Chemiluminescent fingerprints from airborne particulate matter: A luminol-based assay for the characterization of oxidative potential with kinetical implications.

In this study, a new chemiluminescent method based on the dependence of luminol light emission induced by free radicals in airborne particulate matter (PM) is proposed as a screening assay for the rapid characterization of samples from different sources based on their redox properties. This parameter is considered critical for assessing particulate matter toxicity and its impacts on human health. We propose a cell-free, luminescent assay to evaluate the redox potential of particulate matter directly on the filters employed to collect it. A joint chemometric approach based on Principal Component Analysis and Hotelling Analysis was applied to quickly sort out ambient particulate samples with a significantly different light emission profile caused by Luminol reaction. Based on Spearman correlation analysis, the association of the samples light emission intensity with their chemical composition and emission sources was attempted. The overall methodology was tested with certified reference materials and applied to two series of particulate matter samples previously subjected to thorough chemical speciation and subsequent source apportionment. The results show the effectiveness of the luminescent method, allowing the quick assessment of particulate matter oxidative potential, but providing further evidence on the complexity of the oxidative potential determination in this kind of samples. The chemometric processing of the whole dataset clearly highlights the distinct behavior among the two series of samples, the certificate standard reference materials, and the blank controls, supporting the suitability of the approach.

Unburned Tobacco Cigarette Smoke Alters Rat Ultrastructural Lung Airways and DNA.

INTRODUCTION: Recently, the Food and Drug Administration authorized the marketing of IQOS Tobacco Heating System as a Modified Risk Tobacco Product based on an electronic heat-not-burn technology that purports to reduce the risk. METHODS: Sprague-Dawley rats were exposed in a whole-body mode to IQOS aerosol for 4 weeks. We performed the chemical characterization of IQOS mainstream and we studied the ultrastructural changes in trachea and lung parenchyma of rats exposed to IQOS stick mainstream and tissue pro-inflammatory markers. We investigated the reactive oxygen species amount along with the markers of tissue and DNA oxidative damage. Moreover, we tested the putative genotoxicity of IQOS mainstream through Ames and alkaline Comet mutagenicity assays. RESULTS: Here, we identified irritating and carcinogenic compounds including aldehydes and polycyclic aromatic hydrocarbons in the IQOS mainstream as sign of incomplete combustion and degradation of tobacco, that lead to severe remodelling of smaller and largest rat airways. We demonstrated that IQOS mainstream induces lung enzymes that activate carcinogens, increases tissue reactive radical concentration; promotes oxidative DNA breaks and gene level DNA damage; and stimulates mitogen activated protein kinase pathway which is involved in the conventional tobacco smoke-induced cancer progression. CONCLUSIONS: Collectively, our findings reveal that IQOS causes grave lung damage and promotes factors that increase cancer risk. IMPLICATIONS: IQOS has been proposed as a safer alternative to conventional cigarettes, due to depressed concentration of various harmful constituents typical of traditional tobacco smoke. However, its lower health risks to consumers have yet to be determined. Our findings confirm that IQOS mainstream contains pyrolysis and thermogenic degradation by-products, the same harmful constituents of traditional cigarette smoke, and, for the first time, we show that it causes grave lung damage and promotes factors that increase cancer risk in the animal model.

Delta9-THC determination by the EU official method: evaluation of measurement uncertainty and compliance assessment of hemp samples.

Hemp cultivation is living a period of renewed interest worldwide after long years of opposition and abandonment. The European Union (EU) allows and subsidizes the growing of fiber and oilseed cultivars of Cannabis sativa L. with respect to the THC content limit of 0.2%. The EU method for the quantitative determination of Δ9-tetrahydrocannabinol (THC) content in hemp varieties provides to apply a tolerance of 0.03 g of THC per 100 g of sample concerning compliance assessment to that limit. However, the method does not report any precision data, especially useful as a function of THC content to evaluate measurement uncertainty and therefore to establish the conformity of hemp at different THC legal limits. Measurement uncertainty of the method by both bottom-up and top-down approach, besides repeatability and reproducibility, was investigated and estimated in the THC concentration range 0.2-1.0%, which includes the different legal limits set out for hemp around the world. We proposed Decision Rules for conformity of hemp showing that a non-compliant declaration beyond reasonable doubt should be stated when the THC content, as a mean result on a duplicate analysis, exceeds the limit by at least 11-15%, depending on THC limit. We highlighted other issues concerning practical aspects of hemp analysis, from sampling to evaluation of results, as well as the need to carry out collaborative studies on the EU method.

Glucocorticoids in Freshwaters: Degradation by Solar Light and Environmental Toxicity of the Photoproducts.

