selective herbicide, is consistently used worldwide and has been confirmed to be harmful to the health of aquatic organisms. The release of neutrophil extracellular traps (NETs) is one of the newly discovered antimicrobial mechanisms. Although several immune functions have been analyzed under ATR exposure, the effect of ATR on NETs remains mainly unexplored. In the present study, we treated carp neutrophils using 5 μg/ml ATR and 5 μg/ml ATR combined with 100 nM rapamycin to elucidate the underlying mechanisms and to clarify the effect of ATR on phorbol myristate acetate (PMA)-induced NETs. The results of the morphological observation and quantitative analysis of extracellular DNA and myeloperoxidase (MPO) showed that NETs formation were significantly inhibited by ATR exposure. Moreover, we found that in the NETs process, ATR downregulated the expression of the anti-apoptosis gene B-cell lymphoma-2 (Bcl-2), increased the expression of the pro-apoptosis factors Bcl-2-Associated X (BAX), cysteinyl aspartate specific proteinases (Caspase3, 9), and anti-autophagy factor mammalian target of rapamycin (mTOR), decreased the expression of autophagy-related protein light chain 3B (LC3B) and glucose transport proteins (GLUT1, 4), disturbed the activities of phosphofructokinase (PFK), pyruvate kinase (PKM), and hexokinase (HK) and limited reactive oxygen species (ROS) levels, indicating that the reduced NETs release was a consequence of increased apoptosis and diminished ROS burst, autophagy and down-regulated glycolysis under ATR treatment. Meanwhile, rapamycin restored the inhibited autophagy and glycolysis and thus resisted the ATR-suppressed NETs. The present study perfects the mechanism theory of ATR immunotoxicity to fish and has a certain value for human health risk assessment.

Heavy metal contamination of soil in the vicinity of mining sites is a serious environmental problem around the world when mining residue (slag) is dispersed as dust. We conducted an incubation experiment to investigate the effect of a slag containing high levels of Pb and Zn (62.2 and 33.6 g kg⁻¹ slag as PbO and ZnO, respectively, sampled from a site formerly used as a lead and zinc mine) on the nitrogen cycle when mixed with soil (0–0.048 g slag g⁻¹ soil). The nitrogen cycle provides many life supporting-functions. To assess the quality of the soil in terms of the nitrogen cycle we focused on the dynamics of nitrate and ammonium, and bacterial community structure and functions within the soil. After two weeks of pre-incubation, ¹⁵N-labeled urea (500 mg N kg⁻¹) was added to the soil. Changes in soil pH, the concentration and ¹⁵N ratio of nitrate (NO₃⁻-N) and ammonium, and bacterial relative abundance and community structure were measured. Results indicated that increasing the ratio of slag to soil had a stronger negative effect on nitrification than ammonification, as suggested by slower nitrate accumulation rates as the slag:soil ratio increased. In the treatment with the highest amount of slag, the concentration of NO₃⁻-N was 50% of that in the controls at the end of the incubation. Regarding the bacterial community, Firmicutes had a positive and Planctomycetes a negative correlation with increasing slag concentration. Bacterial community functional analysis showed the proportion of bacterial DNA sequences related to nitrogen metabolism was depressed with increasing slag, from 0.68 to 0.65. We concluded that the slag impacted the soil bacterial community structure, and consequently influenced nitrogen dynamics. This study could form the basis of further investigation into the resistance of the nitrogen cycle to contamination in relation to soil bacterial community.

