Atrazine alters early sexual development of the South American silverside, Odontesthes bonariensis.

Atrazine (ATZ) is a frequent contaminant in freshwater ecosystems within agricultural regions. The capacity of this herbicide to interfere with the vertebrate endocrine system is broadly recognized, but the mechanisms and responses usually differ among species. In this study, ATZ effects on hypothalamus-pituitary-gonadal (HPG) axis key genes expression and early gonadal development were evaluated in Odontesthes bonariensis larvae waterborne exposed during the gonadal differentiation period. Fish were treated to 0, 0.7, 7.0, and 70 µg ATZ/L at 25 °C from the 2nd to 6th week after hatching (wah), and a group was kept in clean water until the 12th wah. Parallelly, a group was submitted to 0.05 µg/L of ethinylestradiol (EE2) as a positive estrogenic control. From each treatment, eight larvae were sampled at 6 wah for gene expression analysis and twelve larvae at 12 wah for phenotypic sex histological determination. The expression of gnrh1, lhb, fshb, and cyp19a1b was assessed in the head, and the ones of amha, 11ßhsd2, and cyp19a1a in the trunk. Fish growth was significantly higher in fish exposed to 7 and 70 µg ATZ/L in the 6 wah, but the effect vanished at the 12 wah. The expression of lhb was upregulated in both sex larvae exposed from 7 µg ATZ/L. However, a dimorphic effect was induced on cyp19a1a expression at 70 µg ATZ/L, up or downregulating mRNA transcription in males and females, respectively. Delayed ovarian development and increased number of testicular germ cells were histologically observed from 7 to 70 µg ATZ/L, respectively, and a sex inversion (genotypic male to phenotypic female) was found in one larva at 70 µg ATZ/L. The lhb expression was also upregulated by EE2, but the cyp19a1a expression was not affected, and a complete male-to-female reversal was induced. Further, EE2 upregulated gnrh1 in females and cyp19a1b in both sexes, but it did not alter any assessed gene in the trunk. In conclusion, ATZ disrupted HPG axis physiology and normal gonadal development in O. bonariensis larvae at environmentally relevant concentrations. The responses to ATZ only partially overlapped and were less active when compared to the model estrogenic compound EE2.

Frogs Respond to Commercial Formulations of the Biopesticide Bacillus thuringiensis var. israelensis, Especially Their Intestine Microbiota.

It is generally believed that Bacillus thuringiensis var. israelensis (Bti) biopesticides are harmless to non-target organisms; however, new research shows controversial results. We exposed acutely and chronicallyLithobates sylvaticusandAnaxyrus americanus tadpoles until metamorphic climax to VectoBac 200G (granules) and VectoBac 1200L (aqueous suspension) at 300-20,000 ITU/L covering field-relevant concentrations and higher. The data show that the exposure parameters tested did not affect significantly the survival, total length, total weight, hepatosomatic index, gonadosomatic index, the expression of genes of interest (i.e., related to xenobiotic exposure, oxidative stress, and metamorphosis), and the intestine tissue layer detachment ofL. sylvaticusandA. americanus in a concentration-response pattern. In contrast, VectoBac 200G significantly increased the median time to metamorphosis ofL. sylvaticus tadpoles by up to 3.5 days and decreased the median by up to 1 day inA. americanus. VectoBac 1200L significantly increased the median time to metamorphosis ofL. sylvaticusandA. americanustadpoles by up to 4.5 days. Also, the exposure to VectoBac 200G and 1200L altered the intestine bacterial community composition inA. americanus at application rates recommended by the manufacturer, which led to an increase in the relative abundance of Verrucomicrobia, Firmicutes, Bacteroidetes, and Actinobacteria. Changes in the intestine microbiota might impact the fitness of individuals, including the susceptibility to parasitic infections. Our results indicate that the effect of Bti commercial products is limited; however, we recommend that Bti-spraying activities in amphibian-rich ecosystems should be kept minimal until there is more conclusive research to assess if the changes in the time to metamorphosis and microbiota can lead to negative outcomes in amphibian populations and, eventually, the functioning of ecosystems.