The photodegradation process of seven glucocorticoids (GCs), cortisone (CORT), hydrocortisone (HCORT), betamethasone (BETA), dexamethasone (DEXA), prednisone (PRED), prednisolone (PREDLO) and triamcinolone (TRIAM) was studied in tap and river water at a concentration close to the environmental ones. All drugs underwent sunlight degradation according to a pseudo-first-order decay. The kinetic constants ranged from 0.00082 min-1 for CORT to 0.024 min-1 for PRED and PREDLO. The photo-generated products were identified by high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The main steps of the degradation pathways were the oxidative cleavage of the chain 17 for CORT, HCORT and the rearrangement of the cyclohexadiene moiety for the other GCs. The acute and chronic toxicity of GCs and of their photoproducts was assessed by the V. fischeri and P.subcapitata inhibition assays. The bioassays revealed no significant differences in toxicity between the parent compounds and their photoproducts, but the two organisms showed different responses. All samples produced a moderate acute toxic effect on V. fisheri and no one in the chronic tests. On the contrary, evident hormesis or eutrophic effect was produced on the algae, especially for long-term contact.

The pursuit of good microbiological conditions in domestic softeners: a new improvement.

Effective resin disinfection is mandatory to ensure the microbiological quality of water treated by domestic softeners. The wet and sometimes warm environment inside the softener is ideal for bacteria growth. Our research was focused on the evaluation of the microbial quality of water from softeners sanitized by chlorine solutions or by electrolytic systems. We employed the heterotrophic plate count and specific tests to monitor the presence of opportunistic and pathogenic bacteria (Pseudomonas aeruginosa, Escherichia coli, enterococci, and coliforms). Completely new devices were equipped with a commercially available electrolytic system or with a newly patented one or sanitized by automatic or manual addition of chlorine solutions. In all cases, the contamination was reduced, not completely avoided. In particular, the patented electrolytic system significantly reduced bacterial proliferation in strongly contaminated devices. Our data confirm the difficulties encountered to solve the problem of microbiological quality of softened water and offer encouraging information on new possible solutions.

Airborne particulate matter biotoxicity estimated by chemometric analysis on bacterial luminescence data.

In this work, PM10 samples previously subjected to thorough chemical speciation and receptor modelling, have been investigated for their bio-toxicity using an inhibition test based on bacterial luminescence modulation when in contact with airborne particulate samples. The variation of light emission intensity from a luminescent bacteria strain, the Photobacterium phosphoreum, is proposed as an efficient proxy for the quantification of bio-toxic effects induced by airborne particulate matter. PM10 samples characterized by definite levels of pollutants from the pertaining air shed were found to induce a decrease in the bacterial bioluminescence intensity, expressed as percentage of Inhibition Ratio (IR%). This behaviour suggests the decay of this energy-consuming activity because of a toxic effect. Cluster analysis on chemical composition and IR% data provides evidence of a statistically significant association between the adverse effects on living cells and the range of specific chemical species in PM10.

Sunlight-induced degradation of fluoroquinolones in wastewater effluent: Photoproducts identification and toxicity.

The photodegradation of Ciprofloxacin (CIP), Enrofloxacin (ENR), Danofloxacin (DAN), Marbofloxacin (MAR) and Levofloxacin (LEV), five widely used fluoroquinolones (FQs), was studied in urban WWTP secondary effluent, under solar light. The degradation profiles and the kinetic constants were determined at the micrograms per litre levels (20-50 µg L(-1)). The photo-generated products were identified by high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The toxicity of the photoproducts was assessed by Vibrio fischeri light emission inhibition assay performed on irradiated and not-irradiated FQs solutions, at environmentally significant concentrations. Attention was focused on the evaluation of the photoproducts contribution to the overall biotoxic effect of these emerging pollutants. Data from chronic exposure experiments (24-48 h) were primarily considered. Results confirmed the major usefulness of chronic toxicity data with respect to the acute assay ones and proved the not negligible biotoxicity of the FQs photodegradation products.

Field detection capability of immunochemical assays during criminal investigations involving the use of TNT.

