David A. Cassada

Analysis of oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extraction and liquid chromatography–tandem mass spectrometry

A method using liquid chromatography-tandem mass spectrometry has been developed for determination of trace levels of tetracycline antibiotics in ground water and confined animal feeding operation waste water. Oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) were extracted from water samples using both polymeric and C18 extraction cartridges. The addition of a buffer containing potassium phosphate and citric acid improved tetracycline recoveries in lagoon water. Method detection limits determined in reagent water fortified with 1 microg l(-1) OTC, TC, and CTC were 0.21, 0.20, and 0.28 microg l(-1). Method detection limits in lagoon water samples fortified at 20 microg l(-1) for OTC, TC, and CTC were 3.6, 3.1, and 3.8 microg l(-1). Variability in recovery from laboratory fortified blanks ranged from 86 to 110% during routine analysis.

Enhanced Anthocyanin Extraction from Red Cabbage Using Pulsed Electric Field Processing

This study was conducted to evaluate the effect of pulsed electric field (PEF) treatment on anthocyanin extraction from red cabbage using water as a solvent. Mashed cabbage was placed in a batch treatment chamber and subjected to PEF (2.5 kV/cm electric field strength; 15 micros pulse width and 50 pulses, specific energy 15.63 J/g). Extracted anthocyanin concentrations (16 to 889 microg/mL) were determined using HPLC. Heat and light stabilities of the control and PEF-treated samples, having approximately the same initial concentrations, were studied. PEF treatments enhanced total anthocyanin extraction in water from red cabbage by 2.15 times with a higher proportion of nonacylated forms than the control (P < 0.05). The heat and light stabilities of the PEF-treated samples and control samples were not significantly different (P > 0.05). Practical Application: An innovative pretreatment technology, pulsed electric field processing, enhanced total anthocyanin extraction in water from red cabbage by 2.15 times. Manufacturers of natural colors can use this technology to extract anthocyanins from red cabbage efficiently.

Determination of atrazine, deethylatrazine and deisopropylatrazine in water and sediment by isotope dilution gas chromatography—mass spectrometry

Methods for the trace analyses of atrazine, deethylatrazine (DEA), and deisopropylatrazine (DIA) in water and sediment have been developed by using stable-isotope dilution with gas chromatography-mass spectrometry detection. Water samples are spiked with known amounts of 13C3-atrazine, 13C3-DEA and 13C3-DIA and submitted to solid-phase extraction with C18 bonded silica. Pesticides are eluted from the solid phase with ethyl acetate. Sediment samples are spiked and equilibrated with a known amount of each labeled standard before supercritical fluid extraction (SFE) using a 4% (v/v) methanol-CO2 mobile phase at 43°C and 10 MPa with off-line collection in methanol. A gas chromatograph coupled with a quadrupole mass spectrometer operated in the selected ion monitoring (SIM) mode was used to analyze the concentrated sample extracts. When compared to conventional liquid—liquid extraction methods, these methods decrease extraction time, labor, and solvent volume required. Quantification of the triazines by using isotope dilution compensates for differences in physical recovery for atrazine and its metabolites, especially when large ( > 100 ml) water volumes are extracted. Method detection limits for atrazine, DEA and DIA are 0.02, 0.02 and 0.10 μ g 1−1, respectively, in water. In sediment, the method detection limits are 0.10, 0.20 and 0.50 ng g−1 for atrazine, DEA and DIA, respectively. More than 4000 water and 800 sediment samples have been analyzed by these methods for more than two years. The average accuracy (bias) for atrazine-fortified water samples is +6.4% (n = 200) and the precision from duplicate analyses is ±6.0%. Precision of the SFE method for atrazine is ±11% at the 2 ng g−1 level whereas accuracy is −3.2% (n = 8) for recovery of 13C3-atrazine standard at the 5 ng g−1 level.

