Assessment of heavy metal and metalloid levels and screening potential of tropical plant species for phytoremediation in Singapore.

Heavy metal or metalloid contamination is a common problem in soils of urban environments. Their introduction can be due to unpremeditated anthropogenic activities like atmospheric deposition produced by diffuse sources, construction activities and landscape maintenance. Phytoremediation is a rapidly evolving, sustainable approach to remediate the contaminated lands where metals and metalloids are highly persistent in the environment. The present work sets out to determine the level of 12 heavy metals and metalloids (As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sb and Zn) in soil and their accumulation by plant foliage found in nature parks and industrial sites in Singapore. The latter also involve the investigation of the remediation capacity of selected tropical plant species found at the sampling sites. The study is done using digestion and inductively coupled plasma-optical emission spectrometry. Eleven soil sampling sites across Singapore with 300 sampling points were selected, where soil (0-10 cm) and plant foliage samples were collected. Bioconcentration factors were determined to assess the phytoremediation potential of the collected plant species. Toxicity risk of heavy metals were assessed by comparing the target and intervention values from the soil quality guidelines by the Dutch Standard. Results of the study revealed there were regions where levels of heavy metals and metalloids were relatively high and could affect the environment and the health of flora and fauna in Singapore. Our study discovered that there were available tropical plant species (e.g., wildflowers, ferns and shrubs) which could potentially play a significant role in the remediation of contaminated lands that could open up a huge possibility of developing a sustainable and environmentally-friendly way of managing this emerging urban problem. Results showed that 12 plant species, including hyperaccumulator like Pteris vittata, Centella asiatica, were effective for the accumulation of heavy metals and metalloids.

Phytoremediation: A Promising Approach for Revegetation of Heavy Metal-Polluted Land.

Heavy metal accumulation in soil has been rapidly increased due to various natural processes and anthropogenic (industrial) activities. As heavy metals are non-biodegradable, they persist in the environment, have potential to enter the food chain through crop plants, and eventually may accumulate in the human body through biomagnification. Owing to their toxic nature, heavy metal contamination has posed a serious threat to human health and the ecosystem. Therefore, remediation of land contamination is of paramount importance. Phytoremediation is an eco-friendly approach that could be a successful mitigation measure to revegetate heavy metal-polluted soil in a cost-effective way. To improve the efficiency of phytoremediation, a better understanding of the mechanisms underlying heavy metal accumulation and tolerance in plant is indispensable. In this review, we describe the mechanisms of how heavy metals are taken up, translocated, and detoxified in plants. We focus on the strategies applied to improve the efficiency of phytostabilization and phytoextraction, including the application of genetic engineering, microbe-assisted and chelate-assisted approaches.

Solidification and multi-cytotoxicity evaluation of thermally treated MSWI fly ash.

Municipal solid waste incineration (MSWI) fly ash produced in waste-to-energy plants possesses a serious threat to human health. Although the traditional methods including toxicity characteristic leaching procedure and sequential extraction approach can partially evaluate the reduction of heavy metals leaching from thermally treated MSWI fly ash, the potential threat towards organisms is frequently ignored in previous literature. Considering this, herein we systematically assess the cytotoxicity of heat-treated samples using multiple cells from different biological tissues/organs for the first time. The results indicate that the leachability and transferability of heavy metals are declined after treatment. The biological assays demonstrate that the leachates from the treated residues induce lower phytotoxicity and cytotoxicity compared with the original samples. Moreover, according to the cellular responses of multiple cells to the leachates, normal cells (MC3T3-E1, HUVEC, and L929) are more tolerant to the leachates than cancerous cells (4T1, MG63), and the skin fibroblasts (L929), which often interact with the external circumstance, have the best cellular tolerance. This work provides a novel platform to determine the potential biosecurity of MSWI fly ash-derived products towards organisms, when they are served as secondary building materials in the constructional industry that may be contact with animals and human beings.

Simultaneous extraction, separation, isolation and identification of endogenous components from Etlingera elatior by pressurized matrix solid-phase dispersion using liquid chromatography-mass spectrometry.

