Cyto-genotoxicity evaluation of pyroligneous acid using Allium cepa assay.

Pyroligneous acid (PA) is a highly oxygenated organic condensate obtained by cooling the gases generated from the pyrolysis process. PA has been used in agriculture for several years with multiple beneficial effects, including plant health and yields, pest resilience, and seed germination. It is generally applied to agricultural soils in the dilution of 1:1000 to 1:100, corresponding to 0.1-1% PA concentration. In this study, the cyto-genotoxic potential of PA to Allium cepa meristematic root-tips (where all cells undergo repeated division and form primary root tissues) was examined. Exposure to PA concentrations of 0.1% and above showed a reduction in the mitotic index percentage, and at 5%, a complete arrest in the cell division was recorded. However, chromosomal aberrations at 0.5, 1, and 3% PA were reversible types such as bridges, vagrants, laggards, and multipolar anaphase, with a maximum of only 5.8% chromosomal aberration observed at 3% PA. Comet assay (single-cell gel electrophoresis) for genotoxicity assessment determined using PA exposed A. cepa root tips showed that it was not genotoxic. The absence of cyto-genotoxicity in A. cepa, even at concentrations far above what would be typically encountered in agricultural applications, strongly suggests that PA is unlikely to cause adverse effects on crops and ultimately on the biota and human health.

Exposure to perfluorooctanesulfonate (PFOS) but not perflurorooctanoic acid (PFOA) at ppb concentration induces chronic toxicity in Daphnia carinata.

Widespread environmental contamination of per- and polyfluoroalkyl substances (PFAS) is well established. Nevertheless, few studies have reported on the aquatic toxicity of PFAS, especially in indicator species such as Daphnia. In this study, the toxicity of two major PFAS, namely perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS), was investigated on water flea (Daphnia carinata) using a battery of comprehensive toxicity tests, including a 48 h acute and a 21-day chronic assays. The survival, growth, and reproduction of D. carinata were monitored over a 21-day life cycle. PFOS exhibited higher toxicity than PFOA. The 48 h LC50 values (confidence interval) based on acute toxicity for PFOA and PFOS were 78.2 (54.9-105) mg L-1 and 8.8 (6.4-11.6) mg L-1, respectively. Chronic exposure to PFOS for 21 days displayed mortality and reproductive defects in D. carinata at a concentration as low as 0.001 mg L-1. Genotoxicity assessment using comet assay revealed that exposure for 96 h to PFOS at 1 and 10.0 mg L-1 significantly damaged the organism's genetic makeup. The results of this study have great implications for risk assessment of PFOS and PFOA in aquatic ecosystems, given the potential of PFOS to pose a risk to Daphnia even at lower concentrations (1 µg L-1).

Evaluation of Cyto-genotoxicity of Perfluorooctane Sulfonate (PFOS) to Allium cepa.

Per- and polyfluoroalkyl substances (PFAS) have emerged as contaminants of global concern. Among several PFAS, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are persistent and bioaccumulative compounds. We investigated the cyto-genotoxic potential of PFOS to Allium cepa root meristem cells. The A. cepa root tips were exposed to 6 different concentrations (1-100 mg L-1 ) of PFOS for 48 h. Reduction in mitotic index and chromosomal aberrations was measured as genotoxic endpoints in meristematic root cells. Exposure to PFOS significantly affected cell division by reducing the miotic index at higher concentrations (>10 mg L-1 ). The median effect concentration of PFOS to elicit cytotoxicity based on the mitotic index was 43.2 mg L-1 . Exposure to PFOS significantly increased chromosomal aberrations at concentrations >25 mg L-1 . The common aberrations were micronuclei, vagrant cells, and multipolar anaphase. The alkaline comet assay revealed a genotoxic potential of PFOS with increased tail DNA percentage at concentrations >25 mg L-1 . To our knowledge, this is the first study to report the cyto-genotoxic potential of PFOS in higher plants. Environ Toxicol Chem 2021;40:792-798. © 2020 SETAC.

Rhizodegradation of PAHs differentially altered by C3 and C4 plants.