Co-occurrence of the Cyanotoxins BMAA, DABA and Anatoxin-a in Nebraska Reservoirs, Fish, and Aquatic Plants

Several groups of microorganisms are capable of producing toxins in aquatic environments. Cyanobacteria are prevalent blue green algae in freshwater systems, and many species produce cyanotoxins which include a variety of chemical irritants, hepatotoxins and neurotoxins. Production and occurrence of potent neurotoxic cyanotoxins β-N-methylamino-l-alanine (BMAA), 2,4-diaminobutyric acid dihydrochloride (DABA), and anatoxin-a are especially critical with environmental implications to public and animal health. Biomagnification, though not well understood in aquatic systems, is potentially relevant to both human and animal health effects. Because little is known regarding their presence in fresh water, we investigated the occurrence and potential for bioaccumulation of cyanotoxins in several Nebraska reservoirs. Collection and analysis of 387 environmental and biological samples (water, fish, and aquatic plant) provided a snapshot of their occurrence. A sensitive detection method was developed using solid phase extraction (SPE) in combination with high pressure liquid chromatography-fluorescence detection (HPLC/FD) with confirmation by liquid chromatography-tandem mass spectrometry (LC/MS/MS). HPLC/FD detection limits ranged from 5 to 7 µg/L and LC/MS/MS detection limits were <0.5 µg/L, while detection limits for biological samples were in the range of 0.8–3.2 ng/g depending on the matrix. Based on these methods, measurable levels of these neurotoxic compounds were detected in approximately 25% of the samples, with detections of BMAA in about 18.1%, DABA in 17.1%, and anatoxin-a in 11.9%.

Detection, occurrence, and fate of emerging contaminants in agricultural environments

A total of 43 papers published in 2014 were reviewed ranging from detailed descriptions of analytical methods, to fate and occurrence studies, to measuring and predicting biological effects for a wide variety of emerging contaminants likely to occur in agricultural environments. New methods and studies on veterinary pharmaceuticals, natural and synthetics steroids, and antibiotic resistance genes in agricultural environments continue to expand our knowledge base on the occurrence and potential impacts of these compounds. This review is divided into the following sections: Introduction, Analytical Methods, Occurrence and Fate, Antibiotic Resistance Genes, and Risk Assessment.

In vitro and in vivo safety assessment of edible blue-green algae, Nostoc commune var. sphaeroides Kützing and Spirulina plantensis

Blue-green algae (BGA) have been consumed as food and herbal medicine for centuries. However, safety for their consumption has not been well investigated. This study was undertaken to evaluate in vitro and in vivo toxicity of cultivated Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP). Neither NO nor SP contained detectable levels of microcystin (MC)-LA, MC-RR, MC-LW and MC-LR by LC/MS/MS. Cell viability remained ∼70-80% when HepG2 cells were incubated with 0-500 μg/ml of hexane, chloroform, methanol and water-extractable fractions of NO and SP. Four-week-old male and female C57BL/6J mice were fed an AIN-93G/M diet supplemented with 0%, 2.5% or 5% of NO and SP (wt/wt) for 6 months. For both genders, BGA-rich diets did not induce noticeable abnormality in weight gain and plasma alanine aminotransferase (ALT) and aspartate aminotransferase concentrations except a significant increase in plasma ALT levels by 2.5% NO supplementation in male mice at 6 month. Histopathological analysis of livers, however, indicated that BGA did not cause significant liver damage compared with controls. In conclusion, our results suggest that NO and SP are free of MC and the long-term dietary supplementation of up to 5% of the BGA may be consumed without evident toxic side-effects.