Molecular impacts of dietary exposure to nanoplastics combined with arsenic in Canadian oysters (Crassostrea virginica) and bioaccumulation comparison with Caribbean oysters (Isognomon alatus).

Despite the urge need to address the possible impact of plastic debris, up to now, little is known about the translocation of nanoplastics through the trophic web. Plus, due to their surface reactivity, nanoplastics could sorb and thus increase metals bioavailability to aquatic filter-feeding organisms (e.g., bivalves). In this study, we investigated the dietary exposure route on the oyster Crassostrea virginica through microalgae themselves exposed to three nanoplastic dispersions (PSL, PSC and NPG) at reportedly environmental concentrations combined or not with arsenic. Interactive effects of nanoplastics on arsenic bioaccumulation were studied, along with the expression of key genes in gills and visceral mass. The investigated gene functions were endocytosis (cltc), oxidative stress (gapdh, sod3, cat), mitochondrial metabolism (12S), cell cycle regulation (gadd45, p53), apoptosis (bax, bcl-2), detoxification (cyp1a, mdr, mt), and energy storage (vit). Results showcased that nanoplastic treatments combined with arsenic triggered synergetic effects on gene expressions. Relative mRNA level of 12S significantly increased at 10 and 100 µg L-1 for NPG combined with arsenic and for PSC combined with arsenic. Relative mRNA level of bax increased for PSL combined with arsenic and for PSC combined with arsenic at 10 and 100 µg L-1 respectively. We also observed that relative arsenic bioaccumulation was significantly higher in Crassostrea virginica gills compared to Isognomon alatus'. These results are the first comparative molecular effects of nanoplastics alone and combined with arsenic investigated in farmed C. virginica oysters. Together with I. alatus results we thus shed light on species different sensitivity.

A Review of the Effects of the Biopesticides Bacillus thuringiensis Serotypes israelensis (Bti) and kurstaki (Btk) in Amphibians.

Insecticides are important in agriculture, to reduce human disease, and to decrease the nuisance of biting insects. Despite this, many have the potential for environmental impacts and toxicity in nontarget organisms. We reviewed data on the effects of insecticides based on toxins from Bacillus thuringiensis var. israelensis (Bti) and Bacillus thuringiensis var. kurstaki (Btk) on amphibians. The few peer-reviewed publications that are available for Bti provide variable conclusions, ranging from few observable effects to evidence of acute toxicity at high concentrations. We briefly highlight the current controversies and identify key areas for future investigation.

Alkamides and Piperamides as Potential Antivirals against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has quickly spread globally, infecting millions and killing hundreds of thousands of people. Herein, to identify potential antiviral agents, 97 natural amide-like compounds known as alkamides and piperamides were tested against SARS-CoV-2 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp), and the human angiotensin-converting enzyme 2 (ACE2) using molecular docking and molecular dynamics simulations. The docking results showed that alkamides and dimeric piperamides from Piper species have a high binding affinity and potential antiviral activity against SARS-CoV-2. The absorption, distribution, metabolism, and excretion (ADME) profile and Lipinski's rule of five showed that dimeric piperamides have druglikeness potential. The molecular dynamics results showed that pipercyclobutanamide B forms a complex with Mpro at a similar level of stability than N3-I. Our overall results indicate that alkamides and piperamides, and specifically pipercyclobutanamide B, should be further studied as compounds with SARS-CoV-2 antiviral properties.

Profiling low molecular weight organic compounds from naphthenic acids, acid extractable organic mixtures, and oil sands process-affected water by SPME-GC-EIMS.

