Metabolism of 2,4-D in plants: comparative analysis of metabolic detoxification pathways in tolerant crops and resistant weeds.

The commercialization of 2,4-D (2,4-dichlorophenoxyacetic acid) latifolicide in 1945 marked the beginning of the selective herbicide market, with this active ingredient playing a pivotal role among commercial herbicides due to the natural tolerance of monocots compared with dicots. Due to its intricate mode of action, involving interactions within endogenous auxin signaling networks, 2,4-D was initially considered a low-risk herbicide to evolve weed resistance. However, the intensification of 2,4-D use has contributed to the emergence of 2,4-D-resistant broadleaf weeds, challenging earlier beliefs. This review explores 2,4-D tolerance in crops and evolved resistance in weeds, emphasizing an in-depth understanding of 2,4-D metabolic detoxification. Nine confirmed 2,4-D-resistant weed species, driven by rapid metabolism, highlight cytochrome P450 monooxygenases in Phase I and glycosyltransferases in Phase II as key enzymes. Resistance to 2,4-D may also involve impaired translocation associated with mutations in auxin/indole-3-acetic acid (Aux/IAA) co-receptor genes. Moreover, temperature variations affect 2,4-D efficacy, with high temperatures increasing herbicide metabolism rates and reducing weed control, while drought stress did not affect 2,4-D efficacy. Research on 2,4-D resistance has primarily focused on non-target-site resistance (NTSR) mechanisms, including 2,4-D metabolic detoxification, with limited exploration of the inheritance and genetic basis underlying these traits. Resistance to 2,4-D in weeds is typically governed by a single gene, either dominant or incompletely dominant, raising questions about gain-of-function or loss-of-function mutations that confer resistance. Future research should unravel the physiological and molecular-genetic basis of 2,4-D NTSR, exploring potential cross-resistance patterns and assessing fitness costs that may affect future evolution of auxin-resistant weeds. © 2024 Society of Chemical Industry.

One-Pot Preparation of Ratiometric Fluorescent Molecularly Imprinted Polymer Nanosensor for Sensitive and Selective Detection of 2,4-Dichlorophenoxyacetic Acid.

The development of fluorescent molecular imprinting sensors for direct, rapid, and sensitive detection of small organic molecules in aqueous systems has always presented a significant challenge in the field of detection. In this study, we successfully prepared a hydrophilic colloidal molecular imprinted polymer (MIP) with 2,4-dichlorophenoxyacetic acid (2,4-D) using a one-pot approach that incorporated polyglycerol methacrylate (PGMMA-TTC), a hydrophilic macromolecular chain transfer agent, to mediate reversible addition-fragmentation chain transfer precipitation polymerization (RAFTPP). To simplify the polymerization process while achieving ratiometric fluorescence detection, red fluorescent CdTe quantum dots (QDs) and green fluorescent nitrobenzodiazole (NBD) were introduced as fluorophores (with NBD serving as an enhancer to the template and QDs being inert). This strategy effectively eliminated background noise and significantly improved detection accuracy. Uniform-sized MIP microspheres with high surface hydrophilicity and incorporated ratiometric fluorescent labels were successfully synthesized. In aqueous systems, the hydrophilic ratio fluorescent MIP exhibited a linear response range from 0 to 25 µM for the template molecule 2,4-D with a detection limit of 0.13 µM. These results demonstrate that the ratiometric fluorescent MIP possesses excellent recognition characteristics and selectivity towards 2,4-D, thus, making it suitable for selective detection of trace amounts of pesticide 2,4-D in aqueous systems.

Synergistic degradation of 2,4-dichlorophenoxyacetic acid in water by interfacial pre-reduction enhanced peroxymonosulfate activation derived from novel zero-valent iron/biochar.

