Analysis Of Microsamples Research Articles

RAPID GROWTH EXPLAINS LARGE SIZE OF MOLLUSKS IN THE EOCENE GOSPORT SAND, UNITED STATES GULF COAST

The late–middle Eocene Gosport Sand of the United States Gulf Coastal Plain preserves a spectacularly dense accumulation of fossil mollusks in which shells appear unusually large in comparison to congeners in adjacent units. We use size-frequency distributions of common taxa drawn from bulk samples to quantify size differences between the Gosport Sand and the underlying Lisbon and overlying Moodys Branch Formations. In all but one of the twelve lineages examined, Gosport Sand individuals are larger than those in adjacent units. As taphonomic processes such as sorting or winnowing cannot account for observed differences, the larger body sizes in the Gosport Sand must have resulted from differences in life history. We used sclerochronology and stable isotope geochemistry to assess growth trajectories for twelve lineages common to the three formations. Isotope analyses of microsamples collected along the ontogenetic trajectory reveal seasonal temperature changes, providing a chronometer for growth. Size-age data indicate that, while life spans vary somewhat among the three units, Gosport Sand taxa almost universally grew faster than those in the two adjacent formations. While growth rate can be enhanced by several factors, we hypothesize that rates of primary production were higher during deposition of the Gosport Sand. Ba/Ca ratios along the growth trajectories of shell carbonate exhibit spikes in Gosport Sand mollusks that are indistinguishable from those observed in modern shells that accrete in high productivity settings.

Immunodetection of Proteins in Ancient Paint Media

Diagnostic immunology is a powerful tool, widely used in clinical and biochemical laboratories for detecting molecules. In recent years, the technique has been adapted to materials sciences as a result of the extensive advances achieved in immunology. Today, many companies supply custom antibodies as well as new high-performance bioprobes for virtually any use. The idea of using immunodetection in the field of conservation science is not new. This analytical methodology is, in fact, particularly attractive for investigating biopolymers in painting materials; it is highly sensitive and selective with respect to the biological source of the target molecules. Among biopolymers, proteins have been widely used in the past as painting binders, adhesives, and additives in coating layers. An accurate assessment of these materials is necessary to obtain deeper insights into an artist's technique as well as to design proper restoration and conservation methods. In spite of the diagnostic potential offered by immunodetection-based techniques, some analytical drawbacks had, until recently, limited their use in routine applications in conservation science. In this Account, we highlight the most important results achieved in our research on the development of analytical methodologies based on the use of enzyme-linked immunosorbent assay (ELISA) and immuno-fluorescence microscopy (IFM) techniques for the highly sensitive and specific identification of proteins in artistic and archeological materials. ELISA and IFM offer two alternative analytical routes to this final goal: ELISA provides a fast, cost-effective, quantitative analysis of microsamples put in solution, whereas IFM combines the immunodetection of the targeted molecules with the characterization of their spatial distribution. The latter approach is of great value in the stratigraphic investigation of paintings. We discuss the limits and strengths of these methodologies in the context of the complex matrixes usually found in the investigated materials and the prolonged aging that they have undergone. Immunology is a relatively new technique in conservation science, providing a rich new field for innovation. We see two areas that are particularly ripe for future contributions. The commercial manufacture of antibodies specifically tailored for use in cultural heritage studies holds enormous potential. Moreover, the need for further refinement of detection systems in immuno-fluorescence techniques, especially the suppression of the autofluorescence background in painting materials, offers an abundance of opportunities for researchers. Immunology is a relatively new technique in conservation science, providing a rich new field for innovation.

PAINTED ROCK-CUT TOMBS IN CYPRUS FROM THE HELLENISTIC AND ROMAN PERIODS TO BYZANTIUM: MATERIAL PROPERTIES, DEGRADATION PROCESSES AND SUSTAINABLE PRESERVATION STRATEGIES

The art of wall painting in Cyprus during the transitional period from Greco-Roman to Byzantine times introduced a wide spectrum of painting materials that reflect the social structure and organization of the period, provincial and transregional interactions and technological savoir faire. These materials include natural minerals – locally available or imported – organic fibers, gums, oils, artificially produced, high-fired vitreous composites and other chemically synthesized products. This paper investigates and presents the in situ alteration mechanisms that certain wall paintings have undergone due to physical and chemical processes caused by the adverse microclimate in enclosed rock-cut structures. It also proposes sustainable preservation strategies based on a holistic methodology, integrating novel scientific techniques for non-invasive in situ examination with state-of-the art non-destructive analysis of microsamples at the molecular scale.