Naphthenic acids (NAs) are complex mixtures of carboxylic acids from petroleum that have industrial applications and that may be released to the environment after oil spills. There is significant research on the chemical composition and toxicity of water-soluble NAs derived from oil sands mining in Alberta, Canada. Yet, little is known about low molecular weight organic compounds (LMWOC) from these sources. Headspace solid-phase microextraction coupled to gas chromatography-electron impact mass spectrometry was used for LMWOC profiling of commercial NA blends, and an acid-extractable organics (AEOs) mixture from a tailings pond. From Sigma 1, Sigma 2, Merichem NAs and the AEO extract, 54, 56, 40 and 4 compounds were identified, respectively. These include aliphatic and cyclic hydrocarbons, carboxylic acids, alkylbenzenes, phenols, naphthalene and alkyl-naphthalene, and decalin compounds. A sample of oil sands process-affected water (OSPW) and aqueous solutions of the NA blends were evaluated for matrix effects on LMWOC profiles. Principal component and clustering analyses revealed that LMWOC profiles of commercial extracts were closely related but distinct from the AEO and OSPW samples. Some of the identified LMWOC are reported to be genotoxic or carcinogenic, and therefore the NA mixtures and AEOs should be considered hazardous materials and further evaluated.

Bioautography and GC-MS based identification of piperine and trichostachine as the active quorum quenching compounds in black pepper.

In the search of new and safe antibacterial compounds, the quorum sensing system (QS) modulation by natural products has been studied. As a result, many plant-derived compounds have been identified as potent quorum sensing inhibitors. Piper nigrum L. (black pepper) ethanolic extract inhibits the QS in some Gram-negative bacteria but the active components have not been previously identified. Thus, the objective of this work was to identify the P. nigrum peppercorns main components that block the QS, applying bioassay and chromatographic techniques. Piperine and trichostachine were identified as the main components responsible for the quorum quenching (QQ) activity of P. nigrum peppercorns extract. Piperine at 30 mg/L, decreased the violacein production by Chromobacterium violaceum CV026 by 35%, without affecting bacterial growth. Piperine concentration of 40 mg/L decreases violacein production by C. violaceum CV026 by 70% and growth in only 4.34%. Trichostachine at 50 mg/L decreases violacein production by C. violaceum CV026 by 12%, without affecting bacterial growth. P. nigrum extract concentration of 0.5 g/L decreased violacein production in 40 % and no effects on growth were observed. Neither P. nigrum extract, piperine, nor trichostachine did affect QS of Pseudomonas aeruginosa PAO1. Data here described exhibit the potential of piperamides as modulators of QS, not previously reported.

Neuroendocrine disruption of organizational and activational hormone programming in poikilothermic vertebrates.

In vertebrates, sexual differentiation of the reproductive system and brain is tightly orchestrated by organizational and activational effects of endogenous hormones. In mammals and birds, the organizational period is typified by a surge of sex hormones during differentiation of specific neural circuits; whereas activational effects are dependent upon later increases in these same hormones at sexual maturation. Depending on the reproductive organ or brain region, initial programming events may be modulated by androgens or require conversion of androgens to estrogens. The prevailing notion based upon findings in mammalian models is that male brain is sculpted to undergo masculinization and defeminization. In absence of these responses, the female brain develops. While timing of organizational and activational events vary across taxa, there are shared features. Further, exposure of different animal models to environmental chemicals such as xenoestrogens such as bisphenol A-BPA and ethinylestradiol-EE2, gestagens, and thyroid hormone disruptors, broadly classified as neuroendocrine disrupting chemicals (NED), during these critical periods may result in similar alterations in brain structure, function, and consequently, behaviors. Organizational effects of neuroendocrine systems in mammals and birds appear to be permanent, whereas teleost fish neuroendocrine systems exhibit plasticity. While there are fewer NED studies in amphibians and reptiles, data suggest that NED disrupt normal organizational-activational effects of endogenous hormones, although it remains to be determined if these disturbances are reversible. The aim of this review is to examine how various environmental chemicals may interrupt normal organizational and activational events in poikilothermic vertebrates. By altering such processes, these chemicals may affect reproductive health of an animal and result in compromised populations and ecosystem-level effects.

Analysis of naphthenic acid mixtures as pentafluorobenzyl derivatives by gas chromatography-electron impact mass spectrometry.