Productive coastal and estuarine habitats can be degraded by contaminants including persistent organic pollutants (POPs) such as PCBs, dioxins, and organochlorine insecticides to the extent of official designation as contaminated sites. Top-predatory wildlife may continue to use such sites as the habitat often appears suitable, and thus bioaccumulate POPs and other contaminants with potential consequences on their health and fitness. Victoria and Esquimalt harbours are located on southern Vancouver Island, British Columbia (BC) and are federally designated contaminated sites due mainly to past heavy industrial activities, such as from shipyards and sawmills. We collected scat samples from river otters (Lontra canadensis) throughout an annual cycle, and combined chemical analysis with DNA genotyping to examine whether the harbour areas constituted a contaminant-induced ecological trap for otters. We confirmed spatial habitat use by radio telemetry of a subsample of otters. Fifteen percent of otter scat contained PCB concentrations exceeding levels considered to have adverse effects on the reproduction of mink (Neovison vison), and there were significant positive correlations between concentrations of PCBs and of thyroid (T3) and sex (progesterone) hormones in fecal samples. Radio telemetry data revealed that otters did not show directional movement away from the harbours, indicating their inability to recognize the contaminated site as a degraded habitat. However, analysis and modeling of the DNA genotyping data provided no evidence that the harbour otters formed a sink population and therefore were in an ecological trap. Despite the highly POP-contaminated habitat, river otters did not appear to be adversely impacted at the population level. Our study demonstrates the value of combining chemical and biological technologies with ecological theory to investigate practical conservation problems.

CO2-driven acidification and emerging contaminants, such as pharmaceuticals, pose new threats for the maintenance of natural populations of marine organisms by interfering with their normal biochemical pathways and defences. The combined effects of seawater acidification, as predicted in climate change scenarios, and an emerging contaminant (the non-steroidal anti-inflammatory drug, NSAID, diclofenac) on oxidative stress-related parameters were investigated in the Mediterranean mussel Mytilus galloprovincialis and the Manila clam Ruditapes philippinarum. A flow-through system was used to carry out a three-week exposure experiment with the bivalves. First, the animals were exposed to only three pH values for 7 days. The pH was manipulated by dissolving CO2 in the seawater to obtain two reduced pH treatments (pH −0.4 units and pH −0.7 units), which were compared with seawater at the natural pH level (8.1). Thereafter, the bivalves were concomitantly exposed to the three experimental pH values and environmentally relevant concentrations of diclofenac (0.00, 0.05 and 0.50 μg/L) for an additional 14 days. The activities of superoxide dismutase, catalase and cyclooxygenase, and lipid peroxidation and DNA strand-break formation were measured in both the gills and digestive gland after 7, 14 and 21 days of exposure to each experimental condition. The results show that the biochemical parameters measured in both the mussels and clams were more influenced by the reduced pH than by the contaminant or the pH*contaminant interaction, although the biomarker variation patterns differed depending on the species and tissues analysed. Generally, due to increases in its antioxidant defence, M. galloprovincialis was more resistant than R. philippinarum to both diclofenac exposure and reduced pH. Conversely, reduced pH induced a significant decrease in COX activity in both the gills and digestive gland of clams, possibly resulting in the increased DNA damage observed in the digestive gland tissue.

Mariculture sediment has been recognized as a major contributor of environmental antibiotic resistance genes (ARGs), which are challenging the treatment of infections worldwide. Both antibiotics and fishmeal are used in aquaculture, and each has the potential to facilitate ARG dissemination, however their combined impact on the sediment resistome and their relative contribution remain unclear. In this study, microcosms were exposed to varying concentrations of tetracycline with or without fishmeal (0.1% wt/wt) for 14 days. Sediment genomic DNA was analyzed using high throughput quantitative PCR and 16S rRNA gene amplicon sequencing to compare the contribution of fishmeal and tetracycline to antibiotic resistomes and bacterial communities in mariculture sediment. Sixty-seven ARGs were detected potentially correlating to resistance for several major antibiotics. Fishmeal, but not the dose of tetracycline, contributed to the significant increase of both ARG abundance and diversity in the sediment. Based on principle coordinate analysis and hierarchical clustering, ARGs were clustered into two groups depending on whether fishmeal was added. Aminoglycoside, macrolide–lincosamide–streptogramin b (MLSb) and tetracycline resistance genes were the most abundant when fishmeal was used, while a significant increase in mobile genetic element (MGE) abundance was also detected (P < 0.05). Meanwhile, bacterial community structures were detected with distinct patterns between the two groups (Adonis, P < 0.05). Using the Mantel test and partial least squares path modeling, we identified that sediment resistomes were significantly correlated with microbial community structures (P < 0.05) which were mainly driven by nutrients in fishmeal. Together our findings suggested that fishmeal plays a more important role than tetracycline in proliferation of ARGs in mariculture sediment. This study may provide new insights into the mitigation of ARG propagation in mariculture operations.

