Recycled tire rubber materials in the spotlight. Determination of hazardous and lethal substances.

One way of recycling end-of-life tires is by shredding them to obtain crumb rubber, a microplastic material (<0.5 mm), used as infill in artificial turf sports fields or as playground flooring. There is emerging concern about the health and environmental consequences that this type of surfaces can cause. This research aims to develop an analytical methodology able to determine 11 compounds of environmental and health concern, including antiozonants such as N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine (6PPD) or N, N´-diphenyl-1,4-phenylenediamine (DPPD), and vulcanization and crosslinking agents, such as N-cyclohexylbenzothiazole-2-sulfenamide (CBS), 1,3-di-o-tolylguanidine (DTG) or hexamethoxymethylmelamine (HMMM) from tire rubber. Ultrasound assisted extraction followed by liquid chromatography coupled to tandem mass spectrometry (UAE-LC-MS/MS) is validated demonstrating suitability. The methodology is applied to monitor the target compounds in forty real crumb rubber samples of different origin including, football pitches, outdoor and indoor playgrounds, urban pavements, commercial samples, and tires. Several alternative infill materials, such as sand, cork granulates, thermoplastic elastomers and coconut fibres, are also collected and analysed. All the target analytes are identified and quantified in the crumb rubber samples. The antiozonant 6PPD is present at the highest concentrations up to 0.2 % in new synthetic fields. The tire rubber-derived chemical 6PPD-quinone (2-((4-methylpentan-2-yl)amino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione), recently linked to acute mortality in salmons, is found in all types of crumb rubber samples attaining concentrations up to 40 µg g-1 in football pitches. The crosslinking agent HMMM is detected in most of the playing surfaces, at concentrations up to 36 µg g-1. The tested infill alternatives are free of most of the target compounds. To the best of our knowledge, this study is the largest study considering the target compounds in tire rubber particles and the first to focus on these compounds in playgrounds including the analysis of HMMM, 6PPD-quinone and DTG in crumb rubber used as an infill material.

Assessment of the bioaccessibility of PAHs and other hazardous compounds present in recycled tire rubber employed in synthetic football fields.

Recycled tire crumb rubber (RTCR) surfaces contain harmful and carcinogenic substances, which can be ingested by the users of these facilities, mainly athletes and children. In this work, the potential in-vitro oral bioaccessibility of eighteen polycyclic aromatic hydrocarbons (PAHs) from RTCR employed as infill in synthetic football fields was studied in human synthetic body fluids (saliva, gastric, duodenal and bile), prepared according the Unified Bioaccessibility Method. Solid-phase extraction (SPE) using commercial sorbents and a new green material based on cork (cork industry by-product) were used to isolate the bioaccessible PAHs before gas chromatography-tandem mass spectrometry analysis. The method was optimized and validated attending the analytical figures of merit. The feasibility of cork biosorbent for the extraction of the compounds was demonstrated, as well as the suitability of the UBM method to perform the digestion with good precision. The application to real samples collected from football fields demonstrated the presence of 17 of the 18 target PAHs in the biofluids. Most volatile PAHs such as NAP, ACY, ACE, FLU, PHN and ANC, achieved the highest bioaccessibility percentage levels. The carcinogenic B[a]P was detected in 75 % of the samples at concentrations up to 2.5 ng g-1 (bioaccessible fraction). Children exposure assessment was carried out to identify potential risk. Other hazardous and environmentally problematic compounds such as N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone), recently related with the dead of coho salmon, and hexamethoxymethylmelamine (HMMM), among others, were also detected. This is the first study in which the bioaccesibility from real crumb rubber samples of 15 out of the 16 PAHs considered as priority pollutants by the United States Environmental Protection Agency (EPA) and the presence of 6PPD-quinone and HMMM in the bioaccessible fractions is reported.

Green methodology based on active air sampling followed by solid phase microextraction and gas chromatography-tandem mass spectrometry analysis to determine hazardous substances in different environments related to tire rubber.