Mechanism of DNA depurination by carcinogens in relation to cancer initiation

Depurinating DNA adducts formed by aromatic hydrocarbons and catechol estrogen quinones play a major role in cancer initiation. Most of these adducts depurinate instantaneously, but some guanine adducts depurinate from DNA with half‐lives of hours. We report here, that after 10 h at 37 °C, reaction of estradiol‐3,4‐quinone (E2‐3,4‐Q) with ds‐DNA to yield N7Gua and N3Ade adducts was complete and more efficient than with ss‐DNA. When E2‐3,4‐Q reacted with t‐RNA, no adducts were detected after 10 h, and the level of N3Ade and N7Gua adducts after 10 days was less than half that with ss‐DNA after 10 h. Reaction of E2‐3,4‐Q and dG yielded 4‐OHE2‐1‐N7dG, which spontaneously depurinated to yield 4‐OHE2‐1‐N7Gua. To investigate the mechanism of depurination, E2‐3,4‐Q was reacted with carbocyclicdeoxyguanosine, in which the ring oxygen of the deoxyribose moiety is substituted with CH2, and depurination was observed. The results from this experiment demonstrate that the oxocarbenium ion mechanism plays the major role in depurination and provides the first experimental evidence for this mechanism. A newly discovered β‐elimination mechanism also plays a minor role in depurination. Understanding why the depurinating estrogen‐DNA adducts come from DNA, and not from RNA, underscores the critical role that these adducts play in initiating cancer.

Remediating dinoseb-contaminated soil with zerovalent iron.

Dinoseb, a dinitroherbicide, was once commonly used in aerial crop dusting of agronomic crops in the western United States. Widespread use combined with improper disposal practices at rural air strips has contaminated numerous sites. Our objective was to determine if zerovalent iron (Fe(0)) could remediate dinoseb-contaminated soil. This was accomplished by conducting a series of batch experiments where we first determined if Fe(0) could remove dinoseb in aqueous solutions, then in contaminated soil slurries, and finally, in unsaturated soil microcosms (25 degrees C, theta(g)=0.30 kg H(2)O kg(-1)). Results showed quantitative dinoseb removal in the presence of Fe(0) in all three media (aqueous solutions, soil slurries, moist soils) and that removal increased by including either ferrous or aluminum sulfate with the iron treatment. Incubating contaminated soils with Fe(0) or Fe(0) plus salts (FeSO(4) or Al(2)(SO(4))(3)) resulted in 100% removal of dinoseb within 7 d. Liquid chromatography/mass spectrometry (LC/MS) analysis of degradation products showed the transformations imposed by the iron treatments were reduction of one or both nitro groups to amino groups. These amino degradation products were further transformed to quinonimine and benzoquinone and did not persist. These results support the use of zerovalent iron for on-site treatment of dinoseb-contaminated soil.

Microwave spectrum, structure, and π conjugation of isocyanocyclopropane

The microwave spectrum of the normal isotopic species of isocyanocyclopropane and its three unique mono‐substituted 13C isotopic varieties have been investigated in their natural isotopic abundances. For the normal isotopic species, Stark modulation and RFMDR techniques lead to the following rotational constants: A=15 827.303±0.009 MHz, B=3734.004±0.002 MHz, and C=3515.892±0.002 MHz. These data, along with those for the three isotopic species, lead to the following heavy atom structural parameters: C1C2=1.521±0.007 A, C2C3=1.513±0.005 A, C1N=1.377±0.008 A, and C≡N=1.176±0.005 A. To within a precision of ±1°, the C–N≡C group is linear and is inclined at an angle of 123.4±0.6° to the plane of the ring. The structural parameters are compared to those of related molecules and interpreted in terms of a frontier molecular orbital model for the π conjugation of the cyclopropyl ring.

Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling

The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5 °C for 3 or 24 h after preparation) were transferred to a vacuum bag and inoculated with a three-strain C. perfringens spore cocktail to obtain an inoculum of ca. 2.5 log spores/g. The bags were vacuum-sealed, and the meat was heat treated (75 °C, 20 min) and cooled within 15 h from 54.4 to 7.2 °C. Residual nitrite was determined before and after heat treatment using ion chromatography with colorimetric detection. Cooling of ham (control) stored for 3 and 24 h, resulted in C. perfringens population increases of 1.46 and 4.20 log CFU/g, respectively. For samples that contained low NaNO2 concentrations and were stored for 3 h, C. perfringens populations of 5.22 and 2.83 log CFU/g were observed with or without sodium erythorbate, respectively. Residual nitrite was stable (p > 0.05) for both storage times. Meat processing ingredients (sodium nitrite and sodium erythorbate) and their concentrations, and storage time subsequent to preparation of meat (oxygen content) affect C. perfringens spore germination and outgrowth during abusive cooling of ham.