In vitro bioassays for the study of endocrine-disrupting food additives and contaminants

Endocrine-disrupting chemicals (EDCs) are capable of interfering with normal hormone homeostasis by acting on several targets and through a wide variety of mechanisms. Unwanted exposure to EDCs can lead to a wide spectrum of adverse health effects, especially when exposure is during critical windows of development. Feed and food are considered to be among the main routes of inadvertent exposure to EDCs, so there is an important need for efficient detection of EDCs in these matrices.We describe in vitro bioassays that can complement current analytical chemistry in order to detect unwanted EDCs and describe their action, emphasizing assays that can measure effects on nuclear receptor signaling or hormone production. We outline both validated and unvalidated in vitro assays currently available in the scientific community for detecting and studying the effects of EDCs, and discuss their possible role in the food-safety context. We conclude by identifying gaps in the current battery of in vitro assays available for EDCs and suggest future possibilities for development and validation.     

Trends in the detection of pharmaceuticals and endocrine-disrupting compounds by Field-Effect Transistors (FETs)

Field-effect transistors (FETs) are one of the most widely-used electronic sensors for continuous monitoring and detection of contaminants such as pharmaceuticals and endocrine-disrupting compounds at low concentrations. FETs have been successfully utilized for the rapid analysis of these environmental pollutants due to their advantageous material properties like the disposability, rapid responses and simplicity. This paper presented an up-to-date overview of applied strategies with different bio-based materials in order to enhance the analytical performances of the designed sensors. Comparison and discussion were made between characteristics of recently engineered FET bio-sensors used for the detection of famous and selected pharmaceutical compounds in the literature. The recent progress in environmental research applications, comments on interesting trends, current challenge for future research in endocrine-disrupting chemicals’ (EDCs) detection using FETs biosensors were highlighted.     

Bioassays for the detection of growth-promoting agents, veterinary drugs and environmental contaminants in food

Residues of growth-promoting agents, veterinary drugs and environmental contaminants in food products are routinely analyzed with chemical-analytical methods, using physical and spectrometric properties of a compound. Since residue limits are in general based on biological properties of compounds, bioassays offer in theory a good alternative. As a consequence, these assays are much more suitable for the detection of mixtures of compounds with common biological properties, including possibly unknown agonists. Using modern molecular biological techniques, a new generation of bioassays has been developed, showing in general a higher sensitivity and specificity for the target compounds. The CALUX (chemical activated luciferase expression) assay was developed for the detection of polyhalogenated compounds, based on their affinity for the aryl hydrocarbon (Ah) receptor. This paper focuses on the specificity of the assay. The benzimidazole compounds oxfendazole, fenbendazole, febantel, thiabendazole, mebendazole, omeprazole, lanzoprazole and benomyl were shown to give a positive response in the assay. Similar results were obtained with dexamethasone, corticosterone and cortisol, which in addition were able to enhance the response obtained with TCDD. Similarly to the flavonoids alpha- and beta-naphtoflavone, the reported Ah receptor antagonist 4-amino-3-methoxyflavone showed a strong positive response at a concentration of 400 microM, but failed to inhibit the response obtained with TCDD. It is concluded that the chances of false-negative results appear to be minimal and can be recognized. False-positive or, better, unwanted results are in theory more likely to occur. Possible solutions to avoid or detect these type of results are discussed. In general, these kinds of assays offer great possibilities for screening of food samples. In addition to the further optimization of these assays, future work should be focused on the development of rapid, sample and selective extraction procedures.     

Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications.Over the past 10 years,reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices,such as biological,environmental,food and feed samples,given that high-resolution instrumental analysis techniques ...     

Challenges in the electrochemical (bio)sensing of nonelectroactive food and environmental contaminants

Electrochemical detection of nonelectroactive contaminants can be successfully faced via the use of indirect detection strategies. These strategies can provide sensitive and selective responses often coupled with portable and user-friendly analytical tools. Indirect detection strategies are usually based on the change in the signal of an electroactive probe, induced by the presence of the target molecule at a modified electrode. This critical review aims at addressing the developments in indirect electrosensing strategies for nonelectroactive contaminants in food and environmental analysis in the last few years. Emphasis is given to the strategy design, the electrode modifiers used and the feasibility of technological transfer.     