Pyrosequencing of 16S ribosomal RNA (rRNA) was employed to characterize bacterial communities colonizing the rhizosphere of plants with C3 and C4 photosynthetic pathways grown in soil contaminated with polycyclic aromatic hydrocarbons (PAHs) after 60 and 120 days. The results of this study exhibited a clear difference in bacterial diversity between the rhizosphere and non-rhizosphere samples and between the rhizospheres of the C3 and C4 plants after 120 days. In both C3 and C4 rhizospheres, an incremental change in PAHs degrading bacterial genera was observed in the 120th day samples compared to the 60th day ones. Among the PAHs degrading bacterial genera, Pseudomonas showed good resistance to PAHs in the 120th day rhizosphere of both C3 and C4 plants. Conversely, the genus Sphingomonas showed sensitivity to PAHs in the 120th day rhizosphere soils of C3 plants only. Also, a significant increase in the PAHs degrading genera was observed at 120th day in the C4 rhizosphere in comparison to the C3 rhizosphere, which was reflected in a reduced PAHs concentration measured in the soil remediated with C4 plants rather than C3 plants. These results suggest that the rhizoremediation of PAHs was primarily governed by the plant photosystems, which led to differences in root secretions that caused the variation in bacterial diversity seen in the rhizospheres. This study is the first report to demonstrate the greater effectiveness of C4 plants in enhancing the PAHs degrading bacterial community than C3 plants.

Purification and characterization of a novel fenamiphos hydrolysing enzyme from Microbacterium esteraromaticum MM1.

Fenamiphos is a neurotoxic organophosphorus pesticide used widely to control pests of crops. Fenamiphos and its toxic oxidation products have been detected in surface and groundwaters. A novel enzyme capable of hydrolysing P-O-C bond of fenamiphos is purified from Microbacterium esteraromaticum MM1 total cellular protein using a combination of methods. The purified fenamiphos hydrolysing enzyme (FHE) was identified as enolase (phosphopyruvate hydratase), a housekeeping enzyme with molecular mass and pI value of 45 kDa and 4.5, respectively. The optimum pH and temperature for the activity of the FHE are 7 and 25 °C, respectively. We studied the influence of metal ions and inhibitors on the enzyme activity. The enzyme was strongly activated by Mg2+ whereas Hg2+ and phenylmethyl sulfonyl fluoride (PMSF) inhibited the enzyme. The kinetic parameters, Km and Vmax for fenamiphos hydrolysis were estimated to be 584.15 ± 16.22 µM and 6.46 ± 0.13 µM min-1, respectively. The FHE was functionally active against its original substrate (2-phosphoglycerate) with Km value of 5.82 ± 1.42 µM and Vmax of 4.2 ± 0.1 µM min-1. This enzyme has great potential for its application in the detoxification of fenamiphos and its warfare homologs. To our knowledge, this is the first report on the purification of fenamiphos hydrolysing enzyme.

Phytoremediation efficacy assessment of polycyclic aromatic hydrocarbons contaminated soils using garden pea (Pisum sativum) and earthworms (Eisenia fetida).

Endpoint assessment using biological systems in combination with the chemical analysis is important for evaluating the residual effect of contaminants following remediation. In this study, the level of residual toxicity of polycyclic aromatic hydrocarbons (PAHs) after 120 days of phytoremediation with five different plant species:- maize (Zea mays), Sudan grass (Sorghum sudanense), vetiver (Vetiveria zizanioides), sunflower (Helianthus annuus) and wallaby grass (Austrodanthonia sp.) has been evaluated by ecotoxicological tests such as root nodulation and leghaemoglobin assay using garden pea (Pisum sativum) and acute, chronic and genotoxicity assays using earthworm (Eisenia fetida). The phytoremediated soil exhibited lesser toxicity supporting improved root nodulation and leghaemoglobin content in P. sativum and reducing DNA damage in E. fetida when compared to contaminated soil before remediation. Also, the results of the ecotoxicological assays with the legume and earthworm performed in this study complemented the results obtained by the chemical analysis of PAHs in phytoremediated soil. Therefore, these findings provide a basis for a framework in which remediation efficacy of PAHs-contaminated sites can be evaluated effectively with simple ecotoxicological bioassays using legumes and earthworms.

Low molecular weight organic acids enhance the high molecular weight polycyclic aromatic hydrocarbons degradation by bacteria.

