Highlighting the promise of qPCR-based environmental monitoring: response of the ribosomal RNA:DNA ratio of calanoid copepods to toxic cyanobacteria.

Calanoid copepods are integral to aquatic food webs and may drive the bioaccumulation of toxins and heavy metals, spread of infectious diseases, and occurrence of toxic cyanobacterial harmful algal blooms (HABs) in freshwater aquatic systems. However, interrelationships between copepod and cyanobacterial population dynamics and ecophysiology remain unclear. Insights into these relationships are important to aquatic resource management, as they may help guide mitigation efforts. We developed a calanoid copepod qPCR assay to investigate how copepod abundance and physiological status relate to the abundance of cyanobacteria and the concentration of total microcystin in a HAB-prone freshwater multi-use eutrophic lake. Through in silico and in vitro validation of primers and analyses of time series, we demonstrate that our assay can be used as a reliable tool for environmental monitoring. Importantly, copepod RNA:DNA ratios on and shortly after the day when microcystin concentration was at its highest within the lake were not significantly lower (or higher) than before or after this period, suggesting that copepods may have been tolerant of microcystin levels observed and capable of perpetuating bloom events by consuming competitors of toxic cyanobacteria.

Development of a standardized adsorbable organofluorine screening method for wastewaters with detection by combustion ion chromatography.

Per- and polyfluoroalkyl substances (PFAS) are man-made organofluorine chemicals that can contaminate environmental waters and have gained worldwide attention over the past two decades. PFAS are most frequently detected by mass spectrometric targeted analysis methods which may not detect all the PFAS in samples. This report describes the investigation of adsorbable organofluorine (AOF) with detection by combustion ion chromatography (CIC) for detection of PFAS in surface waters and wastewaters that adsorb to granular activated carbon (GAC) with the recognition that this technique measures more than just PFAS. Overall mean recoveries of 77-120% were obtained in 17 of the 18 tested surface water and wastewater matrices spiked with perfluoropentane sulfonate (PFPeS) and 55-119% mean recoveries were obtained in 11 of the 12 surface water and wastewater matrices spiked with a PFAS mixture. Poor method performance (34-39% mean recoveries) was observed in landfill leachate wastewater. Method detection limits of 1.4-2.2 µg L-1 were achieved using 100 mL sample volumes adsorbed onto commercially available GAC. This report demonstrates that this AOF technique can be a useful screening tool for estimating organofluorine concentrations when PFAS contamination is suspected.

Human serum IgE reacts with a Metarhizium anisopliae fungal catalase.

BACKGROUND: Previous studies have demonstrated that Metarhizium anisopliae extract can induce responses characteristic of human allergic asthma in a mouse model. The study objectives were (1) to identify and characterize the M. anisopliae mycelia extract (MYC) proteins that are recognized by mouse serum IgE, (2) to determine if human serum IgE reacts with these proteins, and (3) to determine if these IgE-reactive proteins are found in other fungi. METHODS: Asthmatic human serum IgE, M. anisopliae crude antigen (MACA) immunized mouse serum IgE, and anti-catalase antibodies were used to probe one- and two-dimensional gel electrophoresis blots of MYC. RESULTS: Mass spectrometry analysis identified catalase as a mouse IgE-reactive protein. This identification was confirmed by assaying catalase activity in the extract and extract immunoblots probed with anti-catalase antibody. Six adult asthmatic sera contained IgE, but not IgG, that was reactive with mycelia extract proteins. A similar protein profile was seen when blots were probed with either mouse anti-MACA IgE or anti-bovine liver catalase antibodies. Furthermore, these mouse anti-MACA and anti-catalase antibodies were cross-reactive with other mold extracts (skin prick testing mix) and Aspergillus niger catalase. CONCLUSIONS: Some human asthmatics have developed IgE that reacts with an M. anisopliae catalase, most likely due to cross-reactivity (minimal IgG development). The cross-reactivity among fungal catalases suggests that IgE-reactive catalase might be useful for exposure assessment. Additionally, the similarity of protein profiles visualized with both human and mouse serum IgE suggests that allergy hazard identification can be facilitated using a mouse model.

Development of a U.S. EPA drinking water method for the analysis of selected perfluoroalkyl acids by solid-phase extraction and LC-MS-MS.