Iron-based biochar exhibits great potential in degrading emerging pollutants and remediation of water environments. In this study, a highly efficient catalytic Fe0/biochar (MZB-800) was synthesized by the co-pyrolysis of poplar sawdust and K2FeO4 at 800 °C. A novel water purification technology of pre-reduction followed by PMS activation for MZB-800 was proposed to degrade the refractory 2,4-dichlorophenoxyacetic acid (2,4-D) pesticide. The corrosive effect of the strong oxidizing potassium salt endowed the MZB-800 surface with more Fe0 and porous structure, achieving greater 2,4-D adsorption binding energy. The removal efficiency of MZB-800 on 2,4-D was greater than that of biochar (BC) and conventional Fe0/biochar (Fe-BC) prepared by FeCl3·6 H2O as the precursor. The proposed novel water purification technology showed the synergistic effect between the interfacial pre-reduction and the PMS activation derived by MZB-800. Regarding 2,4-D degradation and dechlorination performance, the synergistic coefficient between pre-reduction and subsequent PMS activation for MZB-800 were 2 and 1.4 respectively. Based on the normalized kinetic analysis and the Langmuir-Hinshelwood model, we proposed the underlying mechanism of MZB-800 interfacial pre-reduction and subsequent PMS activation for synergistic removal of 2,4-D. The large amount of Fe2+ and hydroxyl density accumulated by the Fe0 and hydroquinone structures on the MZB-800 surface during the pre-reduction stage provided abundant active sites for the subsequent activation of PMS. The improved activation reaction rate generated more reactive oxygen species, further strengthening the removal efficiency of 2,4-D. This work manifested that the novel water purification technology of pre-reduction/PMS activation of iron-based biochar is feasible for removing emerging pollutants in the water environment. ENVIRONMENTAL IMPLICATION: Extensive abuse of 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide with high solubility and refractory degradation has caused environmental pollution and ecological deterioration. This manuscript described a novel water purification technology, centered on high-efficiency Fe0/biochar and utilizing pre-reduction and PMS reactivation strategies to synergistically degrade 2,4-D, which had strong environmental relevance. By elucidating the synergistic removal mechanism, the research provided valuable insights into removing emerging pollutants, thus promoting environmental sustainability and safeguarding ecosystem health. Overall, it is of high importance to provide a feasible and efficient method for removing hazardous 2,4-D from water environments, which contributes to addressing pressing environmental problems.

Effect of Bi2MoO6 Morphology on Adsorption and Visible-Light-Driven Degradation of 2,4-Dichlorophenoxyacetic Acid.

The development of highly efficient and stable visible-light-driven photocatalysts for the removal of herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) from water is still a challenge. In this work, Bi2MoO6 (BMO) materials with different morphology were successfully prepared via a simple hydrothermal method by altering the solvent. The morphology of the BMO material is mainly influenced by the solvent used in the synthesis (H2O, ethanol, and ethylene glycol or their mixtures) and to a lesser extent by subsequent thermal annealing. BMO with aggregated spheres and nanoplate-like structures hydrothermally synthesized in ethylene glycol (EG) and subsequently calcined at 400 °C (BMO-400 (EG)) showed the highest adsorption capacity and photocatalytic activity compared to other synthesized morphologies. Complete degradation of 2,4-D on BMO upon irradiation with a blue light-emitting diode (LED, λmax = 467 nm) was reached within 150 min, resulting in 2,4-dichlorophenol (2,4-DCP) as the main degradation product. Holes (h+) and superoxide radicals (⋅O2-) are assumed to be the reactive species observed for the rapid conversion of 2,4-D to 2,4-DCP. The addition of H2O2 to the reaction mixture not only accelerates the degradation of 2,4-DCP but also significantly reduces the total organic carbon (TOC) content, indicating that hydroxyl radicals are crucial for the rapid mineralization of 2,4-D. Under optimal conditions, the TOC value was reduced by 84.5% within 180 min using BMO-400 (EG) and H2O2. The improved degradation performance of BMO-400 (EG) can be attributed to its particular morphology leading to lower charge transfer resistance, higher electron-hole separation, and larger specific surface area.

The effects of commercial 2,4-D herbicide on game fish species: Natural lake water vs. laboratory system water.