Aflatoxin B1 (AFB1) and microcystin-LR (MC-LR) simultaneously exist in polluted food and water in humid and warm areas, and each has been reported to be genotoxic to liver and associated with hepatocellular carcinoma (HCC). However, the genotoxic effects of the two biotoxins in combination and potential mechanism remain unknown. We treated the human hepatic cell line HL7702 with AFB1 and MC-LR together at different ratios, examined their genotoxic effects using micronuclei and comet assays, and evaluated the possible mechanism by measuring oxidative stress markers and DNA base excision repair (BER) genes. Our data show that co-exposure to AFB1 and MC-LR significantly increased DNA damage compared with AFB1 or MC-LR alone as measured by the levels of both micronuclei and tail DNA. Meanwhile, AFB1 and MC-LR co-exposure showed biphasic effects on ROS production, and a gradual trend towards increased Glutathione (GSH) levels and activity of Catalase (CAT) and Superoxide Dismutase (SOD). Furthermore, MC-LR, with or without AFB1, significantly down-regulated the expression of the base excision repair (BER) genes 8-oxoguanine glycosylase-1 (OGG1) and X-ray repair cross complementing group 1 (XRCC1). AFB1 and MC-LR in combination upregulated the expression of the BER gene apurinic/apyrimidinic endonuclease 1 (APE1), whereas either agent alone had no effect. In conclusion, our studies show that MC-LR exacerbates AFB1-induced genotoxicity and we report for the first time that this occurs through effects on oxidative stress and the deregulation of DNA base excision repair genes.

The present work evaluates the action of nitroreductase enzyme immobilized on Tosylactivated magnetic particles (MP-Tosyl) on three disperse dyes which contain nitro and azo groups. The dyes included Disperse Red 73 (DR 73), Disperse Red 78 (DR 78), and Disperse Red 167 (DR 167). The use of a magnet enabled the rapid and easy removal of the immobilized enzyme after biotransformation; this facilitated the identification of the products generated using high-performance liquid chromatography with diode array detector (HPLC-DAD) and mass spectrometry (LC-MS/MS). The main products formed by the in vitro biotransformation were identified as the product of nitro group reduction to the correspondent amine groups, which were denoted as follows: 50% of 2-(2-(4-((2-cyanoethyl)(ethyl)amino)phenyl)hydrazinyl)-5-nitrobenzonitrile, 98% of 3-((4-((4-amino-2-chlorophenyl) diazenyl)phenyl) (ethyl)amino)propanenitrile and 99% of (3-acetamido-4 - ((4-amino-2-chlorophenyl) diazenyl) phenyl) azanediyl) bis (ethane-2,1-diyl) for DR 73, DR 78 and DR 167, respectively. Based on the docking studies, the dyes investigated were found to be biotransformed by nitroreductase enzyme due to their favorable interaction with the active site of the enzyme. Theoretical results show that DR73 dye exhibits a relatively lower rate of degradation; this is attributed to the cyanide substituent which affects the electron density of the azo group. The docking studies also indicate that all the dyes presented significant reactivity towards DNA. However, Disperse Red 73 was found to exhibit a substantially higher reactivity compared to the other dyes; this implies that the dye possesses a relatively higher mutagenic power. The docking results also show that DR 73, DR 78 and DR 167 may be harmful to both humans and the environment, since the mutagenicity of nitro compounds is associated with the products formed during the reduction of nitro groups. These products can interact with biomolecules, including DNA, causing toxic and mutagenic effects.