A fast, efficient, and simple air sampling methodology was developed to study a high number of volatile and semivolatile organic compounds in air above tire rubber materials and surfaces made of recycled tire rubber. The proposed method, based on active sampling (solid-phase extraction, SPE) using a small quantity of sorbent material (25 mg) followed by solid-phase microextraction (SPME) and gas chromatography-tandem mass spectrometry analysis, was developed with the aim of determining 40 organics substances including polycyclic aromatic hydrocarbons (PAHs), plasticizers, antioxidants, and vulcanization agents. An experimental design was carried out to study the influence of main factors such as type of SPME fibre, solvent addition, headspace volume, stirring, as well as the factor interactions. Method performance showed good linearity in a broad concentration range (0.05 to 200 ng m-3, for most compounds), with coefficients of determination (R2) higher than 0.9900. Whole method precision (≤ 16 %) and accuracy were also satisfactory, obtaining quantitative recoveries (mean values between 80 and 110 % in most cases). Limits of detection and quantification have also been calculated, yielding values of sub ng m-3 for most compounds. The validated method was applied to outdoor and indoor air environments including playgrounds, football pitches and warehouses showing the presence of most target compounds in the samples achieving high levels for some PAHs (concentrations up to 51 ng m-3), benzothiazole (BTZ), diisobutyl- dibutyl- and di-(2-ethylhexyl)- phthalate, among others, reaching concentrations up to hundreds of ng m-3 (BTZ). This is the first time that the combination of techniques SPE and SPME is applied for these families of chemicals, and it is also the first time that this approach is proposed for the simultaneous multiclass compound extraction of substances of different chemical families. The whole sampling and extraction procedure is performed in a short period of time (61 min) allowing high throughput. The elimination of the use of organic solvents and waste generation by using only 25 mg of sorbent and a SPME fibre than are both reused makes the method sustainable and in consonance with the principles of the green chemistry. The method can be implemented in any routine lab and easily automated using a SPME autosampler.

Global evaluation of the chemical hazard of recycled tire crumb rubber employed on worldwide synthetic turf football pitches.

Social and environmental concern about the use of crumb rubber from end-of-life car tires in the construction of different sport and recreational facilities is increasing due to the presence of hazardous compounds. The aim of this research was the assessment of 42 organic chemicals, including polycyclic aromatic hydrocarbons (PAHs), phthalates, adipates, antioxidants and vulcanisation agents in a large number of infill samples (91) from synthetic turf football pitches of diverse characteristics and geographical origin. Samples were taken worldwide, in 17 countries on 4 continents, to show the global dimension of this problem. Ultrasound assisted extraction was employed to extract the target compounds, followed by gas chromatography coupled to tandem-mass spectrometry (UAE-GC-MS/MS). Seventy-eight crumb rubber samples as well as thirteen samples of alternatives materials, such as cork granulates, thermoplastic elastomers and coconut fibre, were analyzed. The results highlight the presence of all target PAH in most rubber samples at concentrations up to µg g-1, including the eight ECHA (European Chemicals Agency) PAHs considered as carcinogenic, and anthracene (ANC), pyrene (PYR) and benzo[ghi]perylene (B[ghi]P), catalogued as substances of very high concern (SVHC). Endocrine disruptors such as some plasticizers (mainly phthalates), and other compounds like benzothiazole (BTZ) and 2-mercaptobenzothiazole (MBTZ) were found reaching the mg g-1 level. This confirms the presence of the hazardous substances in the recycled crumb rubber samples collected all around the world. Three crumb rubber samples exceeded the limit of 20 µg g-1 for the sum of the eight ECHA PAHs. Regarding the chemical composition of other infill alternatives, cork appears to be adequate, while the thermoplastic elastomers contained high levels of some plasticizers. In addition, the plastic infill as well as the crumb rubber both are microplastics. Microplastics are considered contaminants of emerging concern since they do not biodegrade and remain in the environment for a long time.

Evaluating the Presence and Contents of Phytochemicals in Honey Samples: Phenolic Compounds as Indicators to Identify Their Botanical Origin.

Honey is a natural product well known for its beneficial properties. It contains phytochemicals, a wide class of nutraceuticals found in plants, including compounds with highly demonstrated antimicrobial and antioxidant capacities as phenolic compounds and flavonoids. The main goal of this work is the development of a miniaturized and environmentally friendly methodology to obtain the phenolic profile of Galician honeys (Northwest Spain) from different varieties such as honeydew, chestnut, eucalyptus, heather, blackberry and multi-floral. The total phenolic content (TPC) and antioxidant activity (AA) were also evaluated. As regards sample preparation, miniaturized vortex (VE) and ultrasound assisted extraction (UAE) employing aqueous-based solvents were performed. Individual quantification of 41 target phenolic compounds was carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results revealed the presence of 25 phenolic compounds in the 91 analyzed samples, reaching concentrations up to 252 µg g-1. Statistical tools such as analysis of variance (ANOVA) and principal component analysis (PCA) were employed to obtain models that allowed classifying the different honeys according to their botanical origin. Obtained results, based on TPC, AA and ∑phenolic compounds showed that significant differences appeared depending on the honey variety, being several of the identified phenol compounds being responsible of the main differentiation.