Energy-dispersive X-ray fluorescence spectrometer for analysis of conventional and micro-samples: Preliminary assessment

The energy-dispersive X-ray fluorescence spectrometer for analysis of conventional and micro-samples using pinhole collimators of various sizes is developed. The measurements can be performed in the air or, in order to decrease the absorption of long-wavelength radiation of low-Z elements, in helium atmosphere. The sample is excited by the air-cooled Rh target X-ray tube of ca. 100 μm nominal focal spot size and maximum power 75 W. The X-ray spectra of the samples are collected by thermoelectrically cooled Si-PIN detector. The tungsten pinhole collimators of the size holes from 50 to 2000 μm are placed between primary filter and analyzed sample to reduce size of analyzed area. The sample can be moved using the X– Y stage. The position of the sample is monitored by CCD camera and two laser pointers. The beam spot sizes for various collimators are evaluated by the thin-wire and knife-edge methods. Beside the beam spot sizes, the loss of radiation intensity and the changes of spectral distribution of the incident radiation caused by applying various collimators are also investigated. The sample-surface-down geometry in the designed spectrometer allows for a simple analysis of various samples: solutions, loose powders, solid samples of conventional size and micro-samples.

Deposition and remobilization of uranium in the North Baikal region: Evidence from the U-Pb isotopic systems of uranium ores

Based on the study of local volumes of minerals, including their microsampling and subsequent analysis of Pb/Pb and U/Pb isotope ratios with the classic methods of isotopic dilution and thermoionization mass spectrometry (TIMS), U-Pb and Pb-Pb isotopic datings of minerals were carried out in uranium ores from deposits in the Akitkan and Nechera-Nichatka ore districts (North Baikal region). Reliable evidence in favor of the Middle Devonian (384 ± 8 Ma) remobilization of Paleoproterozoic primary uranium concentrations and the redeposition of uranium as pitchblende 2 has been obtained for the first time for ores of the Akitkan district. The Paleoproterozoic (1832 ± 13 Ma) age of uraninite mineralization and the timing of the latest (377 ± 5 Ma) transformation of uranium ores at the Chepok deposit (Nechera-Nichatka district) are substantiated.

A direct temperature-resolved tandem mass spectrometry study of cholesterol oxidation products in light-aged egg tempera paints with examples from works of art

Cholesterol ( 1) constitutes approximately 5% of the lipid fraction of eggs. The compound is therefore abundant in fresh egg tempera paints. The fate of cholesterol upon light ageing of egg tempera paint binding medium was investigated by direct temperature resolved mass spectrometry (DTMS) and tandem mass spectrometry (DTMSMS). Cholesterol oxidation products (COPs) such as 5,6-epoxycholestan-3-ol ( 2) and 3-hydroxycholest-5-en-7-one ( 3) were positively identified in light-aged egg binding medium. Given the fast rate of oxidation of cholesterol, the corresponding oxidation products are better markers for egg tempera than the cholesterol molecule itself. Cholesterol and COPs were discovered in paints on German baroque altar pieces from the 16th and 18th C and in a 20th C glaze on a Mark Rothko Seagram Mural painting at Tate by DTMS fingerprinting analysis of paint microsamples.

Multispectral imaging of paintings

Identifying the materials of a painting is a crucial step in any conservation process. When the objective is to prepare an intervention plan, it is absolutely necessary to understand the matters the restorer is going to encounter. Also, when the aim is a better understanding of the artwork, and perhaps an authenticity check, it is highly relevant to know which materials were employed, since they may differ depending on the period of execution and on the specific artist as well. To identify materials on a painting's surface in a reliable manner, currently the most popular and trustworthy method is the analysis of microsamples of the paint layer. However, chemical analyses although reliable, have a number of drawbacks. The first is linked to the fact that this is an invasive method. The samples used need to be detached from the painting and will not be put back in place. Moreover, the achieved results are in principle - and often also in practice - valid only for that specific specimen and cannot generally be extended to neighboring areas.