A drinking water method for perfluoroalkyl acids (PFAAs) is presented that addresses the occurrence monitoring needs of the U.S. Environmental Protection Agency (EPA) for a future unregulated contaminant monitoring regulation (UCMR). This paper describes the challenges associated with developing an analytical method for 14 PFAAs that will be used for drinking water occurrence monitoring. The method employs solid-phase extraction with analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The final method preservation scheme requires that samples be stored in polypropylene bottles and that they be buffered and free chlorine removed with Trizma buffer. Mean recoveries of chlorinated surface water samples fortified with the PFAAs at 40-100 ng/L (except for the perfluorooctane-sulfonamido-acetic acids at 200 ng/L) are 85-112% with < 5% relative standard deviation. Single laboratory minimum reporting limits of 2.9-14 ng/L are demonstrated with this methodology. The final method meets all of the EPA UCMR survey requirements for sample collection and storage, precision, accuracy, and sensitivity and is expected to be proposed for use under a future UCMR.

LC/MS/MS structure elucidation of reaction intermediates formed during the TiO(2) photocatalysis of microcystin-LR.

Microcystin-LR (MC-LR), a cyanotoxin and emerging drinking water contaminant, was treated with TiO(2) photocatalysts immobilized on stainless steel plates as an alternative to nanoparticles in slurry. The reaction intermediates of MC-LR were identified with mass spectrometry (MS) at pH of Milli-Q water (pH(sq)=5.7). Eleven new [M+H](+) were observed in the liquid chromatography mass spectrometry (LC/MS) chromatogram with some of them giving multiple peaks. Most of these reaction intermediates have not been reported from previous studies employing TiO(2) nanoparticles at acidic conditions (pH=4.0). Investigating the effects of pH (for 3.0

Integrated preservation and sample clean up procedures for studying water ingestion by recreational swimmers via urinary biomarker determination.

The use of cyanuric acid as a biomarker for ingestion of swimming pool water may lead to quantitative knowledge of the volume of water ingested during swimming, contributing to a better understanding of disease resulting from ingestion of environmental contaminants. When swimming pool water containing chlorinated cyanurates is inadvertently ingested, cyanuric acid is excreted quantitatively within 24 h as a urinary biomarker of ingestion. Because the volume of water ingested can be quantitatively estimated by calculation from the concentration of cyanuric acid in 24 h urine samples, a procedure for preservation, cleanup, and analysis of cyanuric acid was developed to meet the logistical demands of large scale studies. From a practical stand point, urine collected from swimmers cannot be analyzed immediately, given requirements of sample collection, shipping, handling, etc. Thus, to maintain quality control to allow confidence in the results, it is necessary to preserve the samples in a manner that ensures as quantitative analysis as possible. The preservation and clean-up of cyanuric acid in urine is complicated because typical approaches often are incompatible with the keto-enol tautomerization of cyanuric acid, interfering with cyanuric acid sample preparation, chromatography, and detection. Therefore, this paper presents a novel integration of sample preservation, clean-up, chromatography, and detection to determine cyanuric acid in 24 h urine samples. Fortification of urine with cyanuric acid (0.3-3.0 mg/L) demonstrated accuracy (86-93% recovery) and high reproducibility (RSD < 7%). Holding time studies in unpreserved urine suggested sufficient cyanuric acid stability for sample collection procedures, while longer holding times suggested instability of the unpreserved urine. Preserved urine exhibited a loss of around 0.5% after 22 days at refrigerated storage conditions of 4 °C.

The development of a matrix-assisted laser desorption/ionization mass spectrometry-based method for the protein fingerprinting and identification of Aeromonas species using whole cells.

This report describes the development of a method to detect the waterborne pathogen Aeromonas using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, MALDI-MS was applied to the characterization of seventeen species of Aeromonas. These seventeen species were represented by thirty-two strains, which included type, reference and clinical isolates. Intact cells from each strain were used to generate a reproducible library of protein mass spectral fingerprints or m/z signatures. Under the test conditions used, peak lists of the mass ions observed in each species revealed that three mass ions were conserved among all the seventeen species tested. These common mass ions having an average m/z of 6301, 12,160 or 12,254, and 13,450, can be potentially used as genus-specific biomarkers to identify Aeromonas in unknown samples. A dendrogram generated using the m/z signatures of all the strains tested indicated that the mass spectral data contained sufficient information to distinguish between genera, species, and strains. There are several advantages of using MALDI-MS based protein mass spectral fingerprinting of whole cells for the identification of microorganisms as well as for their differentiation at the sub-species level: (1) the capability to detect proteins, (2) high throughput, and (3) relatively simple sample preparation techniques. The accuracy and speed with which data can be obtained makes MALDI-MS a powerful tool especially suited for environmental monitoring and detection of biological hazards.

Development of EPA method 535 for the determination of chloroacetanilide and other acetamide herbicide degradates in drinking water by solid-phase extraction and liquid chromatography/tandem mass spectrometry.