From the analytical chemistry point-of-view, an ideal sample preparation method should be simple, rapid, automatic, selective, precise, exhaustive, reproducible and protect the analyte-of-interest from degradation. In this study, a novel sample preparation method, named pressurized matrix solid-phase dispersion (p-MSPD) extraction was developed for simultaneously extracting, separating, purifying, isolating, and analyzing endogenous components in a solid sample matrix. Etlingera elatior, a traditional medicinal plant known as the torch ginger, was applied as a sample matrix to evaluate the p-MSPD process. The entire extraction, separation, isolation, fractionation and detection were performed automatically with a commercial LC-MS system. The novel method was satisfactorily applied for the preparation of real samples without optimization, which had the ability to selectively isolate pure compounds from the solid sample matrix for further NMR analysis. Therefore, the method is recommended for quality control of traditional medicines, research efforts when sample amounts are limited, and laboratories that have ordinary LC-MS instrumentation.

Axonopus compressus (Sw.) Beauv.: A potential biomonitor for molybdenum in soil pollution.

Phytoremediation is an emerging technology that utilizes plants to remediate contaminated environments. In this study, Axonopus compressus (Sw.) Beauv, a fast-growing and hardy groundcover with wide geographical distribution, was exposed to soil Mo treatments ranging from 100 to 1000 mg/kg under tropical greenhouse conditions for five weeks. Generally, Mo accumulation increased as the concentration of Mo in the soil increased. The species was found to accumulate about 4000 mg/kg of Mo without exhibiting severe physiological stress at 600 mg/kg of soil Mo. Maximum accumulation of 6000 mg/kg Mo was observed at the 1000 mg/kg soil Mo treatment, though with severe necrosis and eventual plant mortality. The physiological observations, Mo accumulation behavior, and a bioconcentration factor of about 1 indicated that A. compressus could be a potential biomonitor of Mo.

Solidification and Biotoxicity Assessment of Thermally Treated Municipal Solid Waste Incineration (MSWI) Fly Ash.

In the present work, thermal treatment was used to stabilize municipal solid waste incineration (MSWI) fly ash, which was considered hazardous waste. Toxicity characteristic leaching procedure (TCLP) results indicated that, after the thermal process, the leaching concentrations of Pb, Cu, and Zn decreased from 8.08 to 0.16 mg/L, 0.12 to 0.017 mg/L and 0.39 to 0.1 mg/L, respectively, which well met the limits in GB5085.3-2007 and GB16689-2008. Thermal treatment showed a negative effect on the leachability of Cr with concentrations increasing from 0.1 to 1.28 mg/L; nevertheless, it was still under the limitations. XRD analysis suggested that, after thermal treatments, CaO was newly generated. CaO was a main contribution to higher Cr leaching concentrations owing to the formation of Cr (VI)-compounds such as CaCrO4. SEM/EDS tests revealed that particle adhesion, agglomeration, and grain growth happened during the thermal process and thus diminished the leachability of Pb, Cu, and Zn, but these processes had no significant influence on the leaching of Cr. A microbial assay demonstrated that all thermally treated samples yet possessed strong bactericidal activity according to optical density (OD) test results. Among all samples, the OD value of raw fly ash (RFA) was lowest followed by FA700-10, FA900-10, and FA1100-10 in an increasing order, which indicated that the sequence of the biotoxicity for these samples was RFA > FA700-10 > FA900-10 > FA1100-10. This preliminary study indicated that, apart from TCLP criteria, the biotoxicity assessment was indispensable for evaluating the effect of thermal treatment for MSWI fly ash.

A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury.

Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As3+ and Hg2+. A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As3+ and 45 ppb for Hg2+. While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor.

Progress and development of analytical methods for gibberellins.