The patinas of the Dogon–Tellem statuary: A new vision through physico-chemical analyses

Numerous African art objects collected in the course of colonial or ethnological expeditions during the 20th century are partially or completely covered with a so-called “patina”. These patinas have been formed during religious and ritual ceremonies, where different substances have been spread out at the surface of the objects. The anthropomorphic statuettes from the Dogon culture are well-known examples of this kind of practice. A better understanding of the chemical composition of these patinas could explain details of the ceremonial practices realized throughout the centuries. An important challenge is to extract unequivocal information about the original constituents of these patinas, and the techniques used for their application on ritual objects. The Dogon statuary can be divided into three periods. The oldest one, before 1350 A.D., is called the Tellem period, according to the name of the people who lived in this area before the arrival of the Dogon. The most recent, after 1650 A.D., is the Dogon period. These two periods are separated by an intermediary period, where the attribution to the Tellem or to the Dogon cultures is difficult. During our study, we have worked on the patina of 12 Dogon artifacts from these three different periods. In this paper, we present the results obtained for two representative objects, one of the Tellem period and one of the Dogon period. The aim is to access the chemical composition of the patinas and to see if they can be compared to ethnological reports, as is often done in the literature on ethnological studies. Chemical imaging techniques, based on the combination of microscopy and spectroscopy, are well suited to study both the composition and the spatial organization of heterogeneous complex mixtures of organic and inorganic matter. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), followed by scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDS), and synchrotron radiation-based Fourier transform infrared microscopy (SR-μFTIR) have been applied to non-destructive analyses of micro-samples of the patinas of several Dogon statuettes. A very careful preparation, using ultramicrotomy on embedded samples, allowed us to perform successively all these measurements on a single fragment. Comparison and superposition of the different chemical images lead us to identify minerals (clays, quartz and calcium carbonate), and different organic products (proteins, starch, lipids), as well as to map their spatial distribution. The patina of the most recent statuettes presents a stratigraphy which can be related to successive uses of the objects for ritual purposes, and the results are in agreement with the ethnological reports. For the Tellem objects, the significant quantity of minerals and the lack of stratigraphy suggest that the patina was deposited according to a different recipe. This difference suggests the existence of a diversity of rituals that are more extensive than that described by the ethnologists.

Challenges to metallomics and analytical chemistry solutions

Abstract Metal ions play a fundamental role in the chemistry of life. The understanding of the molecular bases of the living process requires the knowledge of the correlations existing between metal ions and the genome and the derived -omes: transcriptome, proteome, and metabolome. An indispensable step on this way is the characterization of the coordination environment of metal ions present and the identification and quantification of metal-containing chemical species. The ensemble of research activities related to metal ions in biological systems has been recently referred to as "metallomics" [1]. The progress in this field is largely dependent on the high-throughput acquisition of multielement and -species analytical data in biological samples. The paper gives a brief overview of the state of the art of analytical techniques and methods for the multielement quantitative analysis of biological microsamples, and for the detection, identification, and quantitation of metal-containing proteins and low-molecular-weight metabolites. The potential contribution of molecular biology techniques in terms of linking information on metals and metal-species to the genome of an organism is highlighted.

Combined micro-Raman/UV-visible/fluorescence spectrometer for high-throughput analysis of microsamples

Combined micro-Raman/UV-visible (vis)/fluorescence spectroscopy system, which can evaluate an integrated array of more than 10,000 microsamples with a minimuma size of 5 microm within a few hours, has been developed for the first time. The array of microsamples is positioned on a computer-controlled XY translation microstage with a spatial resolution of 1 mum so that the spectra can be mapped with micron precision. Micro-Raman spectrometers have a high spectral resolution of about 2 cm(-1) over the wave number range of 150-3900 cm(-1), while UV-vis and fluorescence spectrometers have high spectral resolutions of 0.4 and 0.1 nm over the wavelength range of 190-900 nm, respectively. In particular, the signal-to-noise ratio of the micro-Raman spectroscopy has been improved by using a holographic Raman grating and a liquid-nitrogen-cooled charge-coupled device detector. The performance of the combined spectroscopy system has been demonstrated by the high-throughput screening of a combinatorial ferroelectric (i.e., BaTi(x)Zr(1-x)O(3)) library. This system makes possible the structure analysis of various materials including ferroelectrics, catalysts, phosphors, polymers, alloys, and so on for the development of novel materials and the ultrasensitive detection of trace amounts of pharmaceuticals and diagnostic agents.