The biodegradation potential of three bacterial cultures isolated from the rhizosphere of maize (Zea mays) and Sudan grass (Sorghum sudanense) grown in PAHs contaminated soils to degrade benzo[a]pyrene (BaP) and pyrene (PYR) was assessed. Of the three bacterial cultures isolated, two belonged to Gram-positive bacteria of phylum Actinobacteria namely Arthrobacter sp. MAL3 and Microbacterium sp. MAL2. The Gram-negative bacterial culture was Stenotrophomonas sp. MAL1, from the phylum Proteobacteria. The cultures were grown in the presence of BaP and PYR as sole carbon sources and with the addition of low molecular weight organic acids (LMWOAs) mixture. After 10-14 days of exposure, all the bacterial isolates exhibited a complete degradation of PYR with the addition of LMWOAs mixture, whereas only 38.7% of BaP was degraded by Stenotrophomonas sp. MAL1 with the addition of LMWOAs mixture. In addition, enhanced PAHs biodegradation by bacterial culture was observed when the PAHs present as mixture (BaP + PYR) with the addition of LMWOAs. Dioxygenase genes were detected in Stenotrophomonas sp. MAL1 (phnAC), and Arthrobacter sp. MAL3 (nidA and PAH-RHDα). Therefore, this study provides new insights on the influence of LMWOAs in enhancing the degradation of high molecular weight (HMW) PAHs in soil by rhizosphere bacterial cultures.

Metabolomics reveals defensive mechanisms adapted by maize on exposure to high molecular weight polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are an important group of persistent organic pollutants. Using plants to remediate PAHs has been recognized as a cost-effective and environmentally friendly technique. However, the overall impact of PAHs on the regulation of plant metabolism has not yet been explored. In this study, we analyzed the alteration in the maize (Zea mays L.) metabolome on exposure to high molecular weight PAHs such as benzo[a]pyrene (BaP) and pyrene (PYR) in a hydroponic medium, individually and as a mixture (BaP + PYR) using GC-MS. The differences in the metabolites were analyzed using XCMS (an acronym for various forms (X) of chromatography-mass spectrometry), an online-based data analysis tool. A significant variation in metabolites was observed between treatment groups and the unspiked control group. The univariate, multivariate and pathway impact analysis showed there were more significant alterations in metabolic profiles between individual PAHs and the mixture of BaP and PYR. The marked changes in the metabolites of galactose metabolism and aminoacyl tRNA biosynthesis in PAHs treated maize leaves exhibit the adaptive defensive mechanisms for individual and PAHs mixture. Therefore, the metabolomics approach is essential for an understanding of the complex biochemical responses of plants to PAHs contaminants. This knowledge will shed new light in the field of phytoremediation, bio-monitoring, and environmental risk assessment.

Comparison of plants with C3 and C4 carbon fixation pathways for remediation of polycyclic aromatic hydrocarbon contaminated soils.

The phytoremediation technique has been demonstrated to be a viable option for the remediation of polycyclic aromatic hydrocarbons (PAHs) contaminated sites. This study evaluated the potential applicability of plants with C3 and C4 carbon fixation pathways for the phytoremediation of recalcitrant high molecular weight (HMW) PAHs contaminated soil. A 60 and 120-day greenhouse study was conducted which showed higher degradation of HMW PAHs in soil grown with C4 plants when compared to C3 plants. Also, no PAHs were detected in the maize cobs, sunflower, wallaby, and Sudan grass seeds at the end of the experiment. The effect of plants in modifying the microbial community and dynamics in the rhizosphere was also examined by measuring soil biochemical properties such as dehydrogenase activity and water-soluble phenols. The results demonstrate a substantial difference in the microbial populations between planted and unplanted soils, which in turn facilitate the degradation of PAHs. To the best of our knowledge, this study for the first time evaluated the phytoremediation efficacy through the A. cepa cyto- and genotoxicity assay which should be considered as an integral part of all remediation experiments.

Impact of plant photosystems in the remediation of benzo[a]pyrene and pyrene spiked soils.

The phytoremediation potential of 14 different plant species belonging to C3 and C4 carbon fixation pathway for soils spiked with polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and pyrene (PYR) was investigated. A glasshouse experiment was conducted to measure the changes in morphological, physiological, biochemical parameters and the bioaccumulation and biodegradation ability of the plants in soils spiked with 48 and 194 mg kg-1 of B[a]P and PYR, respectively. The per cent removal efficacy of B[a]P and PYR by the tested plant species over a period of 50 days was from 6 to 26% and 14 to 40% respectively. The maximum removal of both B[a]P and PYR was observed in Sudan grass (C4), vetiver (C4), maize (C4), and sunflower (C3). In terms of accumulation in root and shoot, the concentration of PYR was higher in both C3 and C4 plant species when compared to B[a]P. Overall the results indicated that C4 plants were more efficient than their C3 counterparts in terms of morphological, physiological, biochemical and degradation ability of PAHs.