Gibberellins, as a group of phytohormones, exhibit a wide variety of bio-functions within plant growth and development, which have been used to increase crop yields. Many analytical procedures, therefore, have been developed for the determination of the types and levels of endogenous and exogenous gibberellins. As plant tissues contain gibberellins in trace amounts (usually at the level of nanogram per gram fresh weight or even lower), the sample pre-treatment steps (extraction, pre-concentration, and purification) for gibberellins are reviewed in details. The primary focus of this comprehensive review is on the various analytical methods designed to meet the requirements for gibberellins analyses in complex matrices with particular emphasis on high-throughput analytical methods, such as gas chromatography, liquid chromatography, and capillary electrophoresis, mostly combined with mass spectrometry. The advantages and drawbacks of the each described analytical method are discussed. The overall aim of this review is to provide a comprehensive and critical view on the different analytical methods nowadays employed to analyze gibberellins in complex sample matrices and their foreseeable trends.

Determination of monomeric composition in polyhydroxyalkanoates by liquid chromatography coupled with on-line mass spectrometry and off-line nuclear magnetic resonance.

Polyhydroxyalkanoates (PHAs) are commercially-valuable biocompatible and biodegradable polymers with many potential medical, pharmaceutical and other industrial applications. The analysis of PHA monomeric composition is especially challenging due to the broad chemical diversity of PHA monomers and lack of analytical standards to represent the chemically-diverse PHA monomer constituents. In this study, a novel strategy based on on-line liquid chromatography-mass spectrometry (LC-MS) and off-line liquid chromatography-nuclear magnetic resonance (LC-NMR) was established to quantify seven PHA monomers with available standards and used to elucidate the structures of unknown PHA monomers. The strategy was successfully applied for the determination of monomeric composition in bacterial PHAs isolated from Pseudomonads cultivated on different carbon sources after hydrolysis. The results of this work demonstrated that the newly-developed strategy was efficient, repeatable, and could have good potential to be employed for detailed analysis of PHA monomeric composition.

Analysis of phytohormones in vermicompost using a novel combinative sample preparation strategy of ultrasound-assisted extraction and solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry.

Vermicompost (VC), a widely used premium organic fertilizer, is the by-product of symbiotic interactions between earthworms and microorganisms living within them. It has been postulated that phytohormones are plausible "magic compounds" in VC that are responsible for making them such good fertilizers. Thus, a novel approach involving ultrasound-assisted extraction (UAE) and solid-phase extraction (SPE) was developed as a fast and efficient sample preparation method to screen for different classes of phytohormones in VC by liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Nine phytohormones from three different classes, including trans-zeatin (tZ), kinetin (K), N(6)-[2-isopentyl]adenine (iP), N(6)-benzyladenine (BA), N(6)-isopentenyladenosine (iPR), indole-3-acetic acid (IAA), 4-[3-indolyl]butyric acid (IBA), 1-naphthaleneacetic acid (NAA) and (+)-abscisic acid (ABA), were simultaneously screened. The extraction parameters influencing UAE efficiency were optimized to provide comparable recovery to the conventional mix-stirring (MSt) method. The optimized UAE method was subsequently applied on the analysis of phytohormones in VC, i.e. phytohormone extract was further pre-concentrated and purified using C18 and MCX SPE cartridges prior to LC-MS/MS analysis. The following phytohormones, namely iP, iPR and IAA, were detected and quantified to be 0.49, 0.53, 79.78ngg(-1), respectively; tZ was found to be below the limit of quantitation. Recoveries of 10.2%, 9.1%, 18.9% and 0.3% for tZ, iP, iPR and IAA were obtained. This is one of the few reported works for the successful detection and quantitation of cytokinins and auxins in VC, that provided the key empirical evidence to explain the growth efficacy of applying VC in promoting plant growth. Additionally, this pioneering work could potentially be applicable for the analysis of other types of organic fertilizers such as composts and activated composted materials awaiting phytohormone analyzes for quality assessment and control.

Bioconversion of styrene to poly(hydroxyalkanoate) (PHA) by the new bacterial strain Pseudomonas putida NBUS12.