The capability to collect timely information about the substances employed on-site at a crime scene is of fundamental importance during scientific investigations in crimes involving the use of explosives. TNT (2,4,6-trinitrotoluene) is one of the most employed explosives in the 20th century. Despite the growing use of improvised explosives, criminal use and access to TNT is not expected to decrease. Immunoassays are simple and selective analytical tests able to detect molecules and their immunoreactions can occur in portable formats for use on-site. This work demonstrates the application of three immunochemical assays capable of detecting TNT to typical forensic samples from experimental tests: an indirect competitive ELISA with chemiluminescent detection (CL-ELISA), a colorimetric lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles label, and a chemiluminescent-LFIA (CL-LFIA). Under optimised working conditions, the LOD of the colorimetric LFIA and CL-LFIA were 1 µg mL(-1) and 0.05 µg mL(-1), respectively. The total analysis time for LFIAs was 15 min. ELISA proved to be a very effective laboratory approach, showing very good sensitivity (LOD of 0.4 ng mL(-1)) and good reproducibility (CV value about 7%). Samples tested included various materials involved in controlled explosions of improvised explosive devices (IEDs), as well as hand swabs collected after TNT handling tests. In the first group of tests, targets covered with six different materials (metal, plastic, cardboard, carpet fabric, wood and adhesive tape) were fixed on top of wooden poles (180 cm high). Samples of soil from the explosion area and different materials covering the targets were collected after each explosion and analysed. In the second group of tests, hand swabs were collected with and without hand washing after volunteers simulated the manipulation of small charges of TNT. The small amount of solution required for each assay allows for several analyses. Results of immunoassays confirmed that they were suitable to detect post-blast residues in soil and target materials and post transfer residues on hands, allowing further confirmation by more selective techniques. ELISA and LFIAs results obtained from the same solution were consistently in good agreement, and were confirmed by gas chromatography coupled to mass spectrometry (GC-MS). The reported immunoassays data demonstrates the suitability of LFIAs as on-site rapid and effective assays to detect TNT traces. The CL-ELISA proved useful in obtaining very sensitive detection in forensic investigations and testing, while CL-LFIA had performances in between LFIA and CL-ELISA.

Bisphenol A determination in baby bottles by chemiluminescence enzyme-linked immunosorbent assay, lateral flow immunoassay and liquid chromatography tandem mass spectrometry.

Two immunoassays, a Lateral Flow ImmunoAssay (LFIA) based on colloidal gold nanoparticle labels and an indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA), were developed and a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was optimized to assess the possible release of bisphenol A (BPA, 4,4'-isopropylidenediphenol) from different plastic baby bottles treated with simulating solutions. Coating conjugate concentration, anti-BPA antibody dilution, incubation time of the primary and secondary antibodies, and tolerance to different organic solvents were optimized to obtain the best performance of the ELISA with chemiluminescent end-point detection. The influence of different buffers on LFIA performance was also evaluated. Both methods showed good repeatability (mean CV value around 13%) and sensitivity. Reproducibility tests for CL-ELISA gave a mean CV value of about 25%. The IC50 and Limit of Detection (LOD) values of CL-ELISA were 0.2 and 0.02 ng mL(-1), respectively. The LOD of LFIA was 0.1 µg mL(-1). A LC-MS/MS method was also optimized. The separation was performed in a C18 column with a triple-quadrupole mass spectrometer with electrospray ionisation interface. The method showed a good linearity in the range 2 to 500 ng mL(-1), with a regression coefficient of 0.998. In the simulating solutions the detection and quantification limits, calculated by the signal to noise level of 3 (S/N = 3), were 5.8 ng mL(-1) and 17.4 ng mL(-1), respectively. This limit of quantification was about 3 and 35 times lower than the permitted limits set by the official method CEN/TS 13130-13 (0.05 µg mL(-1)) and by the Directive 2004/19/EC (0.6 µg mL(-1)), respectively. The methods were applied to determine BPA release from baby bottles, performing repeated procedures according to EU and national regulations. The results demonstrated that no BPA migration from the tested plastic materials occurred with only one exception. The migrated amount, above the regulatory limits, was detected by all the mentioned assays.

Competitive amperometric immunosensor based on covalent linking of a protein conjugate to dendrimer-functionalised nanogold substrate for the determination of 2,4,6-trinitrotoluene.

A new amperometric immunosensor for 2,4,6-trinitrotoluene based on the working principle of competitive enzyme-linked immunosorbent assay was developed and characterised. An electrodeposited nanogold substrate was functionalised by deposition of self-assembled monolayers of 2-aminoethanethiol as linkers for the subsequent immobilisation of polyamidoaminic dendrimers. Our approach makes use of those dendrimers to anchor a trinitrobenzene-ovalbumin conjugate on the electrode surface. The immunosensor was tested and validated for the determination of 2,4,6-trinitrotoluene showing high selectivity with respect to other nitroaromatic compounds, a limit of detection of 4.8 ng/mL and a limit of quantitation of 6 ng/mL. The immunosensor was tested for the quantification of the analyte in spiked soils and in a real sample of post-blast soil, evidencing a good recovery rate (113 %).