Aquatic herbicides with active ingredient 2,4-dichlorophenoxyacteic acid (2,4-D) are commonly used to control and combat aquatic non-native species that cause detrimental impacts including habitat destruction, strained resources among biota, and biodiversity loss. While many (eco)toxicology studies are performed in the laboratory under highly controlled circumstances, these studies may disregard the nuances and disorder that come with the complexity of natural aquatic ecosystems. Therefore, we conducted a series of laboratory experiments using laboratory system water, different lake waters, and different water parameters to determine the effects of ecologically relevant concentrations of 2,4-D (0.00-4.00 ppm a.e.) on the development and survival of two freshwater game species (Sander vitreus and Esox lucius). For 2,4-D exposures using different water sources, there were significant main effects of 2,4-D concentration and water source on walleye embryo and larval survival, however, there was no significant interaction between 2,4-D exposure and water source. For 2,4-D exposures and pH (5-9 pH), there were significant main effects of 2,4-D concentration and pH on walleye and northern pike embryo survival and a significant interaction between 2,4-D exposure and pH. Our results indicate that 2,4-D exposures in controlled laboratory system water can predict similar outcomes as 2,4-D exposures in natural lake water. Moreover, individual water parameters, such as pH, play a significant role in the toxicity of 2,4-D. Taken together, these results suggest that highly controlled laboratory studies are a useful tool for predicting impacts on survival of non-target fish in natural waters, but it is crucial for management agencies to consider individual water sources and specific lake water parameters in herbicide risk assessments to minimize the impacts to non-target organism.

Synthetic auxin herbicide 2,4-D and its influence on a model BY-2 suspension.

2,4-D is a broadly used auxin herbicide. The presence of the 2,4-D synthetic auxin in the medium is imperative for long-term BY-2 tobacco suspension viability. The precise mechanism of this symbiosis of the suspension and the synthetic auxin remains unclear. Our goal was to study the hormonal regulation of the growth of the cell suspension; and to describe the experiments clarifying the interaction between the chosen growth regulators and phytohormones on the cellular level, specifically between the 2,4-D synthetic auxin and the native stress phytohormone - ethylene. This study examined the influence of low 2,4-D concentrations stimulating cell growth in vitro as well as the influence of high herbicide concentrations on the model tobacco BY-2 suspension. The culture took 6 days. Different parameters were evaluated, including the influence of different 2,4-D concentrations on the production of the phytohormone ethylene and its precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) in the tobacco cells. The content of 2,4-D in the cells and the medium was established. The observations of the morphological changes showed that a heavy impregnation of the cell walls taking place depending on the concentration of 2,4-D. A dramatic increase in protective polysaccharides and a remodulation of the cell walls by the formation of a pectin shield in artificial conditions were expected and observed. At the same time, massive production of the stress phytohormone ethylene took place, and, because of that, plant mutagenicity, anomalous tumour-type proliferation growth, and the production of supercells were observed. The hypothesis of the protective shield is discussed.

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

A Ratiometric Biosensor Containing Manganese Dioxide Nanosheets and Nitrogen-Doped Quantum Dots for 2,4-Dichlorophenoxyacetic Acid Monitoring.

Nanomaterials are desirable for sensing applications. Therefore, MnO2 nanosheets and nitrogen-doped carbon dots (NCDs) were used to construct a ratiometric biosensor for quantification of 2,4-dichlorophenoxyacetic acid. The MnO2 nanosheets drove the oxidation of colorless o-phenylenediamine to OPDox, which exhibits fluorescence emission peaks at 556 nm. The fluorescence of OPDox was efficiently quenched and the NCDs were recovered as the ascorbic acid produced by the hydrolyzed alkaline phosphatase (ALP) substrate increased. Owing to the selective inhibition of ALP activity by 2,4-D and the inner filter effect, the fluorescence intensity of the NCDs at 430 nm was suppressed, whereas that at 556 nm was maintained. The fluorescence intensity ratio was used for quantitative detection. The linear equation was F = 0.138 + 3.863·C 2,4-D (correlation coefficient R2 = 0.9904), whereas the limits of detection (LOD) and quantification (LOQ) were 0.013 and 0.040 µg/mL. The method was successfully employed for the determination of 2,4-D in different vegetables with recoveries of 79%~105%. The fluorescent color change in the 2,4-D sensing system can also be captured by a smartphone to achieve colorimetric detection by homemade portable test kit.