Micromachined, planar-geometry, atmospheric-pressure, battery-operated microplasma devices (MPDs) on chips for analysis of microsamples of liquids, solids, or gases by optical-emission spectrometry

Because of their desirable characteristics, for example small size, lightness, low power and gas consumption, and potential for portability, miniaturized plasma sources are receiving significant attention in the scientific literature. To take advantage of these characteristics we micromachined and fabricated new, planar-geometry, self-igniting, atmospheric-pressure microplasma devices (MPDs) on chips. These microplasmas required such low power for their operation they could be operated from a re-chargeable battery (of the type used in cordless power-tools). Despite their advantages, most miniaturized plasma sources reported in the literature have not performed well with liquid samples; analysis of powders or solids that can be converted to a powder (and processed and used as slurries) is even more difficult. To address these shortcomings we coupled an electrothermal, mini-in-torch vaporization (mini-ITV) "dry" sample-introduction system to the low-power planar microplasma devices we developed. In this preliminary investigation, absolute detection limits obtained from microsamples of single-element liquid standards and optical emission spectrometry with photomultiplier-tube detection and a spectral bandpass similar to that of portable, commercially available fiber-optic spectrometers were in the low-pg to ng range, for example 2 pg (for K) to 25 ng (for Pb). Mini-ITV also enabled (as far as we are aware, for the first time) measurement of analyte emission from microsamples of powdered solids (as slurries). In addition to the 3% H2 in Ar mixtures, the ac-operated microplasmas were sustained by use of a variety of electrode materials and different plasma-support gases (e.g. Ar, He and 3% H2 in He) thus indicating fabrication versatility and operational flexibility. Such flexibility has the potential to enable microplasmas to be tailored to analytical problems, and this is demonstrated by using a He MPD and chlorine emission measurements (837.594 nm) from gaseous microsamples as an example.

On‐line preparation of microsamples prior to CE

An experimental setup is presented here for the automated analysis of microsamples, based on the on-line coupling of a capillary SPE module and a CE unit using a two-position six-port valve, an open-closed valve to isolate electrically the sample preparation from the CE unit and a "T" interface. A C18 trapping microcolumn (dimensions 2.5 cm x 100 microm id x 360 microm od) was used for the SPE step. The utility of the proposed experimental setup was demonstrated by applying it to the determination of quinolone antibiotics in serum microsamples, which was efficiently carried out in less than 20 min (4 min for protein denaturation and 15 min for analytes preconcentration and CE-UV separation-determination). A complete optimization study was performed for preconcentration and cleanup of quinolones, the coupling of sample preparation module to the CE unit and electrophoretic separation of quinolones. A preconcentration factor of 10.4 was achieved. The volume injected with the proposed method was 125 nL versus 160 nL introduced by hydrodynamic injection. The volume required for the analysis was 2 microL, which makes the proposed experimental setup very useful for the analysis of microsamples in fields of current interest such as metabolomics or proteomics.

Ultra-low procedural blank and the single-grain mica Rb-Sr isochron dating

Both low procedural blank and high-precision measurements of isotopic ratios are crucial for the analysis of micro samples. This study reports a chemical procedure of ultra-low blank for the high precision measurement of Sr isotopic ratios on micro samples (< 1 ng level) using a new-type thermal ionization mass spectrometer IsoProbe-T, with a case study of single-grain Rb-Sr isochron dating on phlogopite from the Fuxian kimberlite in Liaoning Province. This method can be employed in studies of high resolution Rb-Sr geochronology and Sr isotopic geochemistry and thus will broaden application of the Rb-Sr isotopic system to earth sciences.