U.S. Environmental Protection Agency (EPA) Method 535 has been developed in order to provide a method for the analysis of "Alachlor ESA and other acetanilide degradation products," which are listed on EPA's 1998 Drinking Water Contaminant Candidate List. Method 535 uses solid-phase extraction with a nonporous graphitized carbon sorbent to extract the ethane sulfonic acid (ESA) and oxanilic acid degradates of propachlor, flufenacet, dimethenamid, alachlor, acetochlor, and metolachlor from finished drinking water matrixes. Separation and quantitation of the target analytes are achieved with liquid chromatography/tandem mass spectrometry. Dimethachlor ESA and butachlor ESA were chosen during the method development as the surrogate and internal standard. Drinking water samples were dechlorinated with ammonium chloride without adversely affecting the analyte recoveries. Typical mean recoveries of 92-116% in deionized water and 89-116% in ground water were observed with relative standard deviations of <5%.

Development and Multi-laboratory Verification of US EPA Method 543 for the Analysis of Drinking Water Contaminants by Online Solid Phase Extraction-LC-MS-MS.

A drinking water method for seven pesticides and pesticide degradates is presented that addresses the occurrence monitoring needs of the US Environmental Protection Agency (EPA) for a future Unregulated Contaminant Monitoring Regulation (UCMR). The method employs online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS). Online SPE-LC-MS-MS has the potential to offer cost-effective, faster, more sensitive and more rugged methods than the traditional offline SPE approach due to complete automation of the SPE process, as well as seamless integration with the LC-MS-MS system. The method uses 2-chloroacetamide, ascorbic acid and Trizma to preserve the drinking water samples for up to 28 days. The mean recoveries in drinking water (from a surface water source) fortified with method analytes are 87.1-112% with relative standard deviations of <14%. Single laboratory lowest concentration minimum reporting levels of 0.27-1.7 ng/L are demonstrated with this methodology. Multi-laboratory data are presented that demonstrate method ruggedness and transferability. The final method meets all of the EPA's UCMR survey requirements for sample collection and storage, precision, accuracy, and sensitivity.

Novel chromatographic separation and carbon solid-phase extraction of acetanilide herbicide degradation products.

One acetamide and 5 acetanilide herbicides are currently registered for use in the United States. Over the past several years, ethanesulfonic acid (ESA) and oxanilic acid (OA) degradation products of these acetanilide/acetamide herbicides have been found in U.S. ground waters and surface waters. Alachlor ESA and other acetanilide degradation products are listed on the U.S. Environmental Protection Agency's (EPA) 1998 Drinking Water Contaminant Candidate List. Consequently, EPA is interested in obtaining national occurrence data for these contaminants in drinking water. EPA currently does not have a method for determining these acetanilide degradation products in drinking water; therefore, a research method is being developed using liquid chromatography/negative ion electrospray/mass spectrometry with solid-phase extraction (SPE). A novel chromatographic separation of the acetochlor/alachlor ESA and OA structural isomers was developed which uses an ammonium acetate-methanol gradient combined with heating the analytical column to 70 degrees C. Twelve acetanilide degradates were extracted by SPE from 100 mL water samples using carbon cartridges with mean recoveries >90% and relative standard deviations < or =16%.

Unveiling new degradation intermediates/pathways from the photocatalytic degradation of microcystin-LR.

Mass spectrometry was utilized for structural identification of the intermediates formed during the photocatalytic degradation of the cyanotoxin, microcystin-LR with immobilized TiO2 photocatalysts at neutral pH. Most of the intermediates reported herein have not been found in prior studies. Results indicate that MC-LR degradation is initiated at four sites of the toxin; three on the Adda amino acid (aromatic ring, methoxy group, and conjugated double bonds) and one on the cyclic structure (Mdha amino acid). Several intermediates gave multiple peaks in the TIC (m/z = 1011.5, 1029.5, 1063.5), which were deduced to be geometrical or constitutional isomers. This is the first study that reports the hydroxylation of the aromatic ring and the demethoxylation of MC-LR with TiO2 photocatalysis. The most targeted site was the conjugated diene bonds because of their location in the MC-LR structure. Isomerization at the C4-C5 and C6-C7 of the diene bond of the Adda chain was a direct result of hydroxyl radical addition/substitution. Based on the above, we concluded that oxidation and isomerization of the diene bonds of MC-LR occurred simultaneously. Other steps included hydroxyl substitution, further oxidation, and bond cleavage. As the reaction time progressed, simultaneous oxidation of the Adda chain and the cyclic structure occurred.

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.