Standard reference materials (SRMs) for determination of organic contaminants in environmental samples

For the past 25 years the National Institute of Standards and Technology (NIST) has developed certified reference materials (CRMs), known as standard reference materials (SRMs), for determination of organic contaminants in environmental matrices. Assignment of certified concentrations has usually been based on combining results from two or more independent analytical methods. The first-generation environmental-matrix SRMs were issued with certified concentrations for a limited number (5 to 10) of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Improvements in the analytical certification approach significantly expanded the number and classes of contaminants determined. Environmental-matrix SRMs currently available include air and diesel particulate matter, coal tar, marine and river sediment, mussel tissue, fish oil and tissue, and human serum, with concentrations typically assigned for 50 to 90 organic contaminants, for example PAHs, nitro-substituted PAHs, PCBs, chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs). Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Magnetically confined hydrophobic nanoparticles for the microextraction of endocrine-disrupting phenols from environmental waters

In this article, a solid-phase extraction approach, which takes advantage of the good extraction capabilities of hydrophobic magnetic nanoparticles (MNPs), is presented. The new approach involves the deposition of a thin layer of MNPs in a dedicated stirring unit based on the dual function of a mini-magnet. The system allows the extraction of the analytes in a simple and efficient way. The approach, which reduces the negative effect of the aggregation tendency of hydrophobic MNPs, is characterized for the resolution of a model analytical problem: the determination of some endocrine-disrupting phenols in water by liquid chromatography–photometric detection. All the variables involved in the extraction process have been clearly identified and optimized. The new extraction mode allows the determination of these compounds with limits of detection in the range from 0.15 μg/L (for 4-tert-octylphenol) to 2.7 μg/L (for 4-tert-butylphenol) with a relative standard deviation lower than 5.3 % (for 4-tert-butylphenol).     

Dispersive liquid-liquid microextraction versus single-drop microextraction for the determination of several endocrine-disrupting phenols from seawaters

Two liquid-phase microextraction procedures: single-drop microextraction (SDME) and dispersive liquid-liquid microextraction (DLLME), have been developed for the determination of several endocrine-disrupting phenols (EDPs) in seawaters, in combination with high-performance liquid chromatography (HPLC) with UV detection. The EDPs studied were bisphenol-A, 4-cumylphenol, 4-tertbutylphenol, 4-octylphenol and 4-n-nonylphenol. The optimized SDME method used 2.5 μL of decanol suspended at the tip of a micro-syringe immersed in 5 mL of seawater sample, and 60 min for the extraction time. The performance of the SDME is characterized for average relative recoveries of 102 ± 11%, precision values (RSD) < 9.4% (spiked level of 50 ng mL⁻¹), and detection limits between 4 and 9 ng mL⁻¹. The optimized DLLME method used 150 μL of a mixture acetonitrile:decanol (ratio 15.7, v/v), which is quickly added to 5 mL of seawater sample, then subjected to vortex during 4 min and centrifuged at 2000 rpm for another 5 min. The performance of the DLLME is characterized for average relative recoveries of 98.7 ± 3.7%, precision values (RSD) < 7.2% (spiked level of 20 ng mL⁻¹), and detection limits between 0.2 and 1.6 ng mL⁻¹. The efficiencies of both methods have also been compared with spiked real seawater samples. The DLLME method has shown to be a more efficient approach for the determination of EDPs in seawater matrices, presenting enrichment factors ranging from 123 to 275, average relative recoveries of 110 ± 11%, and precision values (RSD) < 14%, when using a real seawaters (spiked level of 3.5 ng mL⁻¹).     

Biomarkers and bioassays as alternative screening methods for the presence and effects of PCDD, PCDF and PCB

Summary Polyhalogenated aromatic hydrocarbons (PHAH) are wide spread, highly toxic, environmental contaminants. As such they pose risks for both humans and wildlife. For risk assessment purposes, concentrations are generally analyzed by HRGC-HR/LRMS. With the analytical data, mixture toxicity is calculated using the TEF concept. With this method only the defined congeners are taken into account and additivity for all congeners is assumed, whereas synergistic and antagonistic effects for several PCDD/F in combination with PCB have also been reported. To avoid these problems and high analytical costs, bioassays can be used for screening purposes. Cytochrome P450 1 A 1 induction and vitamin A and thyroid hormone levels are shown to be useful markers for PHAH exposure. When bioassays based on cytochrome P450 1A1 induction, in cultured cells, in multi-well culturing plates, are used, 2,3,7,8-TCDD detection limits <0.2 pg are possible. As such these bioassays are highly sensitive, cost effective and time saving. This application can be used as a pre-screening method to determine total dioxin content of environmental samples.     

High-performance ion mobility spectrometry with direct electrospray ionization (ESI-HPIMS) for the detection of additives and contaminants in food

High-performance ion mobility spectrometry (HPIMS) with an electrospray ionization (ESI) source detected a series of food contaminants and additive compounds identified as critical to monitoring the safety of food samples. These compounds included twelve phthalate plasticizers, legal and illegal food and cosmetic dyes, and artificial sweeteners that were all denoted as detection priorities. HPIMS separated and detected the range of compounds with a resolving power better than 60 in both positive and negative ion modes, comparable to the commonly used high-performance liquid chromatography (HPLC) methods, but with most acquisition times under a minute. The reduced mobilities, K₀, have been determined, as have the linear response ranges for ESI-HPIMS, which are 1.5–2 orders of magnitude for concentrations down to sub-ng μL⁻¹ levels. At least one unique mobility peak was seen for two subsets of the phthalates grouped by the country where they were banned. Furthermore, ESI-HPIMS successfully detected low nanogram levels of a phthalate at up to 30 times lower concentration than international detection levels in both a cola matrix and a soy-based bubble tea beverage using only a simplified sample treatment. A newly developed direct ESI source (Directspray) was combined with HPIMS to detect food-grade dyes and industrial dye adulterants, as well as the sweeteners sodium saccharin and sodium cyclamate, with the same good performance as with the phthalates. However, the Directspray method eliminated sources of carryover and decreased the time between sample runs. Limits-of-detection (LOD) for the analyte standards were estimated to be sub-ng μL⁻¹ levels without extensive sample handling or preparation.     

Recombinant cell bioassay systems for the detection and relative quantitation of halogenated dioxins and related chemicals

Proper epidemiological, risk assessment and exposure analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons (HAHs) requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). High-resolution instrumental analysis techniques are established for these chemicals, however, these procedures are very costly and time-consuming and as such, they are impractical for large scale sampling studies (i.e. for epidemiological studies and assessment of areas with widespread contamination). Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take advantage of the ability of these chemicals to activate the aromatic hydrocarbon receptor (AhR) and the AhR signal transduction pathway. Here we review the currently available in vitro AhR-based cell bioassay systems with a focus on recent recombinant reporter gene cell lines that have been developed for detection and relative quantitation of TCDD and related HAHs. Comparison of the relative sensitivities of the various cell bioassays and examples of their use in screening and analysis of environmental, biological, and food and feed samples are presented. Currently available experimental results and validation studies demonstrate the utility of these cell bioassay systems to provide a relatively rapid, accurate, and cost effective screening approach for the detection of TCDD and related HAHs in a variety of environmental, biological, food and feed samples. The availability of these cell bioassay systems will not only facilitate the large scale sampling studies needed for accurate assessment of contamination and exposure to these environmental chemicals, but they provide avenues for the identification of novel classes of TCDD-like chemicals.     

High-performance,synergistically catalytic luminescent nanozyme for the degradation and detection of endocrine-disrupting chemical diethylstilbestrol

A synergistically catalytic luminescent nanozyme was designed and synthesized for the degradation and enzymatic fluorescence detection of diethylstilbestrol, an endocrine-disrupting environmental pollutant. Because of the integration of cocatalytic Cu²⁺ ion and CeO₂ particle, luminescent Tb³⁺ ion, and functional ligand dipicolinic acid through flexible metal-organic framework structure, this nanozyme has not only the dual functions of luminescence and multienzyme such as laccase and horseradish peroxidase but also synergistically catalytic effect via a regeneration of Cu²⁺ oxidized by CeO₂. The synergistically catalytic effect of nanozyme greatly enhances the degradation of diethylstilbestrol. The resultants sensitized the luminescence of Tb³⁺ ions, which was used to sense the pM level of diethylstilbestrol in environmental samples. Such a high-performance catalytic luminescent nanozyme can be used to replace natural enzymes for the enzyme-based degradations and ultrasensitive assays. The strategy of constructing artificial enzymes directly from functional units provides a new way for developing fit-for-purpose multifunctional artificial enzymes.     

Advancement and future challenges of metal–organic coordination polymers: A case study of optical sensor for the detection of the environmental contaminants

Fast industrialization, increasing population, rapid urbanization, and the greediness of creamy layer in the society without the issues of caring the sustainability of ecosystem are the main out of many reasons behind the environmental catastrophe. The ecological balance is disturbed with the noxious materials generated from the uncontrolled use of modern science and technology and also unscientific and unsystematic societal growths. To save the modern civilization, a fast-track task is the monitoring of the toxic materials for the maintenance of sustainable ecological health and their utilization in a circular economy. Accordingly, optical methods are employed for the detection and estimation of environmental contaminants for immediate monitoring. The stability and structural tunability of the coordination polymeric (CPs) materials make them promising optical sensors for easy, low cost, reliable, selective, and sensitive detection of toxic ions/molecules. The crystallinity, thermal, mechanical, magnetic, and optical stability and flexibility of the CPs play a beneficial role for the accurate and stress-free recognition of the toxicants. In this review, the recent developments as well as the outlook on the sensing of pollutants at a very low concentration level using CPs as selective and specific sensors by spectro-fluorometric method are summarized. The progresses and challenges in the fabrication of hybrid materials and understanding their structure property correlations are described with an aim to motivate and facilitate the future researchers to perform research in the area.     

Considerations involved with the use of semipermeable membrane devices for monitoring environmental contaminants

Semipermeable membrane devices (SPMDs) are used with increasing frequency, and throughout the world as samplers of organic contaminants. The devices can be used to detect a variety of lipophilic chemicals in water, sediment/soil, and air. SPMDs are designed to sample nonpolar, hydrophobic chemicals. The maximum concentration factor achievable for a particular chemical is proportional to its octanol-water partition coefficient. Techniques used for cleanup of SPMD extracts for targeted analytes and for general screening by full-scan mass spectrometry do not differ greatly from techniques used for extracts of other matrices. However, SPMD extracts contain potential interferences that are specific to the membrane-lipid matrix. Procedures have been developed or modified to alleviate these potential interferences. The SPMD approach has been demonstrated to be applicable to sequestering and analyzing a wide array of environmental contaminants including organochlorine pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, polychlorinated dioxins and dibenzofurans, selected organophosphate pesticides and pyrethroid insecticides, and other nonpolar organic chemicals. We present herein an overview of effective procedural steps for analyzing exposed SPMDs for trace to ultra-trace levels of contaminants sequestered from environmental matrices.     

Trends in the sample preparation and analysis of nanomaterials as environmental contaminants

Much research on the use of nanomaterials in different applications is being conducted in areas such as water treatment, catalysis, oil processing, medicine, food, sensors, energy storage, building materials, constructions, and others. Nanoparticles are ultra-small particles with exceptional properties, but some nanoparticles and nanomaterials may exhibit harmful properties when leaked into the environment. Due to the lack of analytical methods for the detection and analysis of nanoparticles in complex matrices, not much is known about the potential risks associated with nanomaterials. Therefore, more knowledge is needed of the sampling and analysis of nanomaterials (NMs) as environmental contaminants. This review is undertaken to identify and assess key characteristics in potential sampling and analysis methods for identifying and quantifying the occurrence of NMs in numerous types of environmental media. To select suitable sampling and analysis methods, information on NM sources and transformation in environmental media is essential and thus is also discussed. This provides more information about the negative impacts of NMs on the environment. Challenges and future perspectives on the determination of NMs are also discussed.     

Determination of the pesticides considered as endocrine-disrupting compounds (EDCs) by solid-phase extraction followed by gas chromatography with electron capture and mass spectrometric detection

An SPE method followed by GC-electron capture detection (ECD) with confirmation by MS for the trace determination of four pesticides considered as endocrine-disrupting compounds (EDCs) in natural waters and sediments has been developed. Target analytes, fenarimol, fenvalerate, pendimethalin, and vinclozolin, belong to different chemical groups and are used mainly in agriculture. In the present study, analysis employs an offline SPE step for the extraction of the target analytes from natural waters. Sonication and subsequent SPE clean-up was used for extraction and purification of the sediment samples which were finally treated with activated copper powder. The type of SPE disk, eluents as well as solution parameters including pH value, and concentrations of salts and humic substances were examined for the efficiency of the method. The recoveries of all pesticides were in relatively high levels, ranging from 75 to 97% for waters and 71 to 84% for sediment samples. Both methods were applied to real water and sediment samples and the presence of the tested compounds was investigated.     

Fast isolation of hydrophobic organic environmental contaminants from exposed semipermeable membrane devices (SPMDs) prior to GC analysis

Semipermeable membrane devices (SPMD) represent a passive sampling technology that is becoming widely used for monitoring of surface waters pollution. While "classic" procedures employ dialysis to recover target compounds from exposed SPMDs, in the present study analytes were isolated from cut membrane together with sequestering medium (triolein) using hexane as an extraction solvent. This approach allowed us to reduce the time needed for accomplishment of isolation step from 48 h to only 1 h. Automated gel permeation chromatography (GPC) clean-up is employed in the following step to separate triolein from analytes fraction. Musk compounds (MCs), polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs) and several other persistent organochlorine compounds (OCs) were determined in the respective fraction by GC method employing selective detectors (MSD, ECD). As shown in a series of analyses of SPMDs deployed in various aquatic ecosystems, high recoveries and good repeatability of results together with a possibility to obtain the information on the pollution of sampling site at the day of sample arrival to laboratory make this newly implemented procedure an interesting alternative to time consuming dialysis.     

Improved capillary electrophoretic separation of nine (fluoro)quinolones with fluorescence detection for biological and environmental samples

A capillary electrophoretic method has been developed for the separation of nine (fluoro)quinolones. Detection is done by fluorescence measurement with broad wavelength band excitation between 240 and 400 nm. Best separation is achieved in a carrier electrolyte containing 50 mM H3PO4 adjusted to pH 7.55-acetonitrile (60:40, v/v). Detection limits are in the low microgl(-1) range. The suitability to real samples has been demonstrated by analyzing blood samples and surface water samples. Sample preconcentration and sample clean-up can easily be done by solid-phase extraction. Different phases based on alkyl- or phenyl-modified silica as well as on polymers have been investigated for this purpose. The method should also be useful for determination of residues of (fluoro)quinolones in food or other matrices.     

Recent developments in headspace microextraction techniques for the analysis of environmental contaminants in different matrices

Headspace microextraction procedures such as solid-phase microextraction (SPME) and single drop microextraction (SDME) or liquid-phase microextraction (LPME) are increasingly used for the extraction of environmental organic pollutants from a variety of aqueous, viscous, semisolid and solid environmental and biological matrices. In this article, recent analytical applications of these methodologies when used as an isolation and trace enrichment step prior to the analysis of organic pollutants (pesticides, polycyclic aromatic hydrocarbons, polychlorinated compounds, organotin compounds, phenolic derivatives, aromatic amines, phthalates, etc.) by gas and liquid chromatography are reviewed. The applicability and inherent limitations of headspace microextraction are also discussed. The future direction of research in this field and general trends toward commercial applications are considered.