Elemental analysis of liquid microsamples through inductively coupled plasma spectrochemistry

The elemental analysis of very low liquid sample volumes through plasma spectrometry (i.e. inductively coupled plasma atomic spectrometry (ICP-AES) and ICP mass spectrometry (ICP-MS)) is a challenging research subject because, in many fields, the sample volume is the limiting factor for the analysis. The system for introducing liquid samples has been modified in order to perform this kind of analysis and different choices have appeared. Two general solutions are reviewed: (i) the continuous introduction of the solution into the plasma as an aerosol; and, (ii) the discrete introduction of the sample into the system. Included in the first group are several nebulizers, spray chambers and desolvation systems that have been developed to work at low liquid-flow rates (i.e. below 100 μl/min). The second group includes electrothermal vaporization (ETV) of a low sample volume and its further transport to the plasma. We review these systems and discuss the analytical problems associated with each.

X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis aries) and European red deer (Cervus elaphus)

BackgroundSimple and precise methods for sex determination in animals are a pre-requisite for a number of applications in animal production and forensics. However, some of the existing methods depend only on the detection of Y-chromosome specific sequences. Therefore, the abscence of a signal does not necessarily mean that the sample is of female origin, because experimental errors can also lead to negative results. Thus, the detection of Y- and X-chromosome specific sequences is advantageous.ResultsA novel method for sex identification in mammals (sheep, Ovis aries and European red deer, Cervus elaphus) is described, using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. A partial sequence of the amelogenin gene of sheep and red deer was obtained, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. With a specific pair of primers a DNA fragment of different length between the male and female mammal was amplified.ConclusionPCR amplification using the amelogenin gene primers is useful in sex identification of samples from sheep and red deer and can be applied to DNA analysis of micro samples with small amounts of DNA such as hair roots as well as bones or embryo biopsies.

Lime–pozzolana mortars in Roman catacombs: composition, structures and restoration

Analyses of microsamples collected from Roman catacombs and samples of lime–pozzolana mortars hardened in the laboratory display higher contents in carbonated binder than other subaerial Roman monuments. The measured environmental data inside the Saint Callistus and Domitilla catacombs show a constant temperature of 15–17 °C, a high CO 2 content (1700 to 3500 ppm) and a relative humidity close to 100%. These conditions and particularly the high CO 2 concentration speed-up the lime calcitization roughly by 500% and reduce the cationic diffusion to form hydrous calcium aluminosilicates. The structure of Roman catacomb mortars shows (i) coarser aggregates and thicker beds on the inside, (ii) thin, smoothed, light and fine-grained external surfaces with low content of aggregates and (iii) paintings and frescoes on the outside. The observed high porosity of the mortars can be attributed to cracking after drying linked with the high binder content. Hardened lime lumps inside the binder denote low water/mortar ratios for slaking. The aggregate tephra pyroclasts rich in aluminosilicate phases with accessorial amounts of Ba, Sr, Rb, Cu and Pb were analysed through X-ray diffraction (XRD), electron microprobe analysis (EMPA) and also by environmental scanning electron microscopy (ESEM) to identify the size and distribution of porosity. Results support procedures using local materials, special mortars and classic techniques for restoration purposes in hypogeal backgrounds.

Compensation for matrix effects in ICP-AES by using air segmented liquid microsample introduction. The role of the spray chamber

The combination of sample injection into an air carrier stream (i.e., air segmentation) with a low sample consumption system has been evaluated for the analysis of microsamples through ICP-AES. A PFA micronebulizer has been coupled to: (i), a double pass spray chamber; (ii), a Cinnabar cyclonic spray chamber; and (iii), a torch integrated sample introduction system, TISIS. Three matrices have been studied: in addition to water two concentrated acid solutions (2 mol l−1 nitric acid and 1.7 mol l−1 acetic acid) and Na 5,000 µg ml−1. A simulation of the evolution of the drop size distributions of the aerosols with time was carried out in order to evaluate the extent of solvent evaporation inside the chamber. The total mass of solvent evaporated inside the chamber was estimated and it was concluded that, at 25 °C, about 4–6 s residence time were required to promote the maximum evaporation of the solvent. In order to ensure this, discrete sample introduction into an air carrier stream (i.e., air segmentation) was used. Narrow peaks (i.e., with a full width at half maximum, FWHM, as short as 10 s) were obtained for a 10 µl injected sample. The peaks found for the Cinnabar and TISIS were narrower than those for the double pass spray chamber. More importantly, the interferences caused by inorganic as well as organic matrices were less severe in discrete than in continuous mode. The theoretical simulations allowed explanation of these results in terms of the enhancement of the solvent evaporation both for water and matrices in this operating mode. The enhanced solvent evaporation with respect to the situation in continuous mode minimized differences in analyte transport towards the plasma induced by these compounds. Despite this, in discrete mode a residual matrix effect was found that was attributed to the aerosol transport process. Internal standardization (IS) was applied to transient signals and the interferences were compensated for in virtually all the cases. Good results were obtained for the four emission lines taken as internal standards (i.e., Mg 280.270, Co 228.616, Cr 205.552 and Cu 324.754). However, for acetic acid and a few lines, IS was not efficient for removing interferences. The methodology was validated by analyzing two reference solid samples of foods (i.e., bovine liver and mussel tissue). By using Cd 214.438 as internal standard and under discrete mode 100% recoveries were found.