Differentiation of Aeromonas isolated from drinking water distribution systems using matrix-assisted laser desorption/ionization-mass spectrometry.

The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to analyze the whole cells of both reference strains and unknown Aeromonas isolates obtained from water distribution systems. A library of over 45 unique m/z signatures was created from 40 strains that are representative of the 17 recognized species of Aeromonas, as well as 3 reference strains from genus Vibrio and 2 reference strains from Plesiomonas shigelloides. The library was used to help speciate 52 isolates of Aeromonas. The environmental isolates were broken up into 2 blind studies. Group 1 contained isolates that had a recognizable phenotypic profile and group 2 contained isolates that had an atypical phenotypic profile. MALDI-MS analysis of the water isolates in group 1 matched the phenotypic identification in all cases. In group 2, the MALDI-MS-based determination confirmed the identity of 18 of the 27 isolates. These results demonstrate that MALDI-MS analysis can rapidly and accurately classify species of the genus Aeromonas, making it a powerful tool especially suited for environmental monitoring and detection of microbial hazards in drinking water.

Mesoporous nitrogen-doped TiO2 for the photocatalytic destruction of the cyanobacterial toxin microcystin-LR under visible light irradiation.

The presence of the harmful cyanobacterial toxins in water resources worldwide drives the development of an innovative and practical water treatment technology with great urgency. This study deals with two important aspects: the fabrication of mesoporous nitrogen-doped TiO2 (N-TiO2) photocatalysts and their environmental application for the destruction of microcystin-LR (MC-LR) under visible light. In a nanotechnological sol-gel synthesis method, a nitrogen-containing surfactant (dodecylammonium chloride) was introduced as a pore templating material for tailor-designing the structural properties of TiO2 and as a nitrogen dopant for its visible light response. The resulting N-TiO2 exhibited significantly enhanced structural properties including 2-8 nm mesoporous structure (porosity 44%) and high surface area of 150 m2/g. Red shift in light absorbance up to 468 nm, 0.9 eV lower binding energy of electrons in Ti 2p state, and reduced interplanar distance of crystal lattices proved nitrogen doping in the TiO2 lattice. Due to its narrow band gap at 2.65 eV, N-TiO2 efficiently degraded MC-LR under visible spectrum above 420 nm. Acidic condition (pH 3.5) was more favorable for the adsorption and photocatalytic degradation of MC-LR on N-TiO2 due to electrostatic attraction forces between negatively charged MC-LR and +6.5 mV charged N-TiO2. Even under UV light, MC-LR was decomposed 3-4 times faster using N-TiO2 than control TiO2. The degradation pathways and reaction intermediates of MC-LR were not directly related to the energy source for TiO2 activation (UV and visible) and nature of TiO2 (neat and nitrogen-doped). This study implies a strong possibility for the in situ photocatalytic remediation of contaminated water with cyanobacterial toxins and other toxic compounds using solar light, a sustainable source of energy.

Chromatographic separation and identification of products from the reaction of dimethylarsinic acid with hydrogen sulfide.

The reaction of dimethylarsinic acid (DMAV) with hydrogen sulfide (H2S) is of biological significance and may be implicated in the overall toxicity and carcinogenicity of arsenic. The course of the reaction in aqueous phase was monitored, and an initial product, dimethylthioarsinic acid, was observed by using LC-ICP-MS and LC-ESI-MS. Dimethylarsinous acid was observed as a minor product. A second slower-forming product was identified, and the electrospray mass chromatograms for this species produced ions at m/z 275, 171, and 137 in positive mode. To aid in the identification of this slower-forming product, crystalline standards of sodium dimethyldithioarsinate and dimethylarsino dimethyldithioarsinate were prepared and re-characterized by using improved spectroscopic and structural analysis techniques. An aqueous solution of sodium dimethyldithioarsinate produced a single major chromatographic peak that matched the retention time (7.6 min) of the slower-forming product and contained similar molecular ions at m/z 275, 171, and 137 via LC-ESI-MS. The dimethylarsino dimethyldithioarsinate standard produced four aqueous phase species one of which coeluted with the slower forming product. This coeluting peak also produced the identical ESI-MS ions as the slower-forming product of DMAV + H2S. ESI-MS/MS experiments conducted on sodium dimethyldithioarsinate in deuterated water produced molecular ions at m/z 276, 173, and 137. Subsequent collisionally activated dissociation (CAD) experiments on m/z 276 did not produce a product ion at m/z 173. These data indicate that two different species are present in solution, while NMR data indicate that only dimethyldithioarsinic acid exists in aqueous solutions. This discrepancy was investigated by conducting NMR studies on the acidic solution of sodium dimethyldithioarsinate after taking this solution to dryness. The resolubilized solution produced a proton NMR signal characteristic of dimethylarsino dimethyldithioarsinate. Therefore, it was concluded that the ESI-MS ion at m/z 275 associated with the slowly forming second reaction product and the sodium dimethyldithioarsinate compound is a product of the ESI desolvation process.