Adsorption of 2,4-dichlorophenoxyacetic acid on activated carbons from macadamia nut shells.

This study reports on the application of activated carbons from macadamia nut shells as adsorbents for the removal of 2,4-dichlorophenoxyacetic acid, a commonly used pesticide, from water. Different activating agents (FeCl3, ZnCl2, KOH and H3PO4) were used to obtain adsorbents within a wide range of porous texture and surface properties. The characterization of the resulting activated carbons was performed by N2 adsorption-desorption, elemental analysis, TG and pHPZC. The adsorption experiments were conducted in batch at 25, 45 and 65 °C. The adsorption kinetics on activated carbons obtained with FeCl3 H3PO4 or KOH was well described by the pseudo-second order model, whereas for the resulting from ZnCl2 activation the experimental data fit better the pseudo-first order model. The equilibrium studies were performed with the KOH- and ZnCl2-activated carbons, the two showing higher surface area values. In both cases, high adsorption capacities were obtained (c.a. 600 mg g-1) and the experimental data were better described by the Langmuir and Toth models. The thermodynamic study allows concluding the spontaneous and endothermic character of the adsorption process, as well as an increase of randomness at the solid/liquid interface. Breakthrough curves were also obtained and fitted to the logistic model.

Bifunctional fluorescent molecularly imprinted resin based on carbon dot for selective detection and enrichment of 2,4-dichlorophenoxyacetic acid in lettuce.

The work provided a method for synthesizing a simple fluorescent molecularly imprinted polymer by surface-initiated atom transfer radical polymerization (SI-ATRP) and its application in real sample. Poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) microspheres were selected as a matrix, 4-vinylpyridine, ethylene glycol dimethacrylate, 2,4-dichlorophenoxyacetic acid (2,4-D) as functional monomer, cross-linker and template molecule, respectively, to fabricate MAR@MIP with core-shell structure. For comparison, carbon dot (CD) as a fluorescence source was synthesized with o-phenylenediamine and tryptophan as precursors via hydrothermal method and integrated into MIP to acquire MAR@CD-MIP. MAR@CD-NIP was also prepared without adding the template molecule. The adsorption capacity of MAR@CD-MIP reached 104 mg g-1 for 2,4-D, which was higher than that of MAR@MIP (60 mg g-1). However, the adsorption capacity of MAR@CD-NIP was only 13.2 mg g-1. The linear range of fluorescence detection for 2,4-D was 18-72 µmol/L, and the limit of detection (LOD) was 0.35 µmol/L. The fluorescent MAR@CD-MIP was successfully applied in enrichment of lettuce samples. The recoveries of the three spiked concentrations of 2,4-D in lettuce were tested by fluorescence spectrophotometry and ranged in 97.3-101.7 %. Meanwhile, the results were also verified by HPLC. As a result, bi-functional molecularly imprinted resin was successfully fabricated to detect and enrich 2,4-D in real samples, and exhibited good selectivity, sensitivity and great application prospect in food detection.

Sol-gel and Pechini niobium modified: synthesis, characterization and application in the 2,4-D herbicide degradation.