Optimization of the evaporation cavity in a torch integrated sample introduction system based ICP-AES system. Applications to matrix and transient effects, analysis of microsamples and analysis of certified solid samples

Results corresponding to the optimization of the aerosol evaporation cavity of the previously published torch integrated sample introduction system (TISIS) are shown in the present work. A micronebulizer (i.e., MicroMist) operated at liquid delivery rates in the 20–200 µl min−1 range was used throughout. Theoretical calculations suggested that the TISIS chamber allowed the evaporation of a significant fraction of the solvent contained in primary aerosol, while the remaining droplets first impacted against its inner walls and then evaporated. The cavity dimensions (i.e., length and inner diameter) were optimized in terms of sensitivity and memory effects. The results demonstrated that the longer the cavity, the higher the sensitivity, whereas an increase in its diameter did not significantly modify the aforementioned parameters. The optimized TISIS cavity, with a conical shaped end, proved to significantly shorten the wash-out times with respect to a double pass spray chamber and a low inner volume cyclonic type one (i.e., a Cinnabar spray chamber). Besides, the TISIS was used for discrete sample introduction of very low sample volumes (i.e., from 0.3 to 32 µl). The peaks found were narrower and higher than those provided by a double pass spray chamber. Matrix effects caused by sodium, acetic acid and ethanol solutions were less severe for TISIS than for the double pass and the Cinnabar spray chambers, while transient effects caused by nitric acid solutions almost disappeared when using the TISIS. As a result of the mitigation of the interferences, the recoveries found for three certified solid samples while calibrating with plain water standards were closer to 100% for the TISIS than for double pass and mini cyclonic spray chambers.

An intensity ratio calibration method for the accurate determination of Mg/Ca and Sr/Ca of marine carbonates by ICP‐AES

An inductively coupled plasma‐atomic emission spectroscopy (ICP‐AES) method for the accurate and precise simultaneous measurement of the Mg/Ca and Sr/Ca content of carbonates was established. While a precision of &lt;0.3% (1σ standard deviation (SD)) is easily obtainable for both Mg/Ca and Sr/Ca analysis, a Ca matrix effect complicates achieving similar levels of accuracy with conventional calibration procedures. An alternative ratio calibration procedure is proposed which overcomes the Ca matrix effects and ensures the accuracy of the Mg/Ca and Sr/Ca analysis of marine carbonates to &lt;0.3%, almost an order of magnitude better than conventional calibration methods. The longer‐term precision is &lt;0.1% if the batch run average values are corrected for longer‐term drift. The method is suitable for analysis of foraminiferal calcite and coral aragonite and can easily be adjusted for the analysis of other carbonates or microsamples.

Sensitive micro-sample analysis of bisphenol A in serum by high-performance liquid chromatography with fluorescence detection

A rapid and sensitive HPLC method with fluorescence detection has been developed for the micro-sample analysis of bisphenol A in rat serum. Compounds were separated on a Spherisorb C18 analytical column with acetonitrile-0.1% triethylamine in deionized water, 54:46 (v/v), as mobile phase. Bisphenol A was isolated by one-step extraction with dichloromethane. The calibration plot was linear (r=0.999) over the concentration range 1–1000 ng mL−1. The limits of detection and quantitation were 0.2 ng mL−1 and 1 ng mL−1, respectively, for 100-μL serum samples. The assay was successfully used for a pharmacokinetic study after intravenous administration of a low dose (0.1 mg kg−1) of bisphenol A to rats.