Miniaturized active air sampling method for the analysis of tire rubber pollutants from indoor and outdoor places.

An effective, quick, and sustainable air analysis method was developed to analyze 41 volatiles and semivolatile organic compounds present in tire rubber and crumb rubber materials. The proposed method, based on active sampling using a sorbent material followed by an ultrasound assisted extraction, was developed with the aim of obtaining a fast and simple procedure to determine polycyclic aromatic hydrocarbons, plasticizers, antioxidants, and vulcanization agents in air. A small amount of sorbent (25 mg) was used, and the analytes were recovered in only 1 mL of solvent. An experimental design was applied to study the influence of main factors such as type of sorbent and type of solvent, extraction technique (ultrasound-assisted extraction and vortex extraction), extraction time, as well as the factor interactions. Under optimal conditions, no breakthrough occurs in the studied interval (up to 4 m3 ). Linearity was demonstrated in a wide concentration range. Accuracy of the total sampling-extraction analysis was evaluated obtaining satisfactory recoveries as well as good precision. The method was successfully applied to different outdoor and indoor air environments, including a recycled rubber synthetic turf football pitch.

Hazardous compounds in recreational and urban recycled surfaces made from crumb rubber. Compliance with current regulation and future perspectives.

Crumb rubber obtained from scrap tires is greatly employed for the construction of different facilities for sport, recreational and other uses. However, in recent years the concern about their safety and the related adult and children exposure to these surfaces is growing. This study aims a thorough chemical characterization encompassing 42 hazardous compounds, including polycyclic aromatic hydrocarbons (PAHs), phthalates, adipates, antioxidants and vulcanization agents in a wide range of crumb rubber from different surfaces. For the extraction of the target compounds, a method based on ultrasound-assisted extraction followed by gas chromatography-tandem mass spectrometry (UAE-GC-MS/MS) has been validated. Forty crumb rubber samples coming from synthetic turf football pitches, outdoor and indoor playgrounds, urban pavements, commercial tiles and granulates, and scrap tires, were analyzed. In addition, green alternative materials, such as sand and artificial turf based on cork granulate infill were included to compare the levels of the target compounds with those of crumb rubber. Most of the analyzed recycled surfaces meet the recent limits proposed by the European Commission for rubber granulates and mulches, although they exceed in several cases the maximum levels allowed for rubber consumer products. Besides, most of the other target compounds, including several of them considered as endocrine disruptors, were detected in the analyzed samples, reaching parts per million concentrations.

Evaluation of chemicals of environmental concern in crumb rubber and water leachates from several types of synthetic turf football pitches.

Nowadays concern exists about the safety for both football players and the environment of recycled tire rubber used as infill in synthetic turf football pitches. In this study 40 target compounds, including polycyclic aromatic hydrocarbons (PAHs), plasticizers, antioxidants and vulcanization agents were determined in 50 synthetic football pitches of diverse characteristics to estimate environmental risks. This is the first study of crumb rubber sport facilities in Portugal. Analyses were performed by ultrasound-assisted extraction followed by gas chromatography-tandem mass spectrometry (UAE-GC-MS/MS). To evaluate the transfer of the target chemicals from the crumb rubber to the runoff water, water leachates collected from several football pitches were analyzed by solid-phase microextraction (SPME-GC-MS/MS). In addition, lab-scale runoff simulation experiments were performed to assess whether a persistent inflow of the target compounds from the football pitches into the runoff water wcould exist. Results revealed the presence of most of the target PAHs in crumb rubber at total concentrations up to 57 µg g-1, next to a high number of plasticizers and vulcanization agents. Runoff water collected from the football pitches contained up to 13 PAHs as well as other chemicals of environmental concern. In addition, continuous leaching of chemicals from the crumb rubber to the surrounding water was demonstrated. The transfer of target chemicals into the runoff water poses a potential risk for the aquatic environment.

Galectin-3 alters the lateral mobility and clustering of ß1-integrin receptors.

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5ß1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased ß1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins.

Protecting group-free immobilization of glycans for affinity chromatography using glycosylsulfonohydrazide donors.

A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources.