(Electro)Sensing of Phenicol Antibiotics-A Review.

The presence of residues from frequent antibiotic use in animal feed can cause serious health risks by contaminating products meant for human consumption such as meat and milk. The present paper gives an overview of the electrochemical methods developed for the detection of phenicol antibiotic residues (chloramphenicol, thiamphenicol, and florfenicol) in different kinds of foodstuffs. Electrochemical sensors based on different biomolecules and nanomaterials are described. The detection limit of various developed methods with their advantages and disadvantages will be highlighted.

An improved electrochemical aptasensor for chloramphenicol detection based on aptamer incorporated gelatine.

Because of the biocompatible properties of gelatine and the good affinity of aptamers for their targets, the combination of aptamer and gelatine type B is reported as promising for the development of biosensing devices. Here, an aptamer for chloramphenicol (CAP) is mixed with different types of gelatine and dropped on the surface of disposable gold screen printed electrodes. The signal of the CAP reduction is investigated using differential pulse voltammetry. The diagnostic performance of the sensor is described and a detection limit of 1.83 × 10(-10) M is found. The selectivity and the stability of the aptasensor are studied and compared to those of other CAP sensors described in literature.

The impact of metal pollution on soil faunal and microbial activity in two grassland ecosystems.

In this study the influence of metal pollution on soil functional activity was evaluated by means of Bait lamina and BIOLOG(®) EcoPlates™ assays. The in situ bait lamina assay investigates the feeding activity of macrofauna, mesofauna and microarthropods while the BIOLOG(®) EcoPlate™ assay measures the metabolic fingerprint of a selectively extracted microbial community. Both assays proved sensitive enough to reveal changes in the soil community between the plots nearest to and further away from a metal pollution source. Feeding activity (FA) at the less polluted plots reached percentages of 90% while plots nearer to the source of pollution reached percentages as low as 10%. After 2 and 6 days of incubation average well color development (AWCD) and functional richness (R') were significantly lower at the plots closest to the source of pollution. While the Shannon Wiener diversity index (H') decreased significantly at sites nearer to the source of pollution after 2 days but not after 6 days of incubation. Arsenic, Cu and Pb correlated significantly and negatively with feeding activity and functional indices while the role of changing environmental factors such as moisture percentage could not be ruled out completely. Compared to the Bait lamina method that is used in situ and which is therefore more affected by site specific variation, the BIOLOG assay, which excludes confounding factors such as low moisture percentage, may be a more reliable assay to measure soil functional activity.

Aptasensing of chloramphenicol in the presence of its analogues: reaching the maximum residue limit.

A novel, label-free folding induced aptamer-based electrochemical biosensor for the detection of chloramphenicol (CAP) in the presence of its analogues has been developed. CAP is a broad-spectrum antibiotic that has lost its favor due to its serious adverse toxic effects on human health. Aptamers are artificial nucleic acid ligands (ssDNA or RNA) able to specifically recognize a target such as CAP. In this article, the aptamers are fixed onto a gold electrode surface by a self-assembly approach. In the presence of CAP, the unfolded ssDNA on the electrode surface changes to a hairpin structure, bringing the target molecules close to the surface and triggering electron transfer. Detection limits were determined to be 1.6 × 10(-9) mol L(-1). In addition, thiamphenicol (TAP) and florfenicol (FF), antibiotics with a structure similar to CAP, did not influence the performance of the aptasensor, suggesting a good selectivity of the CAP-aptasensor. Its simplicity and low detection limit (because of the home-selected aptamers) suggest that the electrochemical aptasensor is suitable for practical use in the detection of CAP in milk samples.

Selection and characterization of PCB-binding DNA aptamers.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) that resist natural degradation and bioaccumulate in nature. Combined with their toxicity, this leads them to cause cancer and other health hazards. Thus, there is a vital need for rapid and sensitive methods to detect PCB residues in food and in the environment. In this study, PCB-binding DNA aptamers were developed using PCB72 and PCB106 as targets for aptamer selection. Aptamers are synthetic DNA recognition elements which form unique conformations that enable them to bind specifically to their targets. Using in vitro selection techniques and fluorometry, an aptamer that binds with nanomolar affinity to both the PCBs has been developed. It displayed high selectivity to the original target congeners and limited affinity toward other PCB congeners (105, 118, 153, and 169), suggesting general specificity for the basic PCB skeleton with varying affinities for different congeners. This aptamer provides a basis for constructing an affordable, sensitive, and high-throughput assay for the detection of PCBs in food and environmental samples and offers a promising alternative to existing methods of PCB quantitation. This study therefore advances aptamer technology by targeting one of the highly sought-after POPs, for the first time ever recorded.

Field application of a novel active-passive sampling technique for the simultaneous measurement of a wide range of contaminants in water.

A first test of the field capabilities of a novel in situ sampling technique combining active and passive sampling (APS) was conducted in the sea. The proof-of-concept device uses a pump to draw water into a diffusion cell where dissolved target substances are accumulated onto sorbents which are selective for different classes of contaminants (i.e., metal cations, polar and non-polar organic compounds), simultaneously. A controlled laminar flow established in the diffusion cell enables measurements of contaminant concentrations that are fully independent from the hydrodynamic conditions in the bulk solution. APS measurements were consistent with those obtained using conventional passive sampling techniques such as organic diffusive gradients in thin films (o-DGT) and silicone rubber (SR) samplers (generally < 40% difference), taking into account the prevailing hydrodynamic conditions. The use of performance reference compounds (PRC) for hydrophobic contaminants provided additional information. Field measurements of metal ions in seawater showed large variability due to issues related to the device configuration. An improved field set-up deployed in supplementary freshwater mesocosm experiments provided metal speciation data that was consistent with passive sampling measurements (DGT), taking into account the hydrodynamic conditions. Overall, the results indicate that the APS technique provides a promising approach for the determination of a wide range of contaminants simultaneously, and independently from the hydrodynamic conditions in the bulk solution.

Escherichia coli as a bioreporter in ecotoxicology.

Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.

Characterization of the accumulation of metals and organic contaminants on a novel active-passive sampling device under controlled water flow conditions.

Recently, a new sampling device combining active and passive sampling (APS) was developed for the measurement of time-averaged concentrations of metal species and both polar and non-polar organic contaminants in water. By coupling a diffusion cell (loaded with a set of sorbents selective for different substances) with a small pump and a flow meter, the APS device is able to perform in situ measurements that are independent of the hydrodynamic conditions in the exposure medium. In the present study, the diffusion layer thickness (δ) at the sorbent/solution interface within the diffusion cell was characterised under controlled flow conditions. Laboratory tests indicated that, in the range of flow rates investigated, the average diffusion layer thickness (δ¯) varied from ∼60 to ∼110 µm, depending on the type of substance measured and the position of the sorbent with respect to the flow direction. Due to its ability to maintain an approximately constant δ¯, good to excellent agreement was found between measurements performed with the APS device in non-complexing media and concentrations measured in discrete water samples for all the substances investigated. These results suggest that the APS device could overcome issues affecting the quantitative interpretation of measurements by conventional passive sampling devices and serve as a useful tool for simultaneously monitoring a wide range of contaminants in water.

Mode of action clustering of chemicals and environmental samples on the bases of bacterial stress gene inductions.

Over the years, environment and the human population have seen an increasing exposure to both existing and newly developed chemicals. It is generally accepted that at least some of those are toxic, albeit as pure compound or in combination with others. In response to a growing public awareness and scientific data, the new European chemicals legislation (Registration, Evaluation and Authorization of Chemicals) is under implementation at the moment. As a consequence, during the coming years about 30,000 chemicals have to be assessed on their potential hazard for man and biota. Part of this assessment will be done using existing and new in vitro tests offering insight into the toxicity of chemicals and into their toxicological mode of action. This study presents data on a battery of 14 bacterial reporter gene assay allowing mode of action determination and statistical grouping of chemicals based on their induction profile. Gene induction results are used to group reference chemicals in a statistical cascade employing hierarchical tree and k-means clustering for initial grouping. Both complementary, yet mathematically different, algorithms are consequently confirmed by principal component analysis (PCA). The gene induction profiles of an environmental extract with documented in vivo effects and a chemical with limited toxicological are data available and projected in the PCA vector space. The projection allows correct mode of action grouping and indicates that effect predictions based on the known toxicological effects of the reference compounds can be made.

Mixture toxicity and gene inductions: can we predict the outcome?

As a consequence of the nature of most real-life exposure scenarios, the last decade of ecotoxicological research has seen increasing interest in the assessment of mixture ecotoxicology. Often, mixtures are considered to follow one of two models, concentration addition (CA) or response addition (RA), both of which have been described in the literature. Nevertheless, mixtures that deviate from either or both models exist; they typically exhibit phenomena like synergism, ratio or concentration dependency, or inhibition. Moreover, both CA and RA have been challenged and evaluated mainly for acute responses at relatively high levels of biological organization (e.g., whole-organism mortality), and applicability to genetic responses has not received much attention. Genetic responses are considered to be the primary reaction in case of toxicant exposure and carry valuable mechanistic information. Effects at the gene-expression level are at the heart of the mode of action by toxicants and mixtures. The ability to predict mixture responses at this primary response level is an important asset in predicting and understanding mixture effects at different levels of biological organization. The present study evaluated the applicability of mixture models to stress gene inductions in Escherichia coli employing model toxicants with known modes of action in binary combinations. The results showed that even if the maximum of the dose-response curve is not known, making a classical ECx (concentration causing x% effect) approach impossible, mixture models can predict responses to the binary mixtures based on the single-toxicant response curves. In most cases, the mode of action of the toxicants does not determine the optimal choice of model (i.e., CA, RA, or a deviation thereof).

A novel active-passive sampling approach for measuring time-averaged concentrations of pollutants in water.

Passive sampling with in situ devices offers several advantages over traditional sampling methods (i.e., discrete spot sampling), however, data interpretation from conventional passive samplers is hampered by difficulties in estimating the thickness of the diffusion layer at the sampler/medium interface (δ), often leading to inaccurate determinations of target analyte concentrations. In this study, the performance of a novel device combining active and passive sampling was investigated in the laboratory. The active-passive sampling (APS) device is comprised of a diffusion cell fitted with a pump and a flowmeter. Three receiving phases traditionally used in passive sampling devices (i.e., chelex resin, Oasis HLB, and silicone rubber), were incorporated in the diffusion cell and allowed the simultaneous accumulation of cationic metals, polar, and non-polar organic compounds, respectively. The flow within the diffusion cell was accurately controlled and monitored, and, combined with diffusion coefficients measurements, enabled the average δ to be estimated. Strong agreement between APS and time-averaged total concentrations measured in discrete water samples was found for most of the substances investigated. Accuracies for metals ranged between 87 and 116%, except Cu and Pb (∼50%), whilst accuracies between 64 and 101%, and 92 and 151% were achieved for polar and non-polar organic compounds, respectively. These results indicate that, via a well-defined in situ preconcentration step, the proposed APS approach shows promise for monitoring the concentration of a range of pollutants in water.

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

Hazard characterization and identification of a former ammunition site using microarrays, bioassays, and chemical analysis.

More than 100,000 tons of 2,4,6-trinitrotoluene were produced at the former ammunition site Werk Tanne in Clausthal-Zellerfeld, Germany. The production of explosives and consequent detonation in approximately 1944 by the Allies caused great pollution in this area. Four soil samples and three water samples were taken from this site and characterized by applying chemical-analytical methods and several bioassays. Ecotoxicological test systems, such as the algal growth inhibition assay with Desmodesmus subspicatus, and genotoxicity tests, such as the umu and NM2009 tests, were performed. Also applied were the Ames test, according to International Organization for Standardization 16240, and an Ames fluctuation test. The toxic mode of action was examined using bacterial gene profiling assays with a battery of Escherichia coli strains and with the human liver cell line hepG2 using the PIQOR Toxicology cDNA microarray. Additionally, the molecular mechanism of 2,4,6-trinitrotoluene in hepG2 cells was analyzed. The present assessment indicates a danger of pollutant leaching for the soil-groundwater path. A possible impact for human health is discussed, because the groundwater in this area serves as drinking water.

Assessing in-vitro estrogenic effects of currently-used flame retardants.

Flame retardants are chemicals that are added to nearly all manufactured materials. Additionally, there has been a steady increase in diseases resulting from endocrine-disruption with an aligned increase in use of chemicals. Given the persistence, potential bioaccumulation, limited toxicological understanding, and vast use of flame retardants, there is a need to investigate potential endocrine-disruptive activity associated with these compounds in an effort for better risk assessment. We therefore used the MCF-7 flow-cytometric proliferation assay in an effort to establish potential estrogen-disrupting effects of twelve currently-used flame retardants. Triphenyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tris(butyl) phosphate, hexabromocyclododecane, and tetrabromobisphenol A showed statistically significant estrogenic activity, with hexabromocyclododecane being the most potent of the five (EC20 of 5.5 µM). Tris(2-butoxyethyl) phosphate, tris(1,3-dichloro-2-propyl) phosphate, tri(2-chloroethyl) phosphate, tris(butyl) phosphate, hexabromocyclododecane, tetrabromobisphenol A, and tris(2,3,-dibromopropyl) isocyanurate harboured anti-estrogenic activity when co-treating with 17ß-estradiol, with hexabromocyclododecane showing the highest potency (IC20 of 17.6 µM). Interestingly, some compounds showed both estrogenic and anti-estrogenic effects, indicating both receptor-dependant and -independent mechanisms attributed to some of these compounds, in line with other studies. Multiple currently-used flame retardants may therefore act as xenoestrogens and anti-estrogens, or alter estrogen homeostasis, which could affect endocrine function.

Elucidating toxicological mechanisms of current flame retardants using a bacterial gene profiling assay.

Flame retardants are ubiquitously used chemicals that have been shown to contaminate environments. Toxicological data is largely limited, with little insight into their molecular modes of action that may give rise to their toxic phenotypes. Such insight would aid more effective risk assessments concerning these compounds, while also improving molecular design. We therefore used a bacterial stress-gene profiling assay to screen twelve currently-used flame retardants to obtain mechanistic insights of toxicity. Both brominated and organophosphate flame retardants were tested. All compounds showed statistically significant inductions of several stress genes when compared to control treatments. Triphenyl phosphate, tris(2-butoxyethyl) phosphate, tris(1,3-dichloro-2-propyl)phosphate, tris(butyl)phosphate, and tetrabromobisphenol A elicited (at least) two-fold inductions for any of the stress genes. When looking at absolute induction levels, the promoters induced are indicative of protein perturbation, DNA integrity and membrane integrity. However, normalising for the different induction potentials of the different stress genes and clustering using hierarchical and k-means algorithms indicated that in addition to protein and DNA damage, some compounds also resulted in growth arrest and oxidative damage. This research shows that this assay allows for the determination of toxicological modes-of-action while clustering and accounting for induction potentials of the different genes aids better risk assessment.

Carbon nanotubes based electrochemical aptasensing platform for the detection of hydroxylated polychlorinated biphenyl in human blood serum.

A novel strategy to sense target molecules in human blood serum is achieved by immobilizing aptamers (APTs) on multi-walled carbon nanotubes (MWCNT) modified electrodes. In this work, the aminated aptamer selected for hydroxylated polychlorinated biphenyl (OH-PCB) was covalently immobilized on the surface of the MWCNT-COOH modified glassy carbon electrode through amide linkage. The aptamers function as recognition probes for OH-PCB by the binding induced folding of the aptamer. The developed aptasensing device was characterized by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). The aptasensor displayed excellent performance for OH-PCB detection with a linear range from 0.16 to 7.5 µM. The sensitivity of the developed aptasensing platform is improved (1×10(-8) M) compared to the published report (1×10(-6) M) for the determination of OH-PCB (Turner et al., 2007). The better performance of the sensor is due to the unique platform, i.e. the presence of APTs onto electrodes and the combination with nanomaterials. The aptamer density on the electrode surface was estimated by chronocoulometry and was found to be 1.4×10(13) molecules cm(-2). The validity of the method and applicability of the aptasensor was successfully evaluated by the detection of OH-PCB in a blood serum sample. The described approach for aptasensing opens up new perspectives in the field of biomonitoring providing a device with acceptable stability, high sensitivity, good accuracy and precision.

Application of a multiple endpoint bacterial reporter assay to evaluate toxicological relevant endpoints of perfluorinated compounds with different functional groups and varying chain length.

Perfluorinated compounds are widely distributed in the environment; good knowledge about the toxic mode of action of these compounds can contribute to improved molecular design and risk assessment. The studied compounds were evaluated with a bacterial multiple endpoint reporter assay for responses in four different mode of action classes (oxidative damage, DNA damage, general cell lesions and membrane damage). The results of our study clearly demonstrate that inductions of stress responsive genes occur for the different compounds and confirm some of the known mechanisms of work for well studied compounds like PFOA and PFOS, and in addition provide new information for less studied compounds. Few inductions were observed after exposure to the low carbon number carboxylic acids, PFBtA (CF(3)(CF(2))(2)C(O)O(-)), PFPtA (CF(3)(CF(2))(3)C(O)O(-)), PFHxA (CF(3)(CF(2))(4)C(O)O(-)) and PFHpA (CF(3)(CF(2))(5)C(O)O(-)) at equimolar concentrations (0.0156-1 mM). The induction of membrane damage markers (MicF and OsmY) is prominently present after exposure to PFOS (CF(3)(CF(2))(7)SO(3)(-)) and even more after exposure to PFNA (CF(3)(CF(2))(7)C(O)O(-)). This is the first report describing the mode of action of carboxylic acids with 11 and 12 carbon atoms; they are equally potent inducers relative to PFOS and PFNA. Overall, the effects seen at the level of gene expression were higher for the sulfonic acids than for the carboxylic acids, but the effect of the chain length is more important than the effect of the functional group.