In this work, a comparison was made between the synthesis of niobium-based materials (Nb2O5), both in terms of material characterization and catalytic performance. The methods used were chemical mixtures: modified sol-gel and Pechini. The materials were calcined at different temperatures (753, 873 and 993K) and characterized by the following techniques: photoacousticspectroscopy (PAS), zero charge point (pHPZC), scanning electron microscopy (SEM/EDS), thermogravimetric analysis (TGA/DTG) and X-ray diffraction (XRD). The photocatalytic process was carried out to evaluate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under UV radiation (250 W mercury vapor lamp) and different experimental conditions. In addition, to better understand the influence of parameters such as pH, catalyst concentration (0.2, 0.5 and 0.8 g L-1) and calcination temperature, a Design of Experiments (DoE) was used. The results indicated that despite having similar structures and phases in the XRD analysis, the morphology presents two distinct surfaces, due to the preparation method. Differences in the synthesis method affected the catalytic activity in the parameters studied. Although the zero charge point values are close (6.18-6.36), we observed differences in the band gap depending on the calcination temperature. In the optimal condition studied, the catalyst prepared by the sol-gel method obtained the best results.

UPLC-QTOF mass spectrometry detection of four endocrine disrupting chemicals (Methyl Paraben, 2,4-Dichlorophenoxyacetic acid, Monobutyl Phthalate, and Bisphenol A) in urine of Filipino women

Background and Objective@#Endocrine Disrupting Chemicals (EDCs) are ubiquitously found as low-level contaminants and pose serious threat to women’s health. EDCs may result in various reproductive disorders, fetal birth and developmental abnormalities, and endocrine and metabolic disorders. EDCs can be detected in body fluids of exposed individuals including blood and urine. This study aimed to detect four EDCs — Methyl Paraben (MP), 2,4-Dichlorophenoxyacetic acid (2,4-D), Monobutyl Phthalate (MBP), and Bisphenol A (BPA) in urine samples of women using Ultra-Performance Liquid Chromatography – Quadrupole Time-of-Flight (UPLC-QTOF) mass spectrometry.@*Methods@#Sequential steps of enzymatic deconjugation, liquid-liquid extraction, solid phase extraction, and liquid chromatography separation and mass spectrometry detection were optimized in urine samples. The method was used to analyze 70 urine samples from women of reproductive age.@*Results@#The sample preparation method showed a recovery ranging from 86.6% (MBP) to 100 % (2,4-D). The method demonstrated limits of quantitation ranging from 1.52 ng/m(MP) to 6.46 ng/mL(2,4D). Intra-day precisions expressed as relative standard deviation were all below 15% while accuracy was shown to range from 67.10% (2,4-D) to 102.39% (MBP). MP was detected in nine samples (12.86%) with a geometric mean value of 10.15 ng/ml (range: 3.62-52.39 ng/ml). MBP was detected in 68 samples (97.14%) with a geometric mean value of 97.62 ng/ml (range: 15.32-698.18 ng/ml). BPA was detected only once (9.58 ng/ml) while 2, 4-D was not detected in all samples.@*Conclusion@#A UPLC-QTOF mass spectrometry method to detect four EDCs at parts per billion level (ng/ml) was adapted and applied for analysis of urine samples. This method can find applicability in routine testing of clinical specimens as well as surveillance and other epidemiological studies.

Enhanced removal strategy towards organic matter with low coagulability: Immediate entrapment and complexation of oxidized intermediates by the hybrid ozonation-coagulation process.

The existence of dissolved organic matter (DOM) with low coagulability poses great challenges for conventional coagulation (CC) in water treatment. As a kind of typical organochlorine pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D) cannot be efficiently removed by CC. To enhance the 2,4-D removal, ozonation was applied with coagulation. The hybrid ozonation-coagulation (HOC) achieved 60.61% DOC removal efficiency, which was obviously higher than pre-ozonation coagulation (POC) (45.83%). Synchronous fluorescence spectroscopy revealed stronger complexation between modified 2,4-D and coagulants during the HOC than that in subsequent coagulation of the POC process. During the HOC process, ozone promoted the formation of polymeric Al species, such as Alb. To investigate the 2,4-D removal mechanism, γ-Al2O3/O3 process with the same oxidation ability as the HOC was established. 2,4-D was oxidized step-by-step to 2,4-dichlorophenol, 4,6-dichlororesorcin, 3,5-dichlorocatechol, 2-chlorohydroquinone, 4-chlorocatechol, 1,2,4,5-tetrahydroxybenzene, pentahydroxybenzene and oxalic acid in γ-Al2O3/O3 process. However, during the HOC process, these oxidized intermediates were readily complexed by coagulants and accumulated in flocs. Especially 1,2,4,5-tetrahydroxybenzene and pentahydroxybenzene, completely complexed by AlCl3•6H2O hydrolysates as soon as being formed. Immediate entrapment and complexation between coagulant hydrolysates and 2,4-D oxidized intermediates inhibited the generation of small-molecular-weight organics such as oxalic acid, which enhanced the removal of organics with low coagulability.

Associations of maternal exposure to 2,4-dichlorophenoxyacetic acid during early pregnancy with steroid hormones among one-month-old infants.

BACKGROUND: Exposure to 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used hormonal herbicide, may disrupt steroid hormone homeostasis. However, evidence from population-based studies is limited, especially for one-month-old infants whose steroid hormones are in a state of adjustment to extrauterine life and can be important indicators of endocrine development. This study aimed to explore the associations between maternal 2,4-D exposure during early pregnancy and infant steroid hormone levels. METHODS: The 885 mother-infant pairs were from a birth cohort in Wuhan, China. Maternal exposure to 2,4-D was determined in urine samples from early pregnancy, and nine steroid hormones were determined in infant urine. The associations of maternal 2,4-D exposure with infant steroid hormones and their product-to-precursor ratios were estimated based on generalized linear models, and bioinformatic analysis was conducted with public databases to explore the potential mechanisms involved. RESULTS: The detection frequency of 2,4-D was 99.32 %, and the detection frequency of steroid hormones ranged from 98.42 % to 100.00 %. After adjusting for covariates, an interquartile range increase in 2,4-D concentrations was associated with a 7.84 % decrease in 11-deoxycortisol (95 % confidence interval, CI: -14.12 %, -1.10 %), an 8.09 % decrease in corticosterone (95 % CI: -14.56 %, -1.14 %), an 8.67 % decrease in cortisol (95 % CI: -14.43 %, -2.52 %), a 13.00 % decrease in cortisone (95 % CI: -20.64 %, -4.62 %), and an 11.17 % decrease in aldosterone (95 % CI: -19.62 %, -1.83 %). Maternal 2,4-D was also associated with lower infant cortisol/17α-OH-progesterone, cortisol/pregnenolone, and aldosterone/pregnenolone ratios. In bioinformatic analysis, pathways/biological processes related to steroid hormone synthesis and secretion were enriched from target genes of 2,4-D exposure. CONCLUSIONS: Maternal urinary 2,4-D during early pregnancy was associated with lower infant urinary 11-deoxycortisol, corticosterone, cortisol, cortisone, and aldosterone, reflecting that 2,4-D exposure may interfere with infant steroid hormone homeostasis. Further efforts are still needed to study the relevant health effects of exposure to 2,4-D, particularly for vulnerable populations.

2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.

Background: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss.

The phosphoproteome is a first responder in tiered cellular adaptation to chemical stress followed by proteomics and transcriptomics alteration.

Next-generation risk assessment (NGRA) for environmental chemicals involves a weight of evidence (WoE) framework integrating a suite of new approach methodologies (NAMs) based on points of departure (PoD) obtained from in vitro assays. Among existing NAMs, the omic-based technologies are of particular importance based on the premise that any apical endpoint change indicative of impaired health must be underpinned by some alterations at the omics level, such as transcriptome, proteome, metabolome, epigenome and genome. Transcriptomic assay plays a leading role in providing relatively conservative PoDs compared with apical endpoints. However, it is unclear whether and how parameters measured with other omics techniques predict the cellular response to chemical perturbations, especially at exposure levels below the transcriptomically defined PoD. Multi-omics coverage may provide additional sensitive or confirmative biomarkers to complement and reduce the uncertainty in safety decisions made using targeted and transcriptomics assays. In the present study, we conducted multi-omics studies of transcriptomics, proteomics and phosphoproteomics on two prototype compounds, coumarin and 2,4-dichlorophenoxyacetic acid (2,4-D), with multiple chemical concentrations and time points, to understand the sensitivity of the three omics techniques in response to chemically-induced changes in HepG2. We demonstrated that, phosphoproteomics alterations occur not only earlier in time, but also more sensitive to lower concentrations than proteomics and transcriptomics when the HepG2 cells were exposed to various chemical treatments. The phosphoproteomics changes appear to approach maximum when the transcriptomics alterations begin to initiate. Therefore, it is proximal to the very early effects induced by chemical exposure. We concluded that phosphoproteomics can be utilized to provide a more complete coverage of chemical-induced cellular alteration and supplement transcriptomics-based health safety decision making.

New sensitive LC-MS/MS method for the simultaneous determination of 13 phenolic and carboxylic acid pesticide biomarkers in human urine, including dicamba.

The commercialization in 2016 of genetically engineered seeds tolerant to dicamba and/or 2,4-dichlorophenoxyacetic acid (2,4-D) has caused a rapid increase in the use of these herbicides. New questions about the reproductive and chronic health effects of long-term exposure to these herbicides have been raised. To assess exposure to dicamba and other pesticides of interest in the Heartland Study, a birth cohort study based in the United States, a new analytical method was needed. The present study describes the development and validation of this new solid phase extraction and liquid chromatography-tandem mass spectrometry method that detects simultaneously 13 pesticides or their metabolites in 250 µL of urine. More specifically, the method allows the analysis of dicamba, 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), which are herbicides, of malathion dicarboxylic acid (MDA), para-nitrophenol (PNP), 3,5,6-trichloro-2-pyridinol (TCPy), 2-diethylamino-6-methylpyrimidin-4-ol (DEAMPY) and 2-isopropyl-6-methyl-4-pyrimidinol (IMPY), which are metabolites of organophosphate insecticides, and finally of cis-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA), trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (trans-DCCA), 3-Phenoxybenzoic acid (3-PBA), 4-Fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and cis-3-(2,2-Dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), which are metabolites of synthetic pyrethroids insecticides. The method was validated under ISO/IEC 17025 guidance. The limit of detection (LOD) in urine samples was 0.10 µg/L for dicamba, while the LOD for other analytes ranged between 0.0038 µg/L and 0.091 µg/L. Accuracy was evaluated by analyzing samples from two External Quality Assessment Schemes, namely G-EQUAS and OSEQAS. Preliminary results obtained following the analysis of 91 urine samples taken from pregnant women enrolled in the Heartland Study are presented here. This method is suitable for human biomonitoring studies.

Evolution End Classification of tfd Gene Clusters Mediating Bacterial Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D).

The tfd (tfdI and tfdII) are gene clusters originally discovered in plasmid pJP4 which are involved in the bacterial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) via the ortho-cleavage pathway of chlorinated catechols. They share this activity, with respect to substituted catechols, with clusters tcb and clc. Although great effort has been devoted over nearly forty years to exploring the structural diversity of these clusters, their evolution has been poorly resolved to date, and their classification is clearly obsolete. Employing comparative genomic and phylogenetic approaches has revealed that all tfd clusters can be classified as one of four different types. The following four-type classification and new nomenclature are proposed: tfdI, tfdII, tfdIII and tfdIV(A,B,C). Horizontal gene transfer between Burkholderiales and Sphingomonadales provides phenomenal linkage between tfdI, tfdII, tfdIII and tfdIV type clusters and their mosaic nature. It is hypothesized that the evolution of tfd gene clusters proceeded within first (tcb, clc and tfdI), second (tfdII and tfdIII) and third (tfdIV(A,B,C)) evolutionary lineages, in each of which, the genes were clustered in specific combinations. Their clustering is discussed through the prism of hot spots and driving forces of various models, theories, and hypotheses of cluster and operon formation. Two hypotheses about series of gene deletions and displacements are also proposed to explain the structural variations across members of clusters tfdII and tfdIII, respectively. Taking everything into account, these findings reconstruct the phylogeny of tfd clusters, have delineated their evolutionary trajectories, and allow the contribution of various evolutionary processes to be assessed.

Use of in silico and in vitro methods as a potential new approach methodologies (NAMs) for (photo)mutagenicity and phototoxicity risk assessment of agrochemicals.

The increased use of agrochemicals raises concerns about environmental, animal, and mainly human toxicology. The development of New Approach Methodologies (NAMs) for toxicological risk assessment including new in vitro tests and in silico protocols is encouraged. Although agrochemical mutagenicity testing is well established, a complementary alternative approach may contribute to increasing reliability, with the consequent reduction of false-positive results that lead to unnecessary use of animals in follow-up in vivo testing. Additionally, it is unreasonable to underestimate the phototoxic effects of an accidental dermal exposure to agrochemicals during agricultural work or domestic application in the absence of adequate personal protection equipment, especially in terms of photomutagenicity. In this scenario, we addressed the integration of in vitro and in silico techniques as NAMs to assess the mutagenic and phototoxic potential of agrochemicals. In the present study we used the yno1 S. cerevisiae strain as a biomodel for in vitro assessment of agrochemical mutagenicity, both in the absence and in the presence of simulated sunlight. In parallel, in silico predictions were performed using a combination of expert rule-based and statistical-based models to assess gene mutations and phototoxicity. None of the tested agrochemicals showed mutagenic potential in the two proposed approaches. The Gly and 2,4D herbicides were photomutagenic in the in vitro yeast test despite the negative in silico prediction of phototoxicity. Herein, we demonstrated a novel experimental approach combining both in silico and in vitro experiments to address the complementary investigation of the phototoxicity and (photo)mutagenicity of agrochemicals. These findings shed light on the importance of investigating and reconsidering the photosafety assessment of these products, using not only photocytotoxicity assays but also photomutagenicity assays, which should be encouraged.

The potential of self-activated carbon for adsorptive removal of toxic phenoxyacetic acid herbicide from water.

Phenoxyacetic acid herbicides are widely used in agriculture for controlling weeds. These organic compounds are persistent and recalcitrant, often contaminating water and soil. Therefore, we studied five pristine biochars (BCs), and southern yellow pine (SYP) based self-activated carbon (SAC) for the adsorptive removal of 2,4-Dichlorophenoxyacetic acid (2,4-D) herbicide. Among the tested adsorbents, SYP-SAC-15 demonstrated higher (>90%) 2,4-D removal from water. The SYP-SAC-15 was produced using a facile and green route where the biomass pyrolysis gases worked as activating agents creating a highly porous structure with a surface area of 1499.79 m2/g. Different adsorption kinetics and isotherm models were assessed for 2,4-D adsorption on SYP-SAC-15, where the data fitted best to pseudo-second order (R2 > 0.999) and Langmuir (R2 > 0.991) models, respectively. Consequently, the adsorption process was mainly dominated by the chemisorption mechanism with monolayer coverage of SYP-SAC-15 surface with 2,4-D molecules. At the optimum pH of 2, the maximum 2,4-D adsorption capacity of SYP-SAC-15 reached 471.70 mg/g. Furthermore, an increase in the water salinity demonstrated a positive influence on 2,4-D adsorption, whereas humic acid (HA) showed a negative impact on 2,4-D adsorption. The regeneration ability of SYP-SAC-15 showed excellent performance by retaining 71.09% adsorption capability at the seventh adsorption-desorption cycle. Based on the operating pH, surface area, spectroscopic data, kinetics, and isotherm modeling, the adsorption mechanism was speculated. The 2,4-D adsorption on SYP-SAC-15 was mainly governed by pore filling, electrostatic interactions, hydrogen bonding, hydrophobic and π-π interactions.