Styrene is a toxic pollutant commonly found in waste effluents from plastic processing industries. We herein identified and characterized microorganisms for bioconversion of the organic eco-pollutant styrene into a valuable biopolymer medium-chain-length poly(hydroxyalkanoate) (mcl-PHA). Twelve newly-isolated styrene-degrading Pseudomonads were obtained and partial phaC genes were detected by PCR in these isolates. These isolates assimilated styrene to produce mcl-PHA, forming PHA contents between 0.05±0.00 and 23.10±3.25% cell dry mass (% CDM). The best-performing isolate was identified as Pseudomonas putida NBUS12. A genetic analysis of 16S rDNA and phaZ genes revealed P. putida NBUS12 as a genetically-distinct strain from existing phenotypically-similar bacterial strains. This bacterium achieved a final biomass of 1.28±0.10 g L(-1) and PHA content of 32.49±2.40% CDM. The extracted polymer was mainly comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanoate (C8 ), 3-hydroxydecanoate (C10 ), 3-hydroxydodecanoate (C12 ), and 3-hydroxytetradecanoate (C14 ) monomers at a ratio of 2:42:1257:17:1. These results collectively suggested that P. putida NBUS12 is a promising candidate for the biotechnological conversion of styrene into mcl-PHA.

Lab-on-a-chip for rapid electrochemical detection of nerve agent Sarin.

This paper reports a lab-on-a-chip for the detection of Sarin nerve agent based on rapid electrochemical detection. The chemical warfare agent Sarin (C4H10FO2P, O-isopropyl methylphosphonofluoridate) is a highly toxic organophosphate that induces rapid respiratory depression, seizures and death within minutes of inhalation. As purified Sarin is colourless, odourless, water soluble and a easily disseminated nerve agent, it has been used as a weapon in terrorist or military attacks. To ascertain whether potable water supplies have been adulterated with this extremely potent poison, an inexpensive, sensitive and easy to use portable test kit would be of interest to first responders investigating such attacks. We report here an amperometric-based approach for detecting trace amounts of Sarin in water samples using a screen-printed electrode (SPE) integrated in a microfluidic chip. Enzymatic inhibition was obtained by exposing the immobilised biosensor in the microfluidic platform to Sarin in water samples. With the aid of cobalt phthalocyanine modified SPE, the device could detect Sarin at part-per-billion levels with concentration as low as 1 nM. The detection method reported here represents a significant improvement over the authors'previous optical-based detection method.

Enhanced gas chromatography-mass spectrometry method for bacterial polyhydroxyalkanoates analysis.

A gas chromatography-mass spectrometry method for quantification of polyhydroxyalkanoates (PHAs), containing 4-carbon to 16-carbon monomers, even in the absence of standards, was developed. Strong linear correlations existed between PHA carbon number and retention time/response factor (R(2) ≥ 0.987). Based on the correlations, high recovery values, between 100.5% and 114.3%, were obtained for PHA polymers.

Paper-based enzyme immobilization for flow injection electrochemical biosensor integrated with reagent-loaded cartridge toward portable modular device.

Paper-based enzyme immobilization for a flow injection electrochemical biosensor integrated with a reagent-loaded cartridge toward a portable device was developed. A paper disk was immobilized with enzyme, then it was integrated in a flow cell as an electrochemical biosensor. A silicon tube reagent-loaded cartridge was integrated into the system, a complicated procedure was simplified as a one-click operation toward development for point-of-care applications. In this research, glucose oxidase (GOx) was employed as a model enzyme, silver ion as an inhibition reagent for GOx, and EDTA as a regeneration reagent. When GOx was inhibited by silver ions, glucose was introduced for electrochemical measurements before and after inhibited enzyme regeneration and the difference was caused by silver inhibition. The modular device has great potential for other applications, e.g., detection of enzyme activity and substrate. The platform based on double-test mode provided accurate results due to elimination of an average or control value in comparison with classical routine approaches.

Determination of bisphenol A and naphthols in river water samples by capillary zone electrophoresis after cloud point extraction.