In this study, we report for the first time the efficiency of pentafluorobenzyl bromide (PFBBr) for naphthenic acid (NA) mixtures derivatization, and the comparison in the optimal conditions to the most common NAs derivatization reagents, BF3/MeOH and N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). Naphthenic acids are carboxylic acid mixtures of petrochemical origin. These compounds are important for the oil industry because of their corrosive properties, which can damage oil distillation infrastructure. Moreover, NAs are commercially used in a wide range of products such as paint and ink driers, wood and fabric preservatives, fuel additives, emulsifiers, and surfactants. Naphthenic acids have also been found in sediments after major oils spills in the United States and South Korea. Furthermore, the toxicity of the oil sands process-affected water (OSPW), product of the oil sands extraction activities in Canada's oil sands, has largely been attributed to NAs. One of the main challenges for the chromatographic analysis of these mixtures is the resolution of the components. The derivatization optimization was achieved using surface response analysis with molar ratio and time as factors for derivatization signal yield. After gas chromatography-electron impact mass spectrometry (GC/EIMS) analysis of a mixture of NA standards, it was found that the signal produced by PFB-derivatives was 2.3 and 1.4 times higher than the signal produced by methylated and MTBS-derivatives, respectively. The pentafluorobenzyl derivatives have a characteristic fragment ion at 181m/z that is diagnostic for the differentiation of carboxylic and non-carboxylic acid components within mixtures. In the analysis of a Sigma and a Merichem derivatized oil extract NA mixtures, it was found that some peaks lack the characteristic fragment ion; therefore they are not carboxylic acids. Open column chromatography was used to obtain a hexane and a methanol fraction of the Sigma and Merichem mixtures. The components in the hexane fraction, presumably hydrocarbons that did not react with PFBBr were ~7% by weight. The effectiveness of PFBBr was confirmed when the two NA oil extracts were spiked with 8 distinct NA standards and identified by GC/EIMS in the methanol fraction. Here, we also report retention indices of the methyl, MTBS and PFB derivatives of these 8 NAs. The use of PFBBr increases sensitivity, chromatographic resolution, and identification accuracy for the analysis of standards and mixtures of NAs compared to MTBSTFA and BF3/MeOH. This methodology will have wide applications in the elucidation of NA mixtures.

Dehydroabietic acid cytotoxicity in goldfish radial glial cells in vitro.

Dehydroabietic acid (DHAA) is a resin acid present in aquatic environments shown to induce cellular and molecular damage in aquatic animals. In this study, the cytotoxicity of DHAA on primary cultured goldfish radial glial cells (RGCs), an important component of the central nervous system, was evaluated. Here, it is reported that a concentration of 20mg/L DHAA affected cellular morphology and expression of genes involved in RGC steroidogenesis and metabolism. Higher concentration exposures of DHAA (40mg/L) lead to RGC death based on a lactate dehydrogenase leakage assay. Together, these data have implications in understanding the effects of DHAA on an integral central nervous system cell type important for neurogenesis, steroidogenesis and structural support. Due to the continuous presence of DHAA into water systems, results from this study provide indications as to the potential impacts of DHAA and demonstrate the importance of this class of chemicals on aquatic organisms.

Development of an in vitro Ovary Culture System to Evaluate Endocrine Disruption in Wood Frog Tadpoles.

Gonad-mesonephros complexes from wood frog (Lithobates sylvaticus) tadpoles were incubated in vitro for 1 wk to examine direct effects of naphthenic acids (NA) and 17α-ethinylestradiol (EE2) on ovarian differentiation. Histological images reveal successful preservation of gonadal integrity where cultured oocytes exhibited no significant differences in diameters and areas compared to uncultured conditions. Ovaries exposed to 10 µg/L 17α-EE2 contained oocytes with significantly advanced atresia and diminished areas and diameters, indicating a degree of ovarian regression. A significant reduction in oocyte area was observed in ovaries exposed to 3 mg/L of a commercial extract of petroleum-derived NA. This novel approach has applications for screening direct effects of endocrine-disrupting chemicals on gonadal function in tadpoles.