Chemiluminescent ELISA for the BTEX determination in water and soil.

An indirect competitive chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the determination of benzene, toluene, ethylbenzene, and ortho-, meta-, para-xylenes (BTEX) in soil and water was developed. The assay was optimized concerning the coating conjugate concentration, anti-BTEX antiserum dilution, incubation time effect on primary and secondary antibody incubation, and temperature effect on the competitive step and tolerance to different organic solvents. The IC(50) and lower limit of the detection (LDD(90)) values were 4.6 and 0.5 microg mL(-1), respectively. While water samples could be analyzed directly, soil has to be extracted and diluted prior to immunochemical measurements. BTEX could be recovered from spiked soil with a yield higher than 60% using 5-min ultrasonication with methanol. Finally, the assay was applied to soil and water samples collected at a contaminated site in Italy, and was compared to GC-MS.

Persistence and distribution of pesticide residues in fresh agricultural food consumed in the province of Bologna.

The presence of pesticide residues in fresh vegetables and fruit have been qualitatively and quantitatively determined at the laboratories of the Regional Agency for Environmental Protection (ARPA), Division of the Province of Bologna. More than 1,700 samples have been tested by routine analyses. The possible risks for consumers have been evaluated by various parameters. The most important ones were: the amount of each residue; the respective ADI (Acceptable Daily Intake) limit; the contemporary presence of different residues; an estimation of the daily intake, based on the amount of fruit and vegetables consumed per person. It has been possible to evaluate that the daily intake of pesticide residues in the province of Bologna during the period 2003-06 resulted lower than the ADI limits concerning the vegetables. According to the information on fruit consumption the daily intake of omethoate (O,O-dimethyl S-methylcarbamoylmethyl phosphorothioate) resulted higher than its ADI limit, of dicofol (2,2,2-trichloro-1,1-bis(trichloromethyl)benzenemethanol) very close to the admitted limit, under the respective limits for all the other residues.

Monitoring of environmental pollutants by bioluminescent bacteria.

This review deals with the applications of bioluminescent bacteria to the environmental analyses, published during the years 2000-2007. The ecotoxicological assessment, by bioassays, of the environmental risks and the luminescent approaches are reported. The review includes a brief introduction to the characteristics and applications of bioassays, a description of the characteristics and applications of natural bioluminescent bacteria (BLB), and a collection of the main applications to organic and inorganic pollutants. The light-emitting genetically modified bacteria applications, as well as the bioluminescent immobilized systems and biosensors are outlined. Considerations about commercially available BLB and BLB catalogues are also reported. Most of the environmental applications, here mentioned, of luminescent organisms are on wastewater, seawater, surface and ground water, tap water, soil and sediments, air. Comparison to other bioindicators and bioassay has been also made. Various tables have been inserted, to make easier to take a rapid glance at all possible references concerning the topic of specific interest.

Plasma antioxidant capacity determination: comparative evaluation of chemiluminescent and spectrophotometric assays.

All aerobic organisms have developed different mechanisms for neutralising the free radicals, mostly produced by the monoelectronic reduction of O(2), and preventing the severe damages these can provoke. The efficiency of these mechanisms can be assessed, in different matrices, by a simple and direct chemiluminescent assay (CL) based on luminol oxidation catalysed by horseradish peroxidase. Light emission is mediated by the production of free radicals and it is inhibited after a sample addition in a way that is directly proportional to the sample total content of molecules displaying antioxidant activity. The performances of this chemiluminescent assay were compared with those of two spectrophotometric methods already applied in clinical practice. First spectrophotometric method measures, like CL assay, the total antioxidant capacity, whereas the second one determines free thiol groups content. The chemiluminescent assay has a linearity interval between 0.60 and 9.46 mumol l(-1) of Trolox (y=34.91x+3.10; r=0.999; n=5) with an imprecision, expressed as CV, of 3.8% and an inaccuracy, expressed as percentage recovery, of 109%. The first spectrophotometric method, based on the same reference standard, the Trolox molecule, has a linearity interval between 0.2 and 2.5 mmol l(-1) of Trolox (y=-0.01x+4.54; r=0.95; n=5); the thiol groups assay has a linearity interval between 0.1 and 1 mmol l(-1) of l-cysteine (y=1.68x-47.09; r=0.998; n=5). Different clinical samples of plasma from healthy individuals, obese subjects and patients with liver diseases were tested. Interesting correlations were obtained among the three methods, but no significant correlations emerged between antioxidant capacity and clinical parameters. Significant differences were there only between men and women among obese subjects and between drinkers and non-drinkers among liver disease patients.