As a first attempt, cloud point extraction (CPE) was developed to preconcentrate bisphenol A (BPA), α-naphthol and ß-naphthol prior to performing capillary zone electrophoresis (CZE) analysis. The parameters influencing the CPE efficiency, such as Triton X-114 concentrations, pH value, extraction time and temperature were systematically evaluated. After diluting with acetonitrile, the surfactant-rich phase of CPE can be injected directly into the CE instrument. The CZE baseline separation was achieved with running buffer (pH 9.5) composed of 50mM sodium tetraborate in 30% (v/v) methanol, and an applied voltage of 25 kV. Under the optimized CPE and CZE conditions, an preconcentration factor of 50 times could be obtained and the limit of quantification for the three analytes were found to be 1.67 µg L(-1), 0.80 µg L(-1) and 0.67 µg L(-1) for BPA, α-naphthol and ß-naphthol, respectively. The proposed methods have shown to be a green, rapid and effective approach for determination of three analytes present in river water samples.

Surfactant-assisted pressurized liquid extraction for determination of flavonoids from Costus speciosus by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC), a mode of capillary electrophoresis (CE), is considered an efficient analytical technique allowing for the reduction of organic solvent consumption during the experimental procedure. However, during sample preparation of natural products, the usage of large amount of organic solvent is generally unavoidable. In this article, therefore, a fast, simple, efficient, highly automatic and organic solvent-free sample preparation method, namely surfactant-assisted pressurized liquid extraction (PLE), was developed for the extraction of flavonoids in Costus speciosus flowers before MEKC analysis. The various experimental parameters such as the type and concentration of surfactant, and extraction time were evaluated systematically. Under the optimized conditions, the extraction efficiencies of surfactant-assisted PLE methods were comparable with Soxhlet extraction using organic solvent. The combination of surfactant-assisted PLE and MEKC was shown to be a green, rapid and effective approach for extraction and analysis of flavonoids in C. speciosus flowers.

Determination of metabolites in Uncaria sinensis by HPLC and GC-MS after green solvent microwave-assisted extraction.

Uncaria sinensis (Oliv.) Havil (Rubiaceae) has been used as an important Traditional Chinese Medicine (TCM) herb for the treatment of fevers and various nervous disorders. The major bioactive secondary metabolites from different classes of chemical compounds, i.e. organic acid, flavonoid and alkaloid, present in this TCM herb, namely catechin, caffeic acid, epicatechin and rhynchophylline, were extracted by microwave-assisted extraction (MAE) method with ultra-pure water as the extraction solvent. The optimal extraction conditions for this green solvent MAE method were found to be 100°C for 20 min. The recoveries of the compounds were found to be comparable to that of heating under reflux using ultra-pure water for 60 min. The method precision (RSD, n=6) was found to vary from 0.19% to 5.60% for the proposed method on different days for the secondary metabolites. Simultaneously, the key primary metabolites such as sucrose and phenylalanine for the biosynthesis of bioactive secondary metabolites were successfully characterized by GC-MS. Furthermore, an approach using the combination of primary and secondary metabolite profiling based on their chemical fingerprints with Principal Component Analysis (PCA) was successfully developed to evaluate the quality of U. sinensis obtained from different sources. This approach was shown to be feasible in discriminating U. sinensis from different origins and thus a potential application for the quality control of other medicinal herbs.

Paper disk on screen printed electrode for one-step sensing with an internal standard.

A point-of-care device, an ideal application is to provide a direct answer for an analysis with only a simple sample introduction. In order to meet the requirements, reagents storage, and self-calibration must be integrated on the portable device. This study describes a device with a paper disk impregnated with reagents including an internal standard and buffer on a screen printed electrode leading to accurate result in a single-step assay. We demonstrated on the applicability of this device by detection of trace lead (ppb level) in 10 µL solution based on the simultaneous (in situ) plating of bismuth and heavy metals onto electrode, which formed alloys followed by anodic stripping, Zn(II) was employed as an internal standard. We believe this approach is attractive for use in field based portable devices.