An investigation of the chemical stability of arsenosugars in simulated gastric juice and acidic environments using IC-ICP-MS and IC-ESI-MS/MS.

A more quantitative extraction of arsenic-containing compounds from seafood matrices is essential in developing better dietary exposure estimates. More quantitative extraction often implies a more chemically aggressive set of extraction conditions. However, these conditions may result in undesirable chemical changes in the native arsenicals which may further complicate the toxicological risk assessment. This balance between quantitative extraction and species-specific integrity may be best addressed by using simulated gastric juice as an extraction solvent to mimic 'bioavailability'. This, conceptually, should extract the bioavailable fraction and induce any chemical changes that would occur because of ingestion. The most chemically labile species associated with seafood are thought to be the arsenosugars and for this reason their chemical stability is investigated in this study. Four arsenosugars (3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropylene glycol, As(328); 3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropanesulfonic acid, As(392); 3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxyl-2-hydroxypropyl hydrogen sulfate, As(408); and 3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate, As(482)) were isolated from seaweed extracts and subjected to simulated gastric juice and acidic conditions which mimic the stomach's pH of 1.1. Three acid solutions were used to test the chemical stability of the arsenosugars: simulated gastric juice, 78 mM nitric acid and 78 mM hydrochloric acid. The composition of the solutions was monitored over time (up to 48 h) using IC-ICP-MS for detection. The arsenosugars were found to degrade at the rate of 1.4% per h at 38 degrees C and 12.2% per h at 60 degrees C. The plots of percent conversion versus time were found to be independent of the starting arsenosugar and all had r2 values of greater than 0.97. A single common degradation product was observed in all the stability studies. A mass balance between the starting arsenosugar (As(392), As(408) and As(482)) and the degradation product was conducted with each set of experiments. This mass balance indicated that the degradation process did not produce any unchromatographable species. This degradation product was tentatively identified as As(254) as determined by ESI-MS/MS spectral data. An acid hydrolysis mechanism was proposed for the formation of As(254) from each of the native arsenosugars by hydrolysis at the C-1 carbon on the ribose ring.

An investigation of the chemical stability of arsenosugars in basic environments using IC-ICP-MS and IC-ESI-MS/MS.

This paper evaluates the chemical stability of four arsenosugars using tetramethylammonium hydroxide (TMAOH) as an extraction solvent. This solvent was chosen because of the near quantitative removal of these arsenicals from difficult to extract seafood (oysters and shellfish). Four arsenosugars (3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropylene glycol--As(328), 3-5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropanesulfonic acid--As(392), 3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulfate--As(408), and 3-[5'-deoxy-5'-(dimethylarsinoyl)-beta-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate--As(482)) were evaluated. The stability of these four arsenosugars were studied independently in a solution of 2.5% TMAOH at 60 degrees C over a period of up to 8 h. Two arsenosugars, As(328) and As(392), were found to be relatively stable in this solution for up to 8 h. However, As(408) and As(482) formed detectable quantities of dimethylarsinic acid (DMAA) and As(328) within 0.5 and 2 h, respectively. It was found that 97% of As(408) degrades after 8 h of treatment producing 3.4 times as much DMAA as As(328). This is contrary to As(482), which produces 13 times as much As(328) as DMAA and only 37% of the As(482) was converted by the 8 h treatment at 60 degrees C. These degradation products led to the investigation of weaker TMAOH extraction solvents. Three different concentrations (2.5%, 0.83% and 0.25%) were used to determine the effect of TMAOH concentration on the degradation rate of As(408). By reducing the TMAOH concentration to 0.83%, the conversion of the arsenosugar to As(328) and DMAA is nearly eliminated (less than 5% loss). Arsenosugars, As(408) and As(482), were also studied in 253 mM NaOH to verify the degradation products. The NaOH experiments were conducted to investigate a possible hydroxide based reaction mechanism. Similar degradation plots were found for each arsenosugar when compared to the 2.5% TMAOH data. A mechanism has been proposed for the formation of As(328) from As(408) and As(482) in base via an SN2 reaction (hydroxide attack) at the side chain carbon adjacent to the inorganic ester. The formation of DMAA is observed in all arsenosugars after prolonged exposure. This probably occurs via an SN2 attack